ristocetin and von-Willebrand-Diseases

von Willebrand disease (VWD) is a common autosomally inherited hemorrhagic disorder mainly associated with mucocutaneous bleeding. VWD is due to quantitative (type 1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF), a large multimeric plasma glycoprotein that plays a relevant role in hemostasis. VWF is essential to mediate platelet adhesion and aggregation at the sites of vascular injury under high shear stress conditions. VWF also carries coagulation factor VIII (FVIII), prolonging its half-life and concentrating it at the site of the damaged endothelium. The diagnosis of VWD, in agreement with the International Society for Thrombosis and Hemostasis guidelines, requires several assays that are necessary to evaluate the capacity of VWF to interact with several ligands, e.g. platelet glycoprotein Ibα, collagen and FVIII. Therefore, the differential diagnosis of VWD patients as type 1, 2A, 2B, 2M, 2N or 3 is a prerogative of specialized laboratories, where specific tests, like multimer analysis or ristocetin-induced platelet agglutination, are performed routinely. On the other hand, the basic identification of patients with VWD is nowadays possible in many hemostasis laboratories thanks to the availability of automated tests that measure in patient plasma VWF antigen levels and its platelet-dependent activity. Nevertheless the laboratory investigation for VWD of a subject referred for a hemorrhagic tendency should start only after the attending physician, after evaluation of his/her personal and family bleeding history, confirmed the suspicion for VWD. The purpose of this manuscript is to give an overview of the complex process that leads to the diagnosis of the VWD.

Topics: Female; Hemorrhage; Hemostasis; Humans; Male; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Von Willebrand disease (VWD) is considered the most common inherited bleeding disorder and may also be the most difficult to diagnose. Clinical symptoms of VWD include predominantly mild mucosal bleeding; surgical bleeding may occur with specific challenges and joint bleeding can occur in the most severe forms. A family history either of diagnosed VWD or of bleeding symptoms is typically present. Laboratory diagnosis requires a series of assays of von Willebrand factor (VWF) quantity and function, and factor VIII activity, with no single straightforward diagnostic test available to either confirm or exclude the diagnosis. Newer assays of VWF function are becoming more available and useful in determining the laboratory diagnosis of VWD.

Topics: Clinical Laboratory Techniques; Factor VIII; Humans; Reproducibility of Results; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

Diagnostic tests for von Willebrand disease (VWD) are important for the assessment of VWD, which is a commonly encountered bleeding disorder worldwide. Technical innovations have been applied to improve the precision and lower limit of detection of von Willebrand factor (VWF) assays, including the ristocetin cofactor activity assay (VWF:RCo) that uses the antibiotic ristocetin to induce plasma VWF binding to glycoprotein (GP) IbIXV on target platelets. VWF-collagen-binding assays, depending on the type of collagen used, can improve the detection of forms of VWD with high molecular weight VWF multimer loss, although the best method is debatable. A number of innovations have been applied to VWF:RCo (which is commonly performed on an aggregometer), including replacing the target platelets with immobilized GPIbα, and quantification by an enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric, or chemiluminescent end-point. Some common polymorphisms in the VWF gene that do not cause bleeding are associated with falsely low VWF activity by ristocetin-dependent methods. To overcome the need for ristocetin, some new VWF activity assays use gain-of-function GPIbα mutants that bind VWF without the need for ristocetin, with an improved precision and lower limit of detection than measuring VWF:RCo by aggregometry. ELISA of VWF binding to mutated GPIbα shows promise as a method to identify gain-of-function defects from type 2B VWD. The performance characteristics of many new VWF activity assays suggest that the detection of VWD, and monitoring of VWD therapy, by clinical laboratories could be improved through adopting newer generation VWF assays.

Topics: Hematologic Tests; Humans; Platelet Aggregation; Protein Multimerization; Ristocetin; von Willebrand Disease, Type 2; von Willebrand Diseases; von Willebrand Factor

The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory diagnosis of VWD can be difficult as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2) VWD variants requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity, determining the capacity of VWF to interact with the platelet GPIb-receptor. Knowing the VWF:RCo activity is essential for identifying, subtyping and monitoring VWD, but the assay is poorly standardized and many protocols do not fulfil the clinical need in all situations. This has led to the development of novel activity assays, independent of ristocetin, with enhanced assay characteristics. Results from the first independent clinical evaluations are promising, showing that they are reliable and suitable for VWD diagnosis. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that explore other activities or the size distribution of VWF multimers. These methods are discussed herein. However, in a number of patients it may be difficult to correctly classify the VWD phenotype and genetic analysis may provide the best option to clarify the disorder, through mutation identification.

Topics: Blood Coagulation Tests; Genetic Testing; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (VWD) is considered to be the most common inherited bleeding disorder. VWD is diagnosed following a clinical and physical review, with personal and familial evidence of (primarily mucocutaneous) bleeding, and confirmed by laboratory testing. The latter typically entails initial plasma testing of factor VIII coagulant, von Willebrand factor (VWF) protein ('antigen') and VWF function which has classically been assessed using the ristocetin cofactor (VWF:RCo) assay. More recent attention has focussed on other functional VWF assays, such as collagen binding and so-called 'VWF activity' assays, as possible replacements to the VWF:RCo, or as supplementary tests of VWF 'function'. Additional laboratory testing can comprise a battery of confirmatory and VWD-type assisting assays, including VWF:multimer and von Willebrand factor VIII binding. This review aims to update knowledge of current VWD diagnostics with a particular emphasis on 'functional' VWF assays.

Topics: Biological Assay; Blood Platelets; Collagen; Diagnostic Errors; Factor VIII; Humans; Phenotype; Platelet Aggregation; Protein Binding; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease is caused by either a quantitative or qualitative defect in von Willebrand factor (VWF). Patients may have extensive mucosal bleeding (because of platelet dysfunction) and prolonged bleeding after surgery (because of factor VIII deficiency). Up to 6 different subtypes of the disease have been described, and diagnosis is based on clinical suspicion and laboratory confirmation. Accurate diagnosis is of paramount importance because therapy will vary according to the subtype. Bleeding complications during pregnancy are more frequent when levels of the von Willebrand ristocetin cofactor assay and factor VIII levels are <50 IU/dL. In such cases, therapy before any invasive procedure or delivery must be instituted. The mainstays of therapy are desmopressin and plasma concentrates that contain von Willebrand factor. Delayed postpartum hemorrhage may occur, despite adequate prophylaxis. Frequent monitoring and continued prophylaxis and/or treatment are recommended for at least 2 weeks after delivery.

Topics: Blood Component Transfusion; Chromosomes, Human, Pair 12; Continuity of Patient Care; Deamino Arginine Vasopressin; Delivery, Obstetric; Factor VIII; Female; Hemostatics; Humans; Mutation; Postpartum Hemorrhage; Postpartum Period; Pregnancy; Pregnancy Complications, Hematologic; Ristocetin; Uterine Hemorrhage; von Willebrand Diseases; von Willebrand Factor

Recent multicenter studies have clarified the molecular basis underlying the different von Willebrand disease (VWD) types, all of which are caused by the deficiency and/or abnormality of von Willebrand factor (VWF). These studies have suggested a unifying pathophysiologic concept. The diagnosis of VWD, remains difficult because its clinical and laboratory phenotype is very heterogeneous and may overlap with normal subjects. Stringent criteria are therefore required for a clinically useful diagnosis. In this paper, we delineate a practical approach to the diagnosis and treatment of VWD. Our approach is based on the critical importance of a standardized bleeding history that has been condensed into a final bleeding score and a few widely available laboratory tests, such as VWF ristocetin cofactor activity, VWF antigen and factor VIII. This approach would help identify those subjects who will probably benefit from a diagnosis of VWD. The next step involves performing a trial infusion with desmopressin in all patients who fail to exhibit an enhanced responsiveness to ristocetin. On the basis of these results and through a series of illustrative examples, the clinician will be able to select the best approach for the optimal management of VWD, according to the patient's characteristics and clinical circumstances.

Topics: Adolescent; Adult; Deamino Arginine Vasopressin; Female; Hemorrhage; Hemostatics; Humans; Male; Middle Aged; Multicenter Studies as Topic; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A complete set of laboratory investigations, including bleeding time, PFA-100 closure times, factor VIII (FVIII) coagulant activity (FVIII:C), von Willebrand factor (VWF) ristocetin cofactor (VWF:RCo), collagen binding (VWF:CB), antigen (VWF:Ag) and propeptide (VWFpp), ristocetin-induced platelet aggregation (RIPA), multimeric analysis of VWF and the response of FVIII:C and VWF parameters to desmopressin (DDAVP), is necessary to fully diagnose all variants of von Willebrand disease (VWD) and to discriminate between type 1 and type 2 and between severe VWD type 1 and type 3. The response to DDAVP of VWF parameters is normal in pseudo VWD (mild VWF deficiency due to blood group O), in mild VWD type 1 and in carriers of recessive severe VWD type 1 and 3. The response to DDAVP is rather good but restricted followed by increased clearance in dominant type 1/2E, good but transient in mild type 2A group II, good for VWF:CB, with only poor response for VWF:RCo in 2M and 2U, poor in 2A group I, 2B, 2C and 2D, and very poor or non-responsive in severe recessive VWD type 1 and 3. Homozygosity or double heterozygosity for nonsense (null) mutations in the VWF gene result in recessive VWD type 3. The combination of a nonsense and missense mutation or of two missense mutations (homozygous or double heterozygous) may cause recessive severe VWD type 1. Recessive VWD type 2A subtype IIC (2C) is caused by homozygous or double heterozygous gene defects in the D1-D2 domain. Homozygosity or double heterozygosity for a FVIII binding defect of the VWF is the cause of recessive VWD type 2N (Normandy) characterized by low FVIII:C, mild or moderate VWF deficiency and normal VWF multimers. Dominant VWD type 1/2E is a mixed quantitative and qualitative multimerization defect caused by a heterozygous cysteine mutation in the D3 domain resulting in abnormal multimerization with a secretion and clearance defect of VWF not due to increased proteolysis. Dominant VWD type 1 Vicenza is a qualitative defect with normal secretion but rapid clearance with equally low levels of FVIII:C, VWF:Ag, VWF:RCo, VWF:CB and the presence of unusually large VWF multimers in plasma due to a specific mutation (R1205H) in the D3 domain. Dominant VWD type 2M and 2U are caused by loss-of-function mutations in the A1 domain resulting in quantitative/qualitative deficiencies with a selectively decreased platelet-dependent function with decreased VWF:RCo but normal VWF:CB, a relative decrease in large VWF multimer

Topics: Bleeding Time; Codon, Nonsense; Deamino Arginine Vasopressin; Genes, Dominant; Genes, Recessive; Genotype; Humans; Models, Molecular; Mutation, Missense; Platelet Aggregation; Protein Structure, Quaternary; Protein Structure, Tertiary; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Mild type 1 von Willebrand disease (VWD) is characterized by low to variable penetrance of bleeding, a high (increased) prevalence of blood group O, von Willebrand factor (VWF) values around and above 30% with normal ratios of VWF:ristocetin cofactor activity (RCo)/VWF:antigen (Ag), VWF:collagen binding (CB)/VWF:Ag and factor VIII (FVIII):coagulant activity (C)/VWF:Ag. Within this group of patients, the combination of the C1584 mutation and blood group O is rather frequent. Patients with mild VWD type 1 present good/normal responses of FVIII:C and VWF parameters to desmopressin (DDAVP). With the exclusion of dominant VWD type Vicenza, type 1/2E, recessive 2N and dominant 2M, missense mutations in patients with mild VWD type 1 with normal multimers are mainly located in the regulatory sequence region, the D1/D2 propeptide region, the D' VWF-FVIII binding site region and the D4, B1-B3 and C1-C2 domains but rarely in the D3, A1 or A2 domain. A new category of either dominant or recessive mild VWD type 1 due to mutations in the D4, B1-B3 and C1-C2 domains of the VWF gene consists of two groups: one group with mild VWD with normal VWF multimers and a second group with mild/moderate VWD with smeary multimer pattern.

Topics: ABO Blood-Group System; Bleeding Time; Codon, Nonsense; Deamino Arginine Vasopressin; Dose-Response Relationship, Drug; Genes, Dominant; Genes, Recessive; Genotype; Humans; Models, Molecular; Mutation, Missense; Platelet Aggregation; Protein Structure, Quaternary; Protein Structure, Tertiary; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A complete set of laboratory investigations, including bleeding time, PFA-100 closure time, factor VIII coagulant activity (FVIII:C), von Willebrand factor (VWF) ristocetin cofactor activity (RCo), collagen binding (CB) and antigen concentration (Ag), ristocetin-induced platelet aggregation (RIPA) and multimeric analysis of VWF in low and medium SDS-agarose resolution gels, is warranted to diagnose and classify all variants of von Willebrand disease (VWD). VWD type 2M and 2U are typically characterized by decreased RIPA and a poor response of VWF:RCo to desmopressin (DDAVP), but normal VWF:CB and good responses of VWF:CB, VWF:Ag and FVIII:C to DDAVP. VWF multimeric analysis in patients with VWD 2M and 2U show relative decreases in large VWF multimers with less resolved triplet structure of each of the multimeric bands in low-, medium- or high-resolution gels. VWD type 2M or 2U are caused by a loss-of-function mutation in the A1 domain. The laboratory manifestations and molecular defects in the A1 domain causing VWD type 2M and 2U are clearly distinct from all variants of type 1 VWD and also from all other variants [VWD type 2A, 2B, 2E (IIE) and 2C (IIC)].

Topics: Bleeding Time; Blood Protein Electrophoresis; Collagen; Deamino Arginine Vasopressin; Dose-Response Relationship, Drug; Exons; Genes, Dominant; Genotype; Humans; Models, Molecular; Molecular Weight; Mutation, Missense; Platelet Aggregation; Protein Structure, Quaternary; Protein Structure, Tertiary; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Guidelines and recommendations for the acute and prophylactic treatment of bleeding in von Willebrand disease (VWD) patients with von Willebrand factor (VWF)/factor VIII (FVIII) concentrates should be based on the analysis of the content of VWF/FVIII concentrates and on pharmacokinetic studies in patients with different severity of VWD (type 1, type 2 or type 3). The VW/FVIII concentrates should be assessed using the parameters FVIII:coagulant activity (C), VWF:ristocetin cofactor activity (RCo), VWF:collagen binding and VWF multimeric patterns for the presence of large multimers to determine their predicted efficacy and safety in prospective management studies. As the bleeding tendency is moderate in VWD type 2 and severe in type 3 and because the FVIII:C levels are subnormal in type 2 but very low in type 3 VWD patients, new guidelines using VWF:RCo unit dosing for the acute and prophylactic treatment of bleeding episodes are proposed. Such guidelines should be stratified for the severity of bleeding, the type of surgery (minor or major) and also for the bleeding score in either VWD type 1, 2 or 3.

Topics: Bleeding Time; Blood Loss, Surgical; Collagen; Deamino Arginine Vasopressin; Drug Combinations; Drug Therapy, Combination; Factor VIII; Genotype; Humans; Intraoperative Care; Platelet Aggregation; Postoperative Hemorrhage; Practice Guidelines as Topic; Preanesthetic Medication; Prospective Studies; Protein Structure, Quaternary; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Type 2B von Willebrand disease (VWD) is a qualitative type of VWD with a unique feature among VWD types, resulting from an increased binding of von Willebrand factor (VWF) to its platelet receptor glycoprotein 1b-alpha (GP1BA). This heightened responsiveness takes place in vivo without endothelial injury or shear stress induction, typically resulting in loss of the hemostatically most active high-molecular-weight VWF multimers and leading to a bleeding diathesis. This process also typically leads to clearance of platelets and thus usually mild thrombocytopenia. At least this describes the classic representation of type 2B VWD (i.e., the typical picture we have come to know since the description/classification of this disorder in the early 1990s). Over more recent years, several reports have described individual cases, groups of patients, and families diagnosed with type 2B VWD where this picture was not typical in one or more aspects. This review discusses type 2B-like disorders and sheds light on potential phenotypic modifiers that might be responsible for the variation encountered with the classic picture of type 2B VWD as well as the impact on the diagnostic certainty of type 2B VWD.

Topics: ADAM Proteins; ADAMTS13 Protein; Bleeding Time; Humans; Membrane Glycoproteins; Membrane Proteins; Mutation; Phenotype; Platelet Aggregation; Platelet Function Tests; Platelet Glycoprotein GPIb-IX Complex; Polymorphism, Genetic; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Von Willebrand disease (VWD) is known for its marked heterogeneity which was already recognized by von Willebrand in 1926. The basis of phenotypic differentiation are quantitative and qualitative or functional differences between the different types and subtypes of VWD. Clinical relevant facts for the practioner on diagnosis and therapy of von Willebrand disease and von Willebrand syndrome are presented.

Topics: Deamino Arginine Vasopressin; Factor VIII; Hemostasis; Hemostatics; Humans; Phenotype; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (VWD) is considered to be the most common inherited bleeding disorder. It is diagnosed after a clinical and physical review, with personal and familial evidence of (primarily mucocutaneous) bleeding, and confirmed by laboratory testing. The latter typically entails initial plasma testing of factor VIII coagulant (FVIII:C), von Willebrand factor (VWF) protein (antigen; VWF:Ag), and VWF function, which has classically been assessed using the ristocetin cofactor (VWF:RCo) assay. More recent attention has focused on another functional VWF assay, the collagen binding (VWF:CB) assay, as a possible replacement for the VWF:RCo assay or as a supplementary test of VWF adhesive "activity." Additional laboratory testing can comprise a battery of confirmatory and VWD subtype assisting assays, including assessment of VWF:multimers. This review updates our knowledge of VWD diagnostics with a particular emphasis on the VWF:CB assay. There is good evidence now in place that an optimized VWF:CB assay can significantly reduce the diagnostic error rate otherwise arising from the use of a test panel restricted to including the VWF:RCo assay as the sole functional VWF assay. Nevertheless, the VWF:CB assay should not be used to wholly replace the VWF:RCo assay in phenotypic testing but rather as a supplementary assay. However, with some thought and justification, the VWF:CB assay can be used to partly replace the VWF:RCo assay in some "screening" applications and can also be used to abrogate the need to perform routine VWF:multimers in most test cases.

Topics: Collagen; Diagnostic Errors; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Plasma-derived factor concentrates are important in the management of von Willebrand disease (VWD). We review the current literature regarding pharmacokinetic studies of von Willebrand factor (VWF) concentrates used to treat VWD. Using additional local experience of a crossover pharmacokinetic (PK) study comparing a currently licensed double virally inactivated concentrate, Biostate, with that of its single virally inactivated predecessor, AHF (High Purity), we propose that consideration now be given to the use of new and novel test parameters for future PK studies. In particular, we propose that an evaluation of VWF collagen binding (VWF:CB) using an assay confirmed to be sensitive to high-molecular-weight forms of VWF be used in all future studies in addition to standard parameters such as factor VIII coagulant (FVIII:C), VWF antigen (VWF:Ag), and ristocetin cofactor (VWF:RCo). We further propose the use of calculated ratios of VWF:RCo to VWF:Ag and VWF:CB capacity to VWF:Ag as a measure of delivered VWF "functionality." We also report some new data regarding assessment of VWF:multimers using standard procedures together with densitometry and a novel scoring system to support our proposals. We suggest that these test parameters may be useful in future PK studies to better assess and compare the potential utility and clinical efficacy of VWF factor concentrates for use as therapy for VWD.

Topics: Collagen; Factor VIII; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The correct diagnosis and classification of von Willebrand disease (vWD) is crucial because the presenting biological activity of von Willebrand factor (vWF) determines both the hemorrhagic risk and the subsequent clinical management. A variety of laboratory assays may be employed, not necessarily restricted to assessments of vWF. This article discusses the relative strengths and limitations of various functional or discriminatory vWF assays with a special focus on the vWF:collagen-binding activity (vWF:CBA) assay. This is a functional vWF assay that relies on the property of vWF adhesion to collagen. The vWF:CBA was first described approximately 15 years ago. The journey from that time point has been an interesting one, and the vWF:CBA is now gaining more widespread acceptance. There are now many published studies confirming the superiority of the vWF:CBA over the vWF ristocetin cofactor (vWF:RCof) activity as a functional screening diagnostic test process for vWD. However, both tests may be required in order to appropriately diagnose all forms of vWD. The relationship of these assays with multimer analysis is also discussed. In summary, an optimized vWF:CBA detects primarily high-molecular-weight (HMW) vWF forms and probably only about 30% of the total plasma vWF pool detected by vWF antigen (vWF:Ag). Because these HMW vWF forms are missing in types 2A and 2B vWD, the vWF:CBA is extremely useful in the detection of these qualitative vWD subtypes. In addition, however, concordance of vWF:CBA with vWF:Ag in unison with low vWF levels may alternatively suggest a type 1 vWD, and an absence of both vWF:Ag and vWF:CBA will suggest type 3 vWD. The vWF:CBA is also being investigated in various disease states, as is its usefulness as an effective marker of functional HMW vWF in both desmopressin (DDAVP) and factor-concentrate therapy in vWD.

Topics: Agglutination Tests; Collagen; Dimerization; Humans; Molecular Probe Techniques; Protein Binding; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (vWD), the most common of the hereditary bleeding disorders, arises from quantitative or qualitative defects in von Willebrand factor (vWF). vWF is a multimeric plasma protein that plays a key role in primary and secondary haemostasis. In the current classification scheme, vWD is divided into six subtypes that are based on the nature of the vWF defect. Therapeutic strategies depend on the accurate identification of these subtypes. In most clinical situations, desmopressin is effective treatment for the great majority of patients with mild (type 1) disease, while replacement therapy with factor VIII/vWF concentrates that contain high levels of vWF activity is required for most type 2 and nearly all type 3 vWD patients. Several factor VIII/vWF replacement products are available, one of which (Humate P) has been approved for the treatment of vWD by the US Food and Drug Administration. Preliminary results of recent studies support the hypothesis that treatment with factor VIII/vWF concentrates based upon the content of vWF activity as reflected in the ristocetin cofactor assay is practicable, safe and efficacious. The establishment of optimal treatment regimens with respect to dose intensity and duration will require further study.

Topics: Biological Assay; Drug Monitoring; Factor VIII; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A new variant of von Willebrand's disease has been discovered in 2 members of a Macedonian family of 6. The proband, an 8-year-old boy, showed a prolonged bleeding episode on 1 occasion. Ristocetin-induced platelet aggregation and bleeding time were normal. In plasma, ristocetin cofactor activity (RCo) and von Willebrand factor (vWf) antigen were reduced to the same clearly low level. The determination of vWf antigen of platelets resulted in borderline values, while RCo was clearly reduced. Low- and intermediate-resolution agarose gel electrophoresis showed absence of the largest multimers in plasma vWf, and slight reduction in platelet vWf. High-resolution gels revealed abnormal multimeric structure only in plasma vWf. The smaller multimers could be resolved in a broad central band flanked by 4 fainter satellite bands; however, satellite bands close to the central band were more intense, and more distant ones were fainter, compared to normal plasma. The central band of the fastest-moving multimer was markedly intensified, and the mobility of the whole quintuplet was slightly reduced. Heredity seems to be autosomal-dominant. No mutation was found in exon 28 of the vWf gene. Because there was only 1 mild bleeding episode in the family, this structural variation seems to have only little clinical consequence. We conclude that this vWf abnormality is different from those observed in other type II variants previously described. Based on the revised classification by the International Society on Thrombosis and Haemostasis, we proposed designation type 2A-Bern for this new subtype.

Topics: Base Sequence; Bleeding Time; Blood Platelets; Child; DNA Primers; Female; Genetic Variation; Humans; Male; Molecular Sequence Data; Platelet Aggregation; Polymerase Chain Reaction; Ristocetin; von Willebrand Diseases; von Willebrand Factor

This review summarizes the current knowledge of the structure and function of von Willebrand factor and of the pathophysiologic features, diagnosis, classification, and treatment of von Willebrand's disease, the most common congenital bleeding disorder in humans. Specific regions of the von Willebrand factor subunit that are of functional importance have been identified. The structure of these functional domains of von Willebrand factor, as known to date, is described. A classification of von Willebrand's disease, based on the definition of structural and functional abnormalities of the molecule and initial characterization of genetic mutations, is discussed. With more precise characterization of molecular abnormalities, more selective therapeutic intervention for specific subtypes of von Willebrand's disease should eventuate.

Topics: Binding Sites; Blood Transfusion; Collagen; Deamino Arginine Vasopressin; Factor VIII; Glycoproteins; Heparin; Humans; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Progress has occurred in the past several years in the understanding of the structure and function of von Willebrand factor (vWF). This multimeric glycoprotein exhibits a dual role, that of mediating platelet adhesion and aggregation onto thrombogenic surfaces, and that of functioning as carrier in plasma for the factor VIII procoagulant protein. New insights into the nature of the several functional domains of vWF have led to the identification of the regions of the molecule that interact with factor VIII, heparin, the glycoprotein lb of platelets, and collagen. Alterations of vWF are the cause of von Willebrand disease (vWD), a congenital bleeding disorder. In the majority of patients, the plasma levels of vWF are decreased, but there is no demonstrable structural or functional alteration of the protein. In other patients, however, the structure of vWF is abnormal. This review summarizes the current knowledge on vWF and vWD.

Topics: Chemical Phenomena; Chemistry; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Adenosine Diphosphate; Antibodies, Monoclonal; Bernard-Soulier Syndrome; Blood Platelets; Cell Membrane; Collagen; Endothelium; Epitopes; Glycoproteins; Humans; Immunologic Techniques; Macromolecular Substances; Platelet Adhesiveness; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Ristocetin; Thrombin; von Willebrand Diseases; von Willebrand Factor

Topics: Blood Platelets; Carbohydrates; Endothelium; Factor VIII; Humans; In Vitro Techniques; Platelet Adhesiveness; Platelet Aggregation; Protein Conformation; Ristocetin; Structure-Activity Relationship; von Willebrand Diseases; von Willebrand Factor

Recent developments regarding von Willebrand's disease, a complex disorder of hemostasis, have been helpful in understanding the clinical and laboratory manifestations of the disorder. This article discusses the pathogenesis and classification of the disorder, the role of von Willebrand factor in hemostasis, genetic counseling, and approaches to treatment.

Topics: Adolescent; Adult; Autoimmune Diseases; Blood Platelets; Blood Transfusion; Blood Vessels; Child; Child, Preschool; Epitopes; Estrogens; Factor VIII; Genetic Counseling; Heterozygote; Homozygote; Humans; Lymphoproliferative Disorders; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Antigens; Blood Platelets; Concanavalin A; Factor VIII; Humans; Immunoelectrophoresis, Two-Dimensional; Molecular Weight; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Normal human plasma contains a complex of two proteins that are important in hemostasis and coagulation. The factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related protein (von Willebrand factor) are under separate genetic control, have distinct biochemical and immunologic properties, and have unique and essential physiologic functions. While the nature of their interaction and the details of the biochemical structures remain to be determined, the information now available permits a preliminary understanding of the molecular defects in hemophilia and von Willebrand's diseases.

Topics: Animals; Antibody Formation; Blood Coagulation; Blood Coagulation Factors; Calcium; Epitopes; Factor VIII; Hemophilia A; Hemostasis; Humans; Liver; Molecular Biology; Molecular Weight; Rabbits; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Blood Platelets; Blood Vessels; Epitopes; Factor VIII; Genetic Variation; Humans; Lupus Erythematosus, Systemic; Lymphoproliferative Disorders; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Animals; Blood Coagulation; Blood Proteins; Carrier Proteins; Factor VIII; Female; Hemophilia A; Humans; Methods; Molecular Weight; Platelet Aggregation; Protein Conformation; Ristocetin; Time Factors; von Willebrand Diseases; von Willebrand Factor

Topics: Antigens; Factor VIII; Humans; Molecular Weight; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Antigens; Bleeding Time; Blood Coagulation Tests; Blood Platelets; Blood Transfusion; Factor VIII; Female; Genetic Carrier Screening; Hemophilia A; Humans; Immunoelectrophoresis; Platelet Adhesiveness; Radioimmunoassay; Radiometry; Ristocetin; von Willebrand Diseases

Topics: Binding Sites; Blood Coagulation Factors; Blood Platelets; Factor VIII; Humans; Molecular Weight; Platelet Aggregation; Ristocetin; Thrombocytopenia; von Willebrand Diseases; von Willebrand Factor

Topics: Blood Platelets; Humans; Platelet Aggregation; Ristocetin; Syndrome; Thrombocythemia, Essential; von Willebrand Diseases

Topics: Antibody Specificity; Blood Coagulation Tests; Blood Platelets; Capillary Fragility; Factor VIII; Female; Hemophilia A; Hemorrhage; Humans; Male; Menorrhagia; Plasma; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Factor VIII; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We have examined nine patients with presumed von Willebrand's disease who present the spectrum of that disorder. Two had findings that would be accepted generally as diagnostic of von Willebrand's disease, and seven had variations of the usual pattern. The commonest variation was the combination of borderline and variable levels of coagulant Factor VIII, commensurate levels of Factor VIII-related antigen, and low levels of ristocetin-Willebrand factor.

Topics: Adolescent; Adult; Antigens; Blood Coagulation; Blood Platelets; Child; Child, Preschool; Diagnosis, Differential; Factor VIII; Female; Hemophilia A; Humans; Male; Middle Aged; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Anemia, Megaloblastic; Anti-Inflammatory Agents; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Platelet Disorders; Blood Platelets; Carbenicillin; Collagen; Epinephrine; Hemorrhage; Humans; Isoantibodies; Leukemia; Myeloproliferative Disorders; Platelet Aggregation; Postoperative Complications; Purpura, Thrombocytopenic; Ristocetin; Stress, Psychological; Uremia; von Willebrand Diseases

Topics: Animals; Antibodies; Aspirin; Blood Platelets; Blood Transfusion; Disease Models, Animal; Dogs; Factor VIII; Hemostasis; History, 20th Century; Humans; Platelet Aggregation; Ristocetin; Swine; von Willebrand Diseases; von Willebrand Factor

Topics: Autoantibodies; Diagnosis, Differential; Factor VIII; Hemophilia A; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Animals; Antigens; Cold Temperature; Cross Reactions; Factor VIII; Fibrinolysin; Hemophilia A; Humans; Immune Sera; Molecular Conformation; Platelet Adhesiveness; Rabbits; Ristocetin; Thrombin; von Willebrand Diseases

Topics: Antigens; Blood Platelets; Factor VIII; Hemostasis; Humans; Ristocetin; von Willebrand Diseases

Topics: Antibodies; Antigens; Factor VIII; Humans; Neutralization Tests; Platelet Aggregation; Ristocetin; Serum Albumin; von Willebrand Diseases

Topics: Adsorption; Adult; Animals; Blood Coagulation Factors; Blood Platelets; Blood Transfusion; Chromatography; Depression, Chemical; Factor VIII; Female; Glycoproteins; Hemophilia A; Homozygote; Humans; Male; Platelet Aggregation; Receptors, Drug; Ristocetin; Time Factors; von Willebrand Diseases

Von Willebrand factor (VWF) and factor VIII (FVIII) circulate in a noncovalent complex in blood and promote primary hemostasis and clotting, respectively. A new VWF A1-domain binding aptamer, BT200, demonstrated good subcutaneous bioavailability and a long half-life in non-human primates. This first-in-human, randomized, placebo-controlled, doubleblind trial tested the hypothesis that BT200 is well tolerated and has favorable pharmacokinetic and pharmacodynamic effects in 112 volunteers. Participants received one of the following: a single ascending dose of BT200 (0.18-48 mg) subcutaneously, an intravenous dose, BT200 with concomitant desmopressin or multiple doses. Pharmacokinetics were characterized, and the pharmacodynamic effects were measured by VWF levels, FVIII clotting activity, ristocetin-induced aggregation, platelet function under high shear rates, and thrombin generation. The mean half-lives ranged from 7-12 days and subcutaneous bioavailability increased dose-dependently exceeding 55% for doses of 6-48 mg. By blocking free A1 domains, BT200 dose-dependently decreased ristocetin-induced aggregation, and prolonged collagen-adenosine diphosphate and shear-induced platelet plug formation times. However, BT200 also increased VWF antigen and FVIII levels 4-fold (P<0.001), without increasing VWF propeptide levels, indicating decreased VWF/FVIII clearance. This, in turn, increased thrombin generation and accelerated clotting. Desmopressin-induced VWF/FVIII release had additive effects on a background of BT200. Tolerability and safety were generally good, but exaggerated pharmacology was seen at saturating doses. This trial identified a novel mechanism of action for BT200: BT200 dose-dependently increases VWF/FVIII by prolonging half-life at doses well below those which inhibit VWF-mediated platelet function. This novel property can be exploited therapeutically to enhance hemostasis in congenital bleeding disorders.

Topics: Deamino Arginine Vasopressin; Factor VIII; Humans; Ristocetin; Thrombin; von Willebrand Diseases; von Willebrand Factor

The purpose of this study was to determine the effect of ABO blood groups on von Willebrand factor-ristocetin cofactor activity (vWF-RCo) and on vWF-antigen (vWF-Ag) in children who have no personal or familial history of bleeding.. A survey and testing were performed on 200 children with no personal or familial history of bleeding. In all, 100 of them belonged to blood group O, and the remaining 100 belonged to other blood groups. The blood samples were stored at -80°C for a maximum period of 2 weeks to detect vWF-RCo and vWF-Ag levels.. The mean vWF-Ag (± 2 standard deviation [SD]) level in children with blood group O was 86% (± 20%); and for those with non-O blood group, it was 98.8% (± 25%). There was a significant difference between the 2 groups (P < .001). The mean vWF-RCo (± 2 SD) level in children with blood group O was 89% (± 23%); and for those with non-O blood group, it was 103% (± 17%). There was a significant difference between those in the 2 groups (P < .001). The lowest value of vWF-Ag and vWF-RCo levels in children with blood group O was found to be 50%. In conclusion, we showed that the selection of normal ranges based on the ABO group might influence the clinical diagnosis of vWD and that while the approach of using ABO group ranges for a vWF-Ag level lower than 50 IU/dL is scientifically sound, it might not be useful to assist a clinician in identifying people at increased risk of bleeding.

Topics: ABO Blood-Group System; Adolescent; Blood Coagulation Tests; Child; Child, Preschool; Data Collection; Female; Hemorrhage; Humans; Male; Risk Factors; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We have prospectively evaluated the biologic response to desmopressin in 77 patients with type 1 von Willebrand disease (VWD) enrolled within the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD project. Complete response to desmopressin was defined as an increase of both ristocetin cofactor activity (VWF:RCo) and factor VIII coagulant activity (FVIII:C) to 50 IU/dL or higher and partial response as VWF:RCo or FVIII:C lower than 50 IU/dL after infusion, but at least 3-fold the basal level. Complete response was observed in 83% of patients; partial in 13%; and no response in 4%. Patients with some abnormality of VWF multimeric pattern had significantly lower basal FVIII:C and VWF, lower VWF:RCo/Ag ratio, and less complete responses to desmopressin than patients with a normal multimeric pattern (P=.002). Patients with mutations at codons 1130 and 1205 in the D'-D3 domain had the greatest relative increase, but shortest FVIII and VWF half-lives after infusion. Most partial and nonresponsive patients had mutations in the A1-A3 domains. Response to desmopressin in these VWD patients seemed to be associated with the location of the causative mutation. The presence of subtle multimeric abnormalities did not hamper potential clinically useful responses, as in typical type 1 VWD.

Topics: Adolescent; Adult; Aged; Blood Coagulation Tests; Child; Deamino Arginine Vasopressin; Factor VIII; Female; Genotype; Hemostatics; Humans; Male; Middle Aged; Mutation; Prospective Studies; Protein C; Protein Structure, Tertiary; Ristocetin; von Willebrand Diseases

Even though it is generally held that cryoprecipitate and fraction I-O correct the prolonged bleeding time (BT) in patients with von Willebrand disease (VWD), perusal of reported data indicates that the correction is usually short-lasting and often partial. We decided to do a controlled study of the relationship between the multimeric structure of von Willebrand factor (VWF) and the BT in five patients with severe (type III) VWD after infusion of three plasma concentrates ("wet" cryoprecipitate, lyophilized cryoprecipitate, and fraction I-O) given in random order. The dosage of concentrates was tailored from in vitro measurements to achieve post-infusion levels of ristocetin cofactor above the lower normal limit (50 U/dL) for at least 3 hours. The postinfusion BT became transiently normal in only two of five patients treated with wet cryoprecipitate, whereas it remained prolonged in all five patients treated with lyophilized cryoprecipitate or fraction I-O. For all the concentrates, the proportion of large VWF multimers calculated by scanning the electrophoretic gels were the same as those for normal standard plasmas. An intact multimeric structure was recovered in postinfusion plasmas of patients treated with wet cryoprecipitate, whereas there was a postinfusion loss of large multimers after lyophilized cryoprecipitate and fraction I-O. These findings indicate that the attainment of a normal BT is the exception rather than the rule after the infusion of three plasma fractions used in the treatment of severe VWD, and that an intact multimeric structure in concentrates and postinfusion plasmas is necessary but not sufficient to sustain a normal BT.

Topics: Bleeding Time; Chemical Phenomena; Chemistry; Factor VIII; Humans; Platelet Function Tests; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Platelet-binding Von Willebrand Factor (VWF) strings assemble upon stimulated secretion from endothelial cells.. To investigate the efficiency of platelet binding to multi-molecular VWF bundles secreted from endothelial cells and to investigate the role of osteoprotegerin, a protein located in Weibel-Palade bodies that interacts with the VWF platelet binding domain.. The nanobody VWF/AU-a11 that specifically binds to VWF in its active platelet-binding conformation was used to investigate the conformation of VWF.. Upon stimulated secretion from endothelial cells, VWF strings were only partially covered with platelets, while a VWD-type 2B mutation or ristocetin enhanced platelet binding by 2-3-fold. Osteoprotegrin, reduces platelet adhesion to VWF by 40% ± 18% in perfusion assays. siRNA-mediated down-regulation of endothelial osteoprotegerin expression resulted in a 1.8-fold increase in platelet adhesion to VWF strings. Upon viral infection, there is a concordant rise in VWF and osteoprotegerin plasma levels. Unexpectedly, no such increase was observed in plasma of desmopressin-treated hemophilia A-patients. In a mouse model, osteoprotegerin expression was low in liver endothelial cells of vehicle-treated mice, and concanavalin A-treatment increased VWF and osteoprotegerin expression 4- and 40-fold, respectively. This increase was translated in a 30-fold increased osteoprotegerin/VWF ratio in plasma.. Release of VWF from endothelial cells opens the platelet-binding site, irrespective of the presence of flow. However, not all available platelet-binding sites are being occupied, suggesting some extent of regulation. Part of this regulation involves endothelial proteins that are co-secreted with VWF, like osteoprotegerin. This regulatory mechanism may be of more relevance under inflammatory conditions.

Topics: Animals; Blood Platelets; Endothelial Cells; Humans; Mice; Osteoprotegerin; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Von Willebrand factor (VWF) level and/or function is altered in von Willebrand disease (VWD), the most common heritable bleeding disorder worldwide. Laboratory assessment of VWF is continually evolving. Historically, the primary method for the assessment of VWF platelet-binding activity was the ristocetin cofactor assay (VWF:RCo). Contemporary alternative measures of VWF platelet-binding activity include VWF:GPIbR (recombinant; using ristocetin), VWF:GPIbM (recombinant; gain-of-function mutant), and monoclonal antibody. Recently, the American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia collaboration issued guidelines recommending the use of newer assays of VWF platelet-binding activity (VWF: GPIbM, VWF: GPIbR) over VWF:RCo, given known limitations of the VWF:RCo assay. Despite this recommendation, the newer VWF:GPIbM and VWF:GPIbR assays are not United States Food and Drug Administration cleared, limiting their availability in the United States. We sought to assess assay utilization trends, agreement of VWF testing methods, and imprecision of VWF testing (based on assigned sample type) from the College of American Pathologists Proficiency Testing Surveys. The analysis confirms that, while VWF antigen testing has low imprecision, the various VWF activity assays have significant interassay variability, with VWF:RCo showing greater imprecision than the newer GPIb-binding assays. The overall trends in assay utilization reflect the barriers to complete compliance with modern VWD diagnostic guidelines in North America.

Topics: Antibodies, Monoclonal; Hemophilia A; Humans; Pathologists; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (VWD) is the most common inherited bleeding disorder and caused by an absence, deficiency or defect in von Willebrand factor (VWF). VWD is currently classified into six different types: 1, 2A, 2B, 2N, 2M, 3. Notably, 2M VWD is more often misdiagnosed as 2A or type 1 VWD than properly identified as 2M VWD.. To describe an algorithmic approach to better ensure appropriate identification of 2M VWD, and reduce its misdiagnosis, as supported by sequential laboratory testing.. Comparative assessment of types 1, 2A, 2B and 2M VWD using various laboratory tests, including VWF antigen and several VWF activity assays, plus DDAVP challenge data, ristocetin-induced platelet agglutination (RIPA) data, multimer analysis and genetic testing.. Types 1, 2A, 2B and 2M VWD give characteristic test patterns that can provisionally classify patients into particular VWD types. Notably, type 1 VWD shows low levels of VWF, but VWF functional concordance (VWF activity/Ag ratios >0.6), with both baseline assessment and post-DDAVP. Types 2A, 2B and 2M VWD show VWF functional discordance (low VWF activity/Ag ratio(s)) dependent on the defect, but type 2M separates from 2A/2B VWD based on specific test patterns, especially with collagen binding vs glycoprotein Ib binding assays. RIPA identifies 2B VWD. Multimers separate 2M from 2A/2B.. We provide strategies to improve correct diagnosis of VWD, especially focussed on 2M VWD, and which can be used by most diagnostic haemostasis laboratories, reserving genetic analysis (if required) for confirmation.

Topics: Blood Coagulation Tests; Deamino Arginine Vasopressin; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A number of new assays with different measuring principles are available to measure von Willebrand factor (VWF) glycoprotein Ib (GPIb)-binding activity, but little is known about how these assays might behave differently for subtypes of von Willebrand disease (VWD).. The Comparison of Assays to Measure VWF Activity (COMPASS-VWF) study was designed to compare all available VWF GPIb-binding activity assays for VWF. We specifically searched for particular assay behavior differences.. To sort out random differences from systematic assay behavior deviations, all assays were performed in different laboratories on the same samples in a blinded fashion. Samples from 53 normal controls and 42 well-characterized VWD patients were reanalyzed in this study to dissect assay-specific discrepancies.. No assay behavior differences were found for 53 normal controls. For VWD patients, we found the following systematic assay behavior patterns: (a) All ELISA assays for VWF:GPIbR as well as VWF:GPIbM are insensitive to detect the low VWF activity of VWD type 2B patients with loss of high molecular weight multimers; (b) VWF:Ab assay reports higher activity for the p.V1665E mutation than all other assays; and (c) all ristocetin-based assays (including VWF:RCo using fixed platelets) but the AcuStar assay report discrepantly low VWF activity for the p.P1467S polymorphism. No systematic assay-specific difference was observed for either the particle agglutination VWF:GPIbM assay or the AcuStar assay using magnetic beads.. Different assay principles may lead to discrepant results for certain VWD types or mutations. Therefore, a more extensive study for a large number of patients is needed to better characterize the incidence and relevance of such assay-specific differences.

Topics: Blood Platelets; Enzyme-Linked Immunosorbent Assay; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Hemorrhage and blood loss are still among the main causes of preventable death. Global hemostatic assays are useful point-of-care test (POCT) devices to rapidly detect cumulative effects of plasma factors and platelets on coagulation. Thromboelastography (TEG) and Thromboelastometry (ROTEM) are established methods in many anesthesiological departments for guided hemostatic treatment. However, von Willebrand disease remains undetected by standard ROTEM, especially during emergency care, despite being the most prevalent congenital hemostatic disorder.. In our monocentric cohort pilot study we focused on hemostatic challenges associated with von Willebrand disease. Twenty-seven patients with suspected von Willebrand disease were included. We modified the routine ROTEM assay by adding a preincubation with ristocetin and commercially available plasma-derived von Willebrand factor to identify clinically relevant von Willebrand disease (VWD).. Addition of von Willebrand factor to the ristocetin assay of a VWD type 3 patient restored the reaction of the whole blood probe to match the response of a healthy person. Our modified ROTEM assay with ristocetin (Ricotem) showed that all high responders (n = 7) had VWD. In the low responder group (n = 16) - 10 of 16 had VWD and in the normal responder group (n = 5), 2 of 5 had mild type 1 VWD.. This new modification of the standard ROTEM assay enables the detection of otherwise unnoticed critical von Willebrand disease based on alterations in clot formation and might serve as a novel approach to reliably assess severe VWD patients by platelet-mediated blood clotting in an emergency setting. We recommend incorporating this new VWD-focused screening tool into the current ROTEM-based management algorithm of acute microvascular bleeding.

Topics: Adolescent; Adult; Algorithms; Child; Child, Preschool; Cohort Studies; Emergency Service, Hospital; Female; Hematologic Tests; Humans; Infant; Male; Middle Aged; Pilot Projects; Point-of-Care Systems; Point-of-Care Testing; Ristocetin; Thrombelastography; von Willebrand Diseases; von Willebrand Factor; Young Adult

Laboratory diagnosis of von Willebrand disease (VWD) is made by the measurement of von Willebrand factor (VWF) protein level and its activities. Current VWF activity tests include ristocetin cofactor and collagen binding (VWF:CB) assays.. We have undertaken an evaluation of a new fully automated VWF:CB assay relative to an established enzyme-linked immunosorbent assay (ELISA) method.. The two analytical systems operate with different detection principles: a chemiluminescent method performed on ACL AcuStar Analyzer (the former) and a colorimetric ELISA by Asserachrom Stago (the latter) (type III collagen from human placenta). The HemosIL AcuStar VWF:CB assay is a chemiluminescent 2-step immunoassay that uses magnetic particles coated with a type III collagen triple-helical peptide. VWF:CB levels were determined in 50 healthy subjects and 100 VWD patients (22 type 1, 73 type 2 and 5 type 3).. Eleven VWD samples reported VWF:CB values below the lower detection limit of one or both methods. The new method showed a good correlation with the ELISA method (r > .9, mean bias 3.85 IU/dL) in both healthy and VWD samples. One of 150 samples gave inconsistent results using the two assays, leading to an uncertain diagnosis of VWD type 1 (ELISA method) or type 2 MCB (fully automated method).. The new assay is rapid and simple to use, with its ready-to-use reagent cartridges. This VWF:CB assay, in addition to the measurement of VWF:Ag and VWF:RCo made on the same platform, gives additional information for the diagnosis of VWD in both nonspecialized and reference laboratories.

Topics: Adolescent; Adult; Collagen; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoassay; Limit of Detection; Luminescent Measurements; Male; Middle Aged; Protein Binding; Reagent Kits, Diagnostic; Ristocetin; von Willebrand Diseases; von Willebrand Factor; Young Adult

von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder and can also arise as an acquired syndrome (AVWS). These disorders develop due to defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for these VWF-related disorders requires assessment of both VWF level and VWF activity, the latter requiring multiple assays because of the many functions carried out by VWF to help prevent bleeding. The current paper describes several protocols for assessment of VWF activity by means of VWF ristocetin cofactor (VWF:RCo). These assays identify VWF activity by quantitative assessment of VWF protein adhesion to platelets or other particles and subsequent detection of the adhered VWF as facilitated by inclusion of ristocetin. The most commonly performed assays for VWF:RCo comprise platelet agglutination assays, latex agglutination assays, and chemiluminescent assay (CLIA), with three of these described in this chapter.

Topics: Blood Platelets; Humans; Immunoturbidimetry; Luminescent Measurements; Platelet Function Tests; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Ristocetin-induced platelet aggregation (RIPA) is used as an in vitro test to determine the presence and integrity of the platelet glycoprotein (GP) Ibα-V-IX complex and von Willebrand factor (VWF) interaction and is usually performed using platelet-rich plasma (PRP). Impairment in the response of VWF/GPIbα-V-IX is measured with reference to several established concentrations of ristocetin and may indicate defects in VWF or in GPIbα-V-IX function. RIPA-based mixing studies comprise an additional approach to testing this interaction to help define whether defects identified by RIPA lie in VWF or in GPIbα-V-IX. For example, the correction of an abnormal RIPA trace after mixing PRP with normal plasma and rechallenging with ristocetin at 1.0 mg/mL suggests VWF function/quantity defect. RIPA mixing studies at lower doses of ristocetin (0.5 mg/mL) are recommended for discrimination of von Willebrand disease type 2B (VWD2B) from the rarer platelet-type (PT) VWD and for the phenotypic laboratory diagnosis of VWD2B. The demonstration of a plasma factor capable of inducing platelet aggregation at such low doses of ristocetin represents the hallmark for the phenotypic laboratory diagnosis of VWD2B. Moreover, since both VWD2B and PT-VWD may present with thrombocytopenia, RIPA-based mixing studies are also useful in thrombocytopenic patients in whom RIPA testing is difficult to assess.

Topics: Adolescent; Adult; Blood Platelets; Blood Specimen Collection; Child; Child, Preschool; Equipment Design; Female; Humans; Light; Male; Middle Aged; Platelet Aggregation; Platelet Function Tests; Platelet Glycoprotein GPIb-IX Complex; Reference Values; Ristocetin; von Willebrand Diseases; von Willebrand Factor; Young Adult

von Willebrand disease (VWD) was first described nearly a century ago in 1924 by Erik Adolf von Willebrand. Diagnostic testing at the time was very limited and it was not until the mid to late 1900s that more tests became available to assist with the diagnosis and classification of VWD. Two of these tests are based on ristocetin, one being ristocetin-induced platelet aggregation (RIPA) and the other the von Willebrand factor (VWF) ristocetin cofactor assay (VWF:RCo). The VWF:RCo assay provides functional assessment of in vitro VWF binding to the platelet glycoprotein (Gp) complex, GPIb-IX-V. Despite some advancements and newer technologies utilizing the principles of the original VWF:RCo assay, the original assay is still referred to as the gold standard for measurement of VWF activity. This article will review the history of VWD diagnostic assays, including RIPA and VWF:RCo over the past 40 years, as well as the newer assays that measure platelet binding with or without ristocetin, and which have been developed with the aim to potentially replace platelet-based ristocetin-dependent assays.

Topics: Blood Platelets; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Diagnosis of von Willebrand disease (VWD) requires quantitative as well as qualitative determination of von Willebrand factor (VWF) levels. For functional assessment of VWF, ristocetin cofactor assay by aggregometry is considered to be the gold standard. However, need for technical expertise, labour intensiveness, difficult standardization and high intra- and inter- assay variabilities are some of the limitations of this methodology. Various assays for determination of VWF adhesive function using different methodologies have been developed in recent years.. To evaluate the HemosIL AcuStar chemiluminescence assay (VWF:RCo[Acu]) and the HemosIL latex immunoassay (VWF:act) as diagnostic tests for VWD and identification of type 2 VWD in comparison with the ristocetin cofactor assay performed by aggregometry (VWF:RCo[Agg]).. Results from 96 samples analysed by VWF:RCo[Acu] and 128 samples by VWF:act were compared with VWF:RCo[Agg]. Sixty of these samples (25 normal, 17 type 1 and 18 type 2) were analysed by all three assays.. VWF:RCo[Acu] showed excellent agreement with VWF:RCo[Agg], and readily identified all type 2 VWD samples tested. VWF:act showed reasonable agreement with VWF:RCo[Agg] for most patients, but had a slightly lower sensitivity for detection of type 2 VWD.. VWF:RCo[Acu] assay has the potential to replace VWF:RCo[Agg] for the diagnosis of VWD.

Topics: Humans; Immunoassay; Luminescent Measurements; Microspheres; Plasma; Reference Standards; Reproducibility of Results; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

Von Willebrand Disease (VWD) is the most common inherited bleeding disorder. Measurement of von Willebrand factor (VWF) activity in plasma is often based on platelet agglutination stimulated by the ristocetin cofactor activity. Novel assays, based on latex beads with recombinant glycoprotein Ib instead of platelets, have recently been developed but it is unclear whether these can improve the diagnostic capability for VWD.. To compare four automated VWF activity methods in a mixed population of patients referred for evaluation of bleeding tendency.. The analytical performances of three ristocetin and one non-ristocetin cofactor activity assays were compared in 170 consecutive plasma samples from patients referred for VWD evaluation.. All methods correlated well with concordance correlation coefficients ranging from 0.90-0.95. However, when comparing the VWF activity/antigen ratios in samples classified as having VWD (activity <0.4 IU/mL) the number of samples below a ratio of 0.7 differed between 16 and 8%.. Despite overall correlation between assays we found that differences in classification power might interfere with the interpretation of individual samples.

Topics: Biological Assay; Blood Coagulation Tests; Female; Humans; Male; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The ability of von Willebrand factor (VWF) to bind platelet GP Ib and promote platelet plug formation is measured in vitro using the ristocetin cofactor (VWF:RCo) assay. Automated assay systems make testing more accessible for diagnosis, but do not necessarily improve sensitivity and accuracy.. We assessed the performance of a modified automated VWF:RCo assay protocol for the Behring Coagulation System (BCS(®) ) compared to other available assay methods.. Results from different VWF:RCo assays in a number of specialized commercial and research testing laboratories were compared using plasma samples with varying VWF:RCo activities (0-1.2 IU mL(-1) ). Samples were prepared by mixing VWF concentrate or plasma standard into VWF-depleted plasma. Commercially available lyophilized standard human plasma was also studied. Emphasis was put on the low measuring range. VWF:RCo accuracy was calculated based on the expected values, whereas precision was obtained from repeated measurements.. In the physiological concentration range, most of the automated tests resulted in acceptable accuracy, with varying reproducibility dependent on the method. However, several assays were inaccurate in the low measuring range. Only the modified BCS protocol showed acceptable accuracy over the entire measuring range with improved reproducibility.. A modified BCS(®) VWF:RCo method can improve sensitivity and thus enhances the measuring range. Furthermore, the modified BCS(®) assay displayed good precision. This study indicates that the specific modifications - namely the combination of increased ristocetin concentration, reduced platelet content, VWF-depleted plasma as on-board diluent and a two-curve calculation mode - reduces the issues seen with current VWF:RCo activity assays.

Topics: Automation; Blood Chemical Analysis; Factor VIII; Humans; Limit of Detection; Plasma; Platelet Aggregation; Ristocetin; Treatment Outcome; von Willebrand Diseases; von Willebrand Factor

Topics: Antibody Specificity; Autoantibodies; Diagnosis, Differential; Factor VIII; Female; Humans; Immunosuppressive Agents; Lupus Erythematosus, Systemic; Partial Thromboplastin Time; Platelet Aggregation; Prednisolone; Prothrombin Time; Ristocetin; von Willebrand Diseases; von Willebrand Factor; Young Adult

von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two-centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter-assay imprecision (AcuStar: CV% range 3.3-6.9; TOP500: CV% range 2.6-6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6-173.9 IU dL(-1) ; VWF:RCo 43.1-191.5 IU dL(-1)) and patients (range: VWF:Ag <0.3-115.1 IU dL(-1) ; VWF:RCo <0.5-57.2 IU dL(-1)) showed a good correlation between the two VWF:Ag and VWF:RCo methods (rs = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients' samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL(-1) vs. 2.2 IU dL(-1) and 0.5 IU dL(-1) vs. 4.4 IU dL(-1) respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non-specialized and reference laboratories.

Topics: Blood Coagulation Tests; Humans; Reproducibility of Results; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

Topics: Aged, 80 and over; Gingival Hemorrhage; Humans; Immunoglobulin G; Immunoglobulin lambda-Chains; Isoantibodies; Male; Monoclonal Gammopathy of Undetermined Significance; Platelet Aggregation; Ristocetin; von Willebrand Disease, Type 3; von Willebrand Diseases

Increased endothelial microparticles (EMP) as markers for endothelial activation have been associated with worse outcomes in clinical prothrombotic situations. The procoagulant properties of EMP can be attributed to the expression of phospholipids, tissue factor and von-Willebrand factor on their surface. We therefore investigated whether addition of in-vitro generated EMP modifies hemostasis in plasma from patients with severe von-Willebrand disease (VWD). A large EMP pool was obtained from stimulated endothelial cell lines and EMP concentration was quantified by flow cytometry. The influence of EMP on primary and secondary hemostasis in VWD plasma was assessed using ristocetin-induced platelet aggregation (RIPA) and thrombin generation in a calibrated automated thrombogram (CAT), respectively. After addition of EMP, there was a significant increase in the maximal aggregation level in RIPA as well as a significant shortening of lag time and time-to-peak in CAT in comparison to control buffer. In summary, in vitro-generated EMP have the potential to improve hemostasis in severe VWD plasma and these results warrant further clinical reseach regarding their contribution to the clinical bleeding phenotype as well as their potential to improve replacement therapy.

Topics: Adolescent; Adult; Anti-Bacterial Agents; Blood Platelets; Cell Line, Tumor; Cell-Derived Microparticles; Child; Child, Preschool; Endothelial Cells; Female; Hemostasis; Humans; Male; Platelet Aggregation; Ristocetin; Thrombin; von Willebrand Diseases; von Willebrand Factor; Young Adult

The development of an automated, von Willebrand factor (VWF) activity assay, Innovance(®) VWF Ac (VWF:Ac), which measures VWF binding to the platelet receptor glycoprotein Ibα without ristocetin, led us to evaluate the assay for diagnosing von Willebrand disease (VWD) and monitoring therapy.. After validating that the assay could be performed on an instrument from a different manufacturer, we compared VWF:Ac to VWF ristocetin cofactor activity (VWF:RCo) findings, including ratios of activity/antigen, for 100 healthy controls and 262 consecutive clinical samples from 217 patients (197 adults, 64 children, n = 1 age unknown) referred for VWF testing.. There was excellent correlation (R(2) = 0.96) between VWF:Ac results run at two different sites on two different instruments. VWF:Ac had greater precision and sensitivity to low levels of VWF than the VWF:RCo method. Although there was good correlation between VWF:Ac and VWF:RCo results among healthy controls and patient subjects, VWF:Ac results were undetectable and/or significantly lower than VWF:RCo among patients who had types 2A, 2B, or 2M VWD. Additionally, a higher proportion of patient samples were classified as showing qualitative defects using the VWF:Ac compared with VWF:RCo method. While most samples drawn on VWD therapy had similar VWF levels by VWF:Ac and VWF:RCo, a type 2B VWD subject on replacement had much lower activity estimated by VWF:Ac.. We conclude that Innovance(®) VWF Ac is suitable for the diagnosis, classification, and monitoring of VWD, and that it has a number of advantages over VWF:RCo method.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Automation, Laboratory; Child; Child, Preschool; Female; Hematologic Tests; Humans; Infant; Infant, Newborn; Male; Middle Aged; Quality Control; Reproducibility of Results; Ristocetin; von Willebrand Diseases; von Willebrand Factor; Young Adult

Ristocetin cofactor activity of Von Willebrand factor (VWF:RCo) and the ratio VWF:RCo to its antigen VWF:Ag are used as routine screening to estimate VWF function and to detect types of Von Willebrand disease (VWD) caused by loss of high molecular weight multimers. However, the VWF:RCo test is prone to analytic imprecisions due to various reasons. We compared an assay for VWF activity (VWF:Ac) with VWF:RCo putting emphasis on the ratios to VWF:Ag.. We evaluated 942 samples from 432 patients and evaluated three groups in detail: normal patients (normal multimers, VWF:Ag and VWF:RCo >0.5 U/ml, VWD type 1 excluded; n=258), VWD type 1 (n=76) and acquired Von Willebrand syndrome (AVWS, n=326). In addition, 30 healthy subjects were analysed.. VWF:Ac and VWF:RCo correlated well (Pearson´s r=0.96, p<0.01), so did their ratios to VWF:Ag (Pearson´s r=0.82, p<0.01). We calculated the normal range of VWF:Ac/VWF:Ag for healthy subjects as 0.8-1.16. In comparison, the reference range (mean±2std) derived from normal patient samples was 0.73-1.14. The corresponding ranges for VWF:RCo/VWF:Ag came to 0.74-1.23 (healthy) and 0.62-1.25 (normal patients). The ratios showed similar results regarding VWD type 1. The sensitivity for AVWS was higher with VWF:Ac/VWF:Ag than with VWF:RCo/VWF:Ag (97.5% versus 84.7%).. The data suggest that the results obtained with the VWF:Ac assay are comparable to that of the VWF:RCo assay. An AVWS was more reliably detected by VWF:Ac/VWF:Ag. We assume that the VWF:Ac assay could replace VWF:RCo for routine screening for AVWS, especially when prompt evaluation is required.

Topics: Adult; Antigens; Female; Hematologic Tests; Humans; Male; Middle Aged; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (VWD) is a disorder characterized by deficiency of, or defects in, von Willebrand factor (VWF). VWD was originally identified by Erik Adolf von Willebrand, who in early 1924 investigated a large family suffering from a bleeding disorder that seemed to differ from hemophilia. Erik von Willebrand undertook some initial laboratory investigations to conclude the involvement of a plasma factor, the lack of which prolonged the bleeding time, but failed to impair coagulation times and clot retraction. By the end of the 1960s, VWD was accepted as a combined deficiency of factor VIII (FVIII) and another plasma factor responsible for normal platelet adhesion. Just how these two functions were related to each other was less clear and the diagnostic tests available at the time were poorly reproducible, cumbersome, and unreliable; thus, VWD was poorly delineated from other coagulation and platelet disorders. The early 1970s saw a revolution in diagnostics when ristocetin was identified to induce platelet aggregation, and this formed the basis of the first consistent and reliable VWF "activity" test, permitting quantification of the platelet adhesive function missing in VWD. Concurrently, immunoprecipitating techniques specific for VWF were defined, and the application of such technologies permitted a clearer understanding of both VWF and VWD heterogeneity. Continued exploration of the structure and function of VWF contributed greatly to the understanding of platelet physiology, ligand receptor interaction and pathways of cellular interaction and activation. Recently, additional assays evaluating other functions of VWF, including collagen binding, platelet glycoprotein Ib binding, and FVIII binding, have improved the diagnosis of VWD. The purpose of this narrative review is to explore the history of phenotypic VWD diagnostics, with a focus on laboratory milestones from the past as well highlighting recent and ongoing innovations, and ongoing challenges and possible solutions.

Topics: Blood Coagulation Tests; Collagen; Factor VIII; History, 20th Century; History, 21st Century; Humans; Platelet Aggregation; Protein Binding; Protein Multimerization; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Antigens; Female; Hematologic Tests; Humans; Male; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The ristocetin cofactor activity assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity but remains difficult to perform, and the coefficient of variation of the method is high (about 20-30%). This study evaluated and compared the performance for measuring the VWF activity of two newly commercialised assays [VWF:Ac Innovance (VWF:Ac) and VWF:RCo Acustar (VWF:RCo Acu)] with the reference VWF:RCo aggregation in 123 pathological plasma samples. The correlation and concordance between both new tests (VWF:RCo-Acu and VWF:Ac) and the reference VWF:RCo were good. The results of the VWF activity to VWF antigen ratio were also comparable whatever the method for the classification of VWF deficiency in all patients. Our results showed that both new tests could replace the "gold standard" VWF:RCo in aggregometry with several benefits: they are fully automated, easier and faster to perform, better adapted to emergency situations if necessary.

Topics: Automation; Blood Coagulation; Blood Coagulation Tests; Calibration; Case-Control Studies; Collagen; Enzyme-Linked Immunosorbent Assay; Humans; Platelet Aggregation; Prospective Studies; Reference Values; Reproducibility of Results; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

The functional activity of von Willebrand factor (VWF) is most frequently measured by using the ristocetin cofactor assay (VWF:RCo). However, the method's drawbacks include unsatisfactory precision, sensitivity and availability of automated system applications. We have developed an alternative assay (INNOVANCE VWF Ac) that is based on the binding of VWF to recombinant glycoprotein Ib (GPIb). Two gain-of-function mutations were introduced into a GPIb fragment, allowing an assay format without ristocetin. Fully automated assay applications are available for the BCS/BCS XP systems and the Sysmex CS-2000i, Sysmex CA-7000, Sysmex CA-1500 and Sysmex CA-560 systems.The INNOVANCE VWF Ac assay measuring range extends from 4 to 600% VWF for all systems except the Sysmex CA-560 system. Within-device precision values were found to be between 2 and 7%. The limit of detection was below 2.2% VWF. In a study on the BCS XP system, a total number of 580 sample results yielded a correlation to the VWF:RCo assay of r equal to 0.99 (slope = 0.96). Very similar results were observed when von Willebrand disease samples type 1, 2A, 2B, 2M, 2N and 3 were investigated with the new assay and the VWF:RCo assay. The excellent performance data and comparability to VWF:RCo, together with the ease of use, led us to the conclusion that the ristocetin cofactor assay can be replaced by the new GPIb-binding assay to reliably diagnosing patients with von Willebrand disease.

Topics: Adult; Aged; Automation, Laboratory; Biological Assay; Female; Humans; Limit of Detection; Linear Models; Male; Middle Aged; Models, Molecular; Mutation; Platelet Glycoprotein GPIb-IX Complex; Reagent Kits, Diagnostic; Recombinant Proteins; Reproducibility of Results; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Measuring von Willebrand factor (VWF) activity is essential for the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra- and inter-assay variabilities. A new automated chemiluminescent immunoassay VWF activity has recently become commercially available (HemosIL AcuStar von Willebrand Factor Ristocetin Cofactor Activity). The main objective of this study was to evaluate this new method and to compare it with the VWF:RCo assay as the reference method. We studied 91 samples, 18 healthy volunteers samples and 73 samples from patients (VWF:RCo level <50 IU dL(-1) ): 29 type 1 VWD, 13 type 2A, 5 type 2B, 5 type 2M, 3 type 2N, 5 type 3, 4 type 3 under treatment, 5 type 3 carriers and 4 samples with other pathologies. The HemosIL AcuStar VWF:RCo assay was 96% sensitive and 100% specific for detecting VWF abnormalities. The good analytical performance, and the sensitivity and specificity of HemosIL AcuStar VWF:RCo to detect VWF deficiency renders it a suitable method for VWD screening.

Topics: Automation; Humans; Immunoassay; Luminescent Measurements; Phenotype; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Agglutination Tests; Diagnosis, Differential; Humans; Platelet Adhesiveness; Platelet Aggregation; Platelet Function Tests; Ristocetin; von Willebrand Disease, Type 2; von Willebrand Diseases

Current methods in assessing von Willebrand factor (VWF) ristocetin cofactor activity for Von Willebrand's disease (VWD) diagnosis include platelet agglutination by aggregometer or macroscopic slide examination, which are both time-consuming with suboptimal interassay and intra-assay variation. The purpose of this study is to establish a sensitive assay to detect VWF:RCo activity and evaluate its performance in VWD diagnosis.. We have established a sensitive VWF:RCo-ELISA method using a monoclonal antibody, SZ-151, to immobilize the recombinant fragment of platelet glycoprotein Ib (rfGPIbα). VWF was captured by rfGPIbα in the presence of ristocetin, and then detected by HRP-conjugated rabbit anti-human VWF IgG. We tested the VWF:RCo level by this VWF:RCo-ELISA in 25 patients with different types of VWD and 36 healthy donors, and compared this method to a previously reported ELISA using 2D4 coating antibody.. The sensitivity of VWF:RCo-ELISA was greatly improved with this assay (0.008 IU/dL compared to 0.031 IU/dL by 2D4 antibody). The interassay and intra-assay coefficient variation were 8% and 12%, respectively. The mean values (ranges) of VWF:RCo in patients with type 1, type 2A, type 2B, type 2M, and type 3 of VWD and control group are 31.8 (22.3-56.9), 4.8 (0.6-11.8), 8.6 (1.6-19.7), 3.9 (1.0-6.8), 1.0 (0.5-1.6), and 91.5 (47.3-169.2) IU/dL, respectively. The corresponding ratios (ranges) of VWF:RCo/VWF:Ag are 0.83 (0.70-1.16), 0.27 (0.08-0.58), 0.31 (0.15-0.40), 0.18 (0.14-0.21), 0.52 (0.13-1.19), and 0.92 (0.62-1.26).. The VWF:RCo-ELISA using monoclonal anti-rfGPIbα antibody SZ-151 showed improved sensitivity and reliability in detecting VWF:RCo activity, and its clinical application would facilitate the diagnosis and classification of VWD.

Topics: Enzyme-Linked Immunosorbent Assay; Humans; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases

von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF 'activity' assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen-binding (VWF:CB) and VWF mAb-based (VWF activity [VWF:Act]) assays are used by some laboratories.. To perform a cross-laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B.. A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays.. The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within-method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers.. We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura.

Topics: Antibodies, Monoclonal; Biomarkers; Calibration; Clinical Laboratory Techniques; Collagen; Drug Monitoring; Enzyme-Linked Immunosorbent Assay; Humans; Molecular Weight; Observer Variation; Predictive Value of Tests; Reference Standards; Reproducibility of Results; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (VWD) is the most common inherited bleeding disorder, but variable severity and several classification types mean that diagnosis is often not straightforward. In many countries, the assays are not readily available and/or are not well standardized. The latest methods and the basis of VWD are discussed here, together with information from the international quality assessment programme (IEQAS). Factor XIII deficiency is a rare, but important bleeding disorder, which may be missed or diagnosed late. A discussion and update on this diagnosis is considered in the final section of our review.

Topics: Clinical Laboratory Techniques; Collagen; Factor XIII Deficiency; Hemagglutinins; Hemophilia A; Humans; Platelet Aggregation; Quality Control; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)-based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα(235Y;239V)) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.

Topics: Amino Acid Substitution; Blood Chemical Analysis; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Humans; In Vitro Techniques; Membrane Glycoproteins; Mutant Proteins; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoproteins; Polymorphism, Genetic; Protein Binding; Protein Multimerization; Recombinant Proteins; Ristocetin; von Willebrand Disease, Type 1; von Willebrand Disease, Type 2; von Willebrand Disease, Type 3; von Willebrand Diseases; von Willebrand Factor

The effective diagnosis and monitoring of Von Willebrand Disease (VWD) requires an accurate assessment of ristocetin co-factor activity (VWF:RCo). Current methodologies include automated platelet aggregometry and manual visual agglutination both of which are laborious to perform and notoriously subject to a high degree of inter and intra assay variation.. We have evaluated an automated VWF:RCo assay (BC Von Willebrand Reagent, Siemens, Marberg, Germany) for use on the Sysmex CS2100i analyser (Milton Keynes, UK) and retrospectively compared the results with an in-house manual visual agglutination assay and VWF antigen (Siemens) in normal subjects and in 53 patients with various types of VWD and 23 patients following VWF therapeutic treatment.. The intra and interassay CV was improved with the automated assay (2.3% and 3.8% respectively) compared to 7% with the manual VWF:RCo assay. Good correlation was found between the two assays (r=0.91) in 53 patients with VWD. The mean manual VWF:RCo was 0.25IU/ml and mean automated VWF:RCo was 0.27IU/ml. A comparable increase in VWF:RCo following treatment, mostly with Desmopressin, was found in 13 patients with type 1 VWD (mean 3.9 fold increase with manual VWF:RCo and 3.1 fold with the automated VWF:RCo). In 13 patients with type 2 or 3 VWD following treatment mostly with concentrate , a higher increase was found with the automated VWF:RCo assay than the manual assay (mean 11.9 fold manually and mean 20.3 automated).. The automated VWF:RCo assay shows enhanced precision and analysis time in this difficult and time consuming laboratory test and its introduction should greatly improve the reliability of VWF testing.

Topics: Blood Coagulation Tests; Blood Platelets; Deamino Arginine Vasopressin; Hemostatics; Humans; Platelet Aggregation; Platelet Function Tests; Ristocetin; Time Factors; von Willebrand Diseases; von Willebrand Factor

Acquired von Willebrand syndrome (AvWS) is an uncommon complication of multiple myeloma (MM), and it is believed to be connected with paraprotein. The aim of this study was to determine the incidence of AvWS in patients with MM, and estimate the role of paraprotein in its occurrence. The study included 40 patients with MM. The plasma level of paraprotein, platelet adhesion on glass pearls, plasma von Willebrand factor antigen concentration, and ristocetin-induced platelet aggregation (RIPA) were measured initially. Absence of RIPA was found in six patients with MM (15%); however, all six of them had normal levels of von Willebrand factor antigen. Paraprotein was isolated from the serum of these patients. Platelet aggregation was measured in six healthy donors before and after addition of the isolated paraprotein. RIPA was significantly decreased in healthy donors in the presence of paraprotein (P<0·001). The same test was repeated with added human immunoglobulins for intravenous use without any change in RIPA. A significant negative correlation between plasma paraprotein level and RIPA was found (P<0·001). These investigations have shown that paraprotein is associated with AvWS in patients with MM.

Topics: Adult; Aged; Aged, 80 and over; Blood Coagulation Tests; Female; Humans; Male; Middle Aged; Multiple Myeloma; Paraproteins; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Heyde's syndrome is characterized by iron deficiency anemia due to gastrointestinal bleeding and calcific aortic stenosis. Patients with this syndrome have a bleeding diathesis due to a loss of the largest multimers of von Willebrand factor (vWF). Here we present a case of Heyde's syndrome diagnosed with abnormal closure times and normal vWF Ristocetin cofactor activity (vWF:Rco). In this case, a 79-year-old man with known aortic stenosis and recurrent gastrointestinal bleeding was cured of a life-threatening hemorrhage after the replacement of his stenotic aortic valve. Factor VIII activity (1.53  IU/ml), vWF antigen (1.26  IU/ml), vWF:Rco (1.11  IU/ ml), and the ratio of vWF antigen/vWF:Rco (0.88) were all within normal limits. Instead, to prove a defect in platelet aggregation, closure times measured with collagen/ADP and collagen/epinephrine were abnormal (>300  s). Postoperatively, these closure times normalized. What is unique about our current report is that we measured both vWF:Rco and closure times, the two readily available assays in most coagulation laboratories. vWF:Rco is a standard assay for measuring platelet activity but may miss defects in platelet aggregation that are only seen under high shear stress. As the closure times can detect such defects, it is perhaps more representative than traditional assays, and in situations such as our case, closure times may be the only method by which subtle abnormalities in vWF function could be detected.

Topics: Adenosine Diphosphate; Aged; Aortic Valve; Aortic Valve Stenosis; Blood Coagulation Tests; Collagen; Epinephrine; Factor VIII; Gastrointestinal Hemorrhage; Humans; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605-Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596-Ala1669 sequence (VWF74) and (2) VWF Asp1596-Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.

Topics: ADAM Proteins; ADAMTS13 Protein; Adult; Binding Sites; Blood Platelets; Case-Control Studies; Diabetes Mellitus, Type 2; Female; Humans; Hydrolysis; Male; Methionine; Middle Aged; Oxidative Stress; Peptide Fragments; Peroxynitrous Acid; Platelet Aggregation; Protein Multimerization; Ristocetin; Tyrosine; von Willebrand Diseases; von Willebrand Factor

Topics: Blood Platelets; Membrane Glycoproteins; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Ristocetin; von Willebrand Disease, Type 2; von Willebrand Diseases

The diagnosis of von Willebrand disease relies on abnormalities in specific tests of von Willebrand factor (VWF), including VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo). When examining healthy controls enrolled in the T. S. Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease, we, like others, found a lower mean VWF:RCo compared with VWF:Ag in African American controls and therefore sought a genetic cause for these differences. For the African American controls, the presence of 3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, was associated with a significantly lower VWF:RCo/VWF:Ag ratio, whereas the presence of D1472H alone was associated with a decreased ratio in both African American and Caucasian controls. Multivariate analysis comparing race, SNP status, and VWF:RCo/VWF:Ag ratio confirmed that only the presence of D1472H was significant. No difference was seen in VWF binding to collagen, regardless of SNP status. Similarly, no difference in activity was seen using a GPIb complex-binding assay that is independent of ristocetin. Because the VWF:RCo assay depends on ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of "VWF activity" by this assay and may not reflect a functional defect or true hemorrhagic risk.

Topics: Black or African American; Crotalid Venoms; Exons; Humans; Platelet Function Tests; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Ristocetin; von Willebrand Diseases; von Willebrand Factor

BACKGROUND, OBJECTIVES AND METHODS: An accurate, sensitive and precise assay for reliable determination of the ristocetin cofactor activity of von Willebrand factor (VWF:RCo) in plasma and von Willebrand Factor (VWF)-containing concentrates has been evaluated. The assay is based on a commercially available automated protocol with modifications including a combination of adding additional ristocetin and the use of two calibration curves for the high and low measuring ranges..  Addition of extra ristocetin resulted in improved measurement of VWF recoveries from various VWF-containing concentrates that were underestimated using the standard automated protocol. The modifications resulted in improved assay performance over an extended measuring range (2.00-0.03 IUmL(-1) ). Accuracy was tested using VWF deficiency plasma spiked with the 1st international standard (IS) for VWF concentrate. Seven dilutions, ranging from 1.80 to 0.05IUmL(-1) , were analyzed and resulted in measured concentrations between 80% and 100% of the assigned potency of the standard. Linearity was determined from the regression plot of the same concentrate dilutions and resulted in a correlation coefficient of 0.998. The repeatability, expressed as coefficient of variation, was 2% in the normal range (0.90IUmL(-1) ) and 8% at the level of 0.05IUmL(-1) . The corresponding reproducibility results were 2% and 15% at the normal and low measuring ranges, respectively..  Analysis of patients with von Willebrand disease (VWD) indicates that the modified automated BCS(®) protocol has a superior discrimination power compared with the standard protocol. This is especially true in samples with low VWF, as in patients with type 3 VWD.

Topics: Algorithms; Automation; Blood Coagulation Tests; Calibration; Humans; Reference Standards; Reproducibility of Results; Ristocetin; Temperature; von Willebrand Diseases; von Willebrand Factor

Type 2B von Willebrand disease (VWD2B) and platelet-type von Willebrand disease (PT-VWD) are rare bleeding disorders characterised by an increased ristocetin-induced platelet aggregation (RIPA) at low dose of ristocetin. It was the objective of this study to detect children with VWD2B and PT-VWD using RIPA at low dose of ristocetin (0.5 mg/ml) in the screening evaluation of bleeding disorders, and to analyse the phenotypic data along with the molecular findings. Over a 14-year period, 641 children with personal and family bleeding symptoms or bleeding from birth with previously uncharacterised haemostatic disorders were prospectively studied. Six unrelated patients (0.93%) showed RIPA at low dose of ristocetin. RIPA-based mixing studies identified that the plasma of the six probands and at least one parent from five unrelated families induced aggregation of normal platelets with the addition of low-dose ristocetin. None of the probands' platelets showed aggregation with cryoprecipitate. Low ristocetin cofactor activity/VWF antigen ratio with absent collagen binding activity or thrombocytopenia were detected respectively in only two patients. Molecular analysis of exon 28 of the VWF gene identified mutations in only three patients. No mutation in the GP1BA gene was found. In this large prospective paediatric study, the screening approach including RIPA at low dose of ristocetin permitted the detection of patients with VWD2B that would otherwise have been missed. No patient with phenotype or genotype of PT-VWD was identified. Heterogeneity of bleeding symptoms and phenotypic parameters were found among members of the same family.

Topics: Adolescent; Argentina; Blood Coagulation Tests; Child; Child, Preschool; DNA Mutational Analysis; Enzyme-Linked Immunosorbent Assay; Female; Genotype; Humans; Infant; Male; Mutation; Phenotype; Platelet Aggregation; Platelet Function Tests; Predictive Value of Tests; Prognosis; Prospective Studies; Ristocetin; von Willebrand Disease, Type 2; von Willebrand Diseases; von Willebrand Factor

Over the last decade, considerable progress has been made in the laboratory diagnosis of VWD. Precise, sensitive and automated VWF:Ag assays became widely available. The VWF:RCo performance was improved to a certain degree. However, the sensitivity, precision and general availability of automated applications is not yet optimal. Nevertheless, this type of assay is still recognized as superior to other activity assays, e. g. VWF:CBA assays and antibody-binding "activity" assays, for the detection of defects in VWF function. A decision limit of either 30 or 40 IU dl-1 VWF (VWF:RCo or VWF:Ag) is recommended for a diagnosis of type 1 VWD. Type 2 VWD can be differentiated from type 1 by calculating the VWF:RCo/VWF:Ag ratio. Improved and easier to perform multimer analysis and genetic testing are beginning to facilitate the diagnosis of the VWD type 1, 2A, 2B, 2N, 2M or 3. Within type 1 or 2, a decreased VWF survival can be detected by the VWFpp assay and its ratio to VWF:Ag. A new type of VWF activity assay, based on the binding of VWF to a GPIbα-fragment, has been developed. One assay variant does not need ristocetin as a cofactor anymore. The performance investigations presented so far are very promising. It is probable that these GPIbα-binding assays will detect functional VWF defects as the VWF:RCo assay, but are much more sensitive and precise. Fully automated applications on routine analyzers are expected to be commercialized soon.

Topics: Blood Platelets; Clinical Laboratory Techniques; Enzyme-Linked Immunosorbent Assay; Fibrinogen; Genetic Testing; Humans; Immunoassay; Ristocetin; Thrombin Time; von Willebrand Diseases; von Willebrand Factor

Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in von Willebrand factor (VWF) and diagnosed by a disproportionate decrease in VWF ristocetin cofactor activity (VWF:RCo) as compared with VWF antigen (VWF:Ag).. We report here on the spurious diagnosis of VWD in a patient with a sequence variation in the ristocetin-binding domain of VWF.. The index case had a VWF:RCo of 11 IU dL(-1), with VWF:RCo/VWF:Ag ratio of 0.09. DNA sequencing revealed a novel P1467S mutation in a known ristocetin-binding region of the A1 domain. Because of the discrepancy between the laboratory findings, consistent with type 2M VWD, and the patient's lack of bleeding symptoms, further studies were performed to determine whether this mutation affected VWF function or merely reduced its ability to interact with ristocetin.. Studies with recombinant VWF showed normal platelet binding with botrocetin, but a significant decrease in binding in response to ristocetin. Ristocetin-induced binding to recombinant GPIb was also absent, but normal binding was seen when a gain-of-function GPIb construct was used in the absence of ristocetin. VWF function under shear stress was normal when analyzed with a cone and plate(let) analyzer.. The decreased VWF:RCo seen with the P1467S sequence variation likely represents an artifact as a result of the use of ristocetin to measure VWF activity. The normal VWF function in other assays correlates with the lack of hemorrhagic symptoms, and suggests the need for more physiologically relevant assays of VWF function.

Topics: Binding Sites; Child; Female; Humans; Mutation, Missense; Platelet Function Tests; Protein Binding; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (VWD) is a common inheritable bleeding disorder caused by deficiency of von Willebrand Factor (VWF), which is involved in platelet adhesion and aggregation. We report a family consisting of three patients with VWD characterized by an apparently normal multimeric pattern, moderately decreased plasma factor VIII (FVIII) and VWF levels, and disproportionately low-plasma VWF:RCo levels. The patients were found to be heterozygous for the novel N1421K mutation, caused by a 4263C > G transversion in exon 28 of the VWF gene coding for the A1 domain. Botrocetin- and ristocetin-mediated binding of plasma VWF to GPIb were reduced in the patients. In vitro mutagenesis and expression in COS-7 cells confirmed the impairment of the mutant in botrocetin- and ristocetin-mediated VWF binding to GPIb. VWF collagen binding capacity was unaffected in plasma from the heterozygous individuals as well as in medium from transfected COS-7 cells. Our findings indicate that the N1421K substitution in the VWF affects the GPIb binding site or a recognition element by a conformational change of the A1 domain.

Topics: Amino Acid Substitution; Animals; Anti-Bacterial Agents; Binding Sites; Blood Platelets; Chlorocebus aethiops; Collagen; COS Cells; Crotalid Venoms; Factor VIII; Family; Female; Heterozygote; Humans; Male; Mutation, Missense; Pedigree; Platelet Adhesiveness; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Protein Structure, Tertiary; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Acquired loss of functional von Willebrand factor (VWF) has been termed the acquired von Willebrand syndrome (AVWS). AVWS is a rare adult-onset bleeding diathesis that is clinically similar to congenital von Willebrand disease (VWD), and occurs with a variety of autoimmune, lymphoproliferative, or myeloproliferative disorders. We have identified four patients with AVWS in association with immunoglobulin light chain (AL) amyloidosis. These patients, lacking any pre-existing or family history of abnormal bleeding, developed cutaneous, mucosal, or gastrointestinal bleeding in the course of their disease without deficiency of clotting factor X or other factors; the activated partial thromboplastin time (aPTT) was prolonged in three out of the four cases. Despite normal VWF antigen levels, VWF ristocetin cofactor activity (VWF:RCo) was low. Electrophoresis patterns of high molecular weight (HMW) VWF multimers were abnormal in two of the four cases. Two of the patients were treated with high-dose intravenous melphalan followed by autologous stem cell transplantation (HDM/SCT) and achieved hematologic remission. In these two patients, the bleeding diathesis improved and the coagulation parameters normalized, confirming a causal relationship between the plasma cell dyscrasia and the AVWS. AVWS should be considered in AL amyloidosis patients with hemorrhagic diatheses and normal clotting factor levels.

Topics: Adult; Amyloidosis; Antigens; Blood Protein Electrophoresis; Electrophoresis, Agar Gel; Hemorrhage; Humans; Immunoglobulin Light Chains; Male; Melphalan; Molecular Weight; Partial Thromboplastin Time; Peripheral Blood Stem Cell Transplantation; Remission Induction; Ristocetin; Transplantation, Autologous; von Willebrand Diseases; von Willebrand Factor

Topics: Adult; Agglutination Tests; Child, Preschool; Diagnosis, Differential; DNA Mutational Analysis; Female; Humans; Middle Aged; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Ristocetin; von Willebrand Diseases

von Willebrand's disease (VWD) is a heterogeneous bleeding disorder caused by quantitative or qualitative defects in von Willebrand factor (VWF). The diagnosis of VWD requires several laboratory tests. The aim of our study was to validate a flow cytometric test for the diagnosis of VWD and for monitoring the effects of desmopressin therapy.. Flow cytometric analysis of ristocetin-induced VWF binding to platelets was performed in platelet-rich plasma (PRP) samples from patients with VWD and from control subjects and in samples of formalin-fixed platelets in the presence of plasma from patients or controls. In 12 VWD patients the test was conducted before and 1 hour after desmopressin infusion. Results were compared with VWF:Ag, VWF:RCo, VWF:CB, RIPA, PFA-100 and the skin bleeding time.. Ristocetin-induced VWF binding to platelets, evaluated by both flow cytometry-based assays, was significantly reduced in patients with type1, 2A and 2M VWD as compared with that in healthy subjects. Patients with type 2B VWD showed reduced binding of VWF to formalin-fixed platelets, but increased binding to autologous platelets in PRP, similar to RIPA. VWF binding to platelets assessed by both flow cytometric assays correlated significantly with VWF:Ag, VWF:RCo, VWF:CB, RIPA, PFA100 and bleeding time. VWF binding to platelets increased after desmopressin infusion.. The measurement of ristocetin-induced binding of VWF to platelets by flow cytometry is a sensitive, simple and rapid test for the diagnosis of VWD and for the monitoring of the effects of desmopressin therapy. The flow cytometric assay performed with autologous platelets is useful in the identification of type 2B VWD patients.

Topics: Adolescent; Adult; Aged; Anti-Bacterial Agents; Bleeding Time; Blood Platelets; Child; Child, Preschool; Deamino Arginine Vasopressin; Female; Flow Cytometry; Hemostatics; Humans; Male; Middle Aged; Monitoring, Physiologic; Platelet Function Tests; Ristocetin; Time Factors; von Willebrand Diseases; von Willebrand Factor

Mutations in the A1 domain of von Willebrand factor (VWF) may be associated with gain of function in the VWF-platelet GPIb interaction and consumption of large VWF multimers, as seen in type 2B von Willebrand disease (VWD). We report a new VWF abnormality associated with greater VWF-GPIb interaction in the presence of all VWF multimers. The index case is a woman with a lifelong history of bleeding, found hyperresponsive to ristocetin with spontaneous platelet aggregation (SPA). She had normal factor VIII, VWF:Ag, VWF:RCo and VWF:CB levels, normal VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios, and a full panel of plasma and platelet VWF multimers. A missense mutation (4115T>G) was found in exon 28 of the VWF gene, which replaced a isoleucine with a serine at position 1372 of pre-pro-VWF (I1372S) at heterozygous level. Recombinant VWF carrying the I1372S mutation and showing a normal VWF multimer organisation was capable of inducing SPA on normal platelet-rich plasma (unlike wild-type VWF), as well as a hyper-response to ristocetin in the same platelets (0.6 mg/ml ristocetin vs. 1.2 of wild-type VWF). The new I1372S VWF mutation, characterized by SPA and hyper-responsiveness to ristocetin thus has some of the features of type 2B VWD, but not the lack of large VWF multimers, so we defined this variant as type 2B-like VWD. Why I1372S VWF is associated with bleeding symptoms, despite normal VWF levels and multimer organisation, remains to be seen.

Topics: Adult; Animals; Blood Platelets; Cell Line; Cricetinae; DNA Mutational Analysis; Exons; Female; Genotype; Hemorrhage; Humans; Mutation, Missense; Pedigree; Phenotype; Platelet Aggregation; Platelet Function Tests; Platelet Glycoprotein GPIb-IX Complex; Protein Structure, Quaternary; Protein Structure, Tertiary; Ristocetin; Transfection; von Willebrand Diseases; von Willebrand Factor

Increased affinity of Von Willebrand factor (VWF) for its platelet receptor GPIb-GPIX complex is responsible of an hemorrhagic disease, which is the Von Willebrand disease (VWD) type 2B when the molecular abnormality is located on the VWF, and the platelet-type 2B VWD when the mutation concern the platelet receptor. Haemostatic abnormalities in these bleeding disorders are similar; prolonged bleeding time, fluctuating thrombocytopenia, decreased factor VIII-VWF complex, and an increased response to low dose of ristocetin in platelets rich plasma. High molecular weight VWF multimers are decreased. We report here 2 cases of type 2B VWD and 1 case of platelet type 2B VWD. The distinction between these 2 diseases was established by studying platelet aggregation with weak doses of ristocetin in mixtures of washed platelets (of normal control or patient)+poor platelets plasma (normal or patient). In one case, VWD 2B was discovered late in a 49 years old man, and the factor VIIIC-VWF complex was not diminished. The distinction between these two congenital diseases is important for the treatment of bleeding manifestations which need VWF concentrates infusions in type 2B VWD and administration of platelets concentrates in pseudo type 2B VWD.

Topics: Adult; Electrophoresis, Polyacrylamide Gel; Female; Humans; Male; Middle Aged; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoproteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A quantitative description of bleeding symptoms in type 1 von Willebrand disease (VWD) has never been reported.. The aim was to quantitatively evaluate the severity of bleeding symptoms in type 1 VWD and its correlation with clinical and laboratory features.. Bleeding symptoms were retrospectively recorded in a European cohort of VWD type 1 families, and for each subject a quantitative bleeding score (BS) was obtained together with phenotypic tests.. A total of 712 subjects belonging to 144 families and 195 controls were available for analysis. The BS was higher in index cases than in affected family members (BS 9 vs. 5, P < 0.0001) and in unaffected family members than in controls (BS 0 vs. -1, P < 0.0001). There was no effect of ABO blood group. BS showed a strong significant inverse relation with either von Willebrand ristocetin cofactor (VWF:RCo), von Willebrand antigen (VWF:Ag) or factor VIII procoagulant activity (FVIII:C) measured at time of enrollment, even after adjustment for age, sex and blood group (P < 0.001 for all the four upper quintiles of BS vs. the first quintile, for either VWF:RCo, VWF:Ag or FVIII:C). Higher BS was related with increasing likelihood of VWD, and a mucocutaneous BS (computed from spontaneous, mucocutaneous symptoms) was strongly associated with bleeding after surgery or tooth extraction.. Quantitative analysis of bleeding symptoms is potentially useful for a more accurate diagnosis of type 1 VWD and to develop guidelines for its optimal treatment.

Topics: ABO Blood-Group System; Adolescent; Adult; Aged; Aged, 80 and over; Child; Child, Preschool; Cohort Studies; Europe; Factor VIII; Family Health; Female; Hemorrhage; Humans; Infant; Male; Middle Aged; Phenotype; Retrospective Studies; Ristocetin; Surveys and Questionnaires; von Willebrand Diseases; von Willebrand Factor

Type 2B von Willebrand disease (VWD) is characterised by an increased affinity of von Willebrand factor (VWF) for its platelet receptor glycoprotein Ib (GPIb). This feature is usually studied in vitro by a ristocetin-dependent VWF platelet-binding assay, which has some limitations as it requires [e.g. (radio)-labelled anti-VWF antibodies and normal formaldehyde-fixed platelets]. We, here, extended the applicability of an enzyme-linked immunosorbent assay-based method previously described for the measurement of ristocetin co-factor activity that used a recombinant fragment of GPIb (rfGPIb alpha) and horseradish peroxidase-labelled rabbit anti-human VWF antibodies for measuring the captured ristocetin-VWF complexes on the rfGPIb alpha. Thirty-one type 2B VWD patients from 15 families with eight different known mutations were studied. VWF in plasma from 28 of these patients bound better than normal VWF at 0.2 mg/ml ristocetin, with the ratio, optical density (OD) patient/OD normal pool plasma, higher than 1.8. For two of the three other patients with no enhanced response of plasma VWF, the platelet lysate VWF showed an enhanced binding capacity; for the last patient, the results in other members of the family are unequivocal. We conclude that, this new method for measurement of plasma or platelet VWF-binding capacity offers great advantages for correct type 2B VWD diagnosis.

Topics: Adolescent; Adult; Aged; Blood Platelets; Child; Child, Preschool; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Humans; Infant; Middle Aged; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We have previously demonstrated that the von Willebrand factor ristocetin cofactor activity (VWF:RCo), used in the diagnosis of vonWillebrand disease (VWD), can be accurately determined via ELISA by measuring the ristocetin-induced binding of VWF to a captured recombinant fragment of GPIbalpha (rfGPIbalpha, AA 1-289) (Vanhoorelbeke et al., Thromb Haemost 2000; 83: 107-13). This ELISA is more reliable than the currently used platelet agglutination test. Normal plasma contains relatively high concentrations of glycocalicin, a proteolytic fragment of GPIbalpha. We therefore studied whether non-purified plasma glycocalicin can replace rfGPIbalpha in our ELISA. Of 42 anti-GPIbalpha monoclonal antibodies (MAbs) capable of binding plasma glycocalicin, only one MAb captured glycocalicin in a spatial orientation exposing the VWF-binding site in glycocalicin, allowing a specific and dose-dependent ristocetin-mediated VWF-binding. Intra- and interassay variability were comparable with those for the rfGPIbalpha based VWF:RCo ELISA. The VWF:RCo activity of plasma from 33 normal individuals, 19 type 1, 16 type 2A, 9 type 2B, 8 type 2M and 7 type 3VWD patients was determined with this ELISA and allowed a clear identification of VWD patients. Furthermore, determination of the VWF:RCo/VWF:Ag ratio resulted in the discrimination between type 1 and type 2 VWD patients. Results for the glycocalicin based and the rfGPIbalpha based VWF:RCo ELISAs were in good agreement (r=0.943). There was also a good correlation between the glycocalicin based ELISA and the standard platelet agglutination test (r=0.963). In conclusion, to diagnose VWD, a VWF:RCo ELISA based on antibody immobilized plasma glycocalicin can be performed reliably.

Topics: Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; Humans; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Reproducibility of Results; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

Topics: Adult; Deamino Arginine Vasopressin; Female; Hemostasis, Surgical; Hemostatics; Humans; Mammaplasty; Risk Factors; Ristocetin; von Willebrand Diseases

von Willebrand disease (VWD) is one of the most common inherited bleeding disorders in the west. Limited studies from India showed a prevalence of approximately 10 per cent of VWD among the cases with hereditary bleeding disorders. VWD remains an underdiagnosed entity in India. The prevalence of different subtypes of VWD is also not known which is essential for a proper management of these cases. The present study was thus undertaken to know the prevalence of VWD and its various subtypes in the western part of our country.. A total of 796 consecutive patients presented with various bleeding manifestations were analysed. The initial screening and confirmation tests for the diagnosis of VWD included bleeding time (BT), screening coagulation tests i.e., prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), factor VIII: C assay, ristocetin-induced platelet aggregation (RIPA) and VWF antigen (VWF:Ag) estimations. VWF multimer analysis, ristocetin cofactor activity (RCOF), VWF collagen binding assay (VWF: CBA), factor VIII : VWF binding assay were also done to classify and subtype these cases.. The patients were subtyped as per the International Society of Thrombosis and Haemostasis (ISTH) criteria. Of the 796 patients screened, 58 were diagnosed as VWD. Of the 15 families with a positive family history of bleeding, 26 additional cases were diagnosed as VWD. Majority of the patients were type 3 (59.5%) with severe clinical manifestations, about 18 per cent of type 1 VWD patients were detected in this group while the prevalence of the qualitative variants of VWD i.e., type 2 VWD was found to be 19 per cent and the prevalence of various subtypes were type 2A (9.52%), type 2B (4.76%), type 2M (1.2%), type 2N (3.6%).. The high prevalence of type 3 and a low prevalence of type 1 VWD which is in contrast to the western reports, suggests the low awareness of the disease as also the underdiagnosis of the mild cases in our country.

Topics: Adult; Blood Coagulation Tests; Child; Collagen; Female; Humans; India; Male; Partial Thromboplastin Time; Prevalence; Prothrombin Time; Ristocetin; Thrombin Time; von Willebrand Diseases; von Willebrand Factor

Endothelial microparticles (EMP) released from activated or apoptotic endothelial cells (EC) are emerging as useful markers for detection of EC dysfunction. Our recent observation that EMP carry von Willebrand factor (vWf) led us to investigate their interaction with platelets. EMP were incubated with normal washed platelets in the presence or absence of ristocetin, then platelet aggregates were measured by flow cytometry. In the absence of ristocetin, negligible EMP conjugated with platelets (< 5%) but in the presence of ristocetin (1 mg mL(-1)), EMP induced up to 95% of platelets to aggregate. EMP-platelet interaction was 80% blocked by anti-CD42b, or by 0.1 microm filtration to remove EMP. Platelet aggregates induced by normal plasma or high molecular weight vWf (Humate-P) dissociated 50% within 15-25 min following 1:20 dilution. In contrast, aggregates formed with EMP persisted two- to threefold longer with the same treatment, indicating greater stability. A similar degree of prolongation of dissociation was observed using plasma from thrombotic thrombocytopenic purpura (TTP) patients compared with normal plasma. Addition of EMP to plasma from severe von Willebrand disease restored his ristocetin-induced platelet aggregation. Multimer analysis of vWf on EMP showed unusually large vWf (ULvWf). In summary, EMP carries ULvWf multimers, promote platelet aggregates, and increase the stability of the aggregates thus formed.

Topics: Cells, Cultured; Dimerization; Endothelium, Vascular; Humans; Macromolecular Substances; Platelet Aggregation; Protein Binding; Purpura, Thrombotic Thrombocytopenic; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Regular multilaboratory surveys of laboratories primarily in Australia, New Zealand, and Southeast Asia have been conducted over the past 8 years to evaluate testing proficiency in the diagnosis of von Willebrand disorder (VWD). We have reassessed the findings of these surveys with a particular emphasis on the diagnostic errors and error rates associated with particular tests or test panel limitations. The 37 plasma samples dispatched to survey participants include 9 normal samples, 4 type 1 VWD samples, 8 type 2 VWD samples (2A x 3, 2B x 3, 2M x 1, and 2N x 1), and 4 type 3 VWD samples. In addition to providing numerical test results, participant laboratories (average, n = 35) were asked to provide diagnostic interpretations of their test results regarding whether VWD was evident and, if so, the probable subtype. Although laboratories usually provided correct interpretative responses, diagnostic errors occurred in a substantial number of cases. On average, type 1 VWD plasma was misidentified as type 2 VWD plasma in 11% of cases, and laboratories that performed the ristocetin cofactor assay for von Willebrand factor (VWF:RCo) without performing the collagen-binding activity assay for VWF (VWF:CB) were 6 times more likely to make such an error than those that did perform the VWF:CB. Similarly, type 2 VWD plasma samples were misidentified as type 1 or type 3 VWD in an average of 20% of cases, and laboratories that performed the VWF:RCo without the VWF:CB were 3 times more likely to make such an error than those that performed the VWF:CB. Finally, normal plasma was misidentified as VWD plasma in an average of 5% of cases, and laboratories that performed the VWF:RCo without the VWF:CB were 10 times more likely to make such an error than those that performed the VWF:CB. We conclude that laboratories are generally proficient in their testing for VWD and that diagnostic error rates are substantially reduced when test panels are more comprehensive and include the VWF:CB.

Topics: Asia, Southeastern; Australia; Clinical Laboratory Techniques; Collagen; Data Collection; Diagnostic Errors; Humans; Practice Guidelines as Topic; Quality Control; Ristocetin; von Willebrand Diseases

von Willebrand disease (VWD), the most common inherited bleeding disorder, is very heterogeneous, both in its phenotype and genotype. One particular molecular mechanism of VWD is due to recombination events between the true gene and its pseudogene on chromosome 22. We assessed the frequency and extension of such events in 50 multi-ethnic index patients with severe VWD type 3 and in five index patients with VWD type 2M Vicenza. One additional unclassified patient had been diagnosed with possible VWD in Russia solely on a clinical basis. Gene conversions, previously thought to be rare events, were identified in >10% of our study population: in six multi-ethnic patients with severe VWD type 3, in one patient with VWD type 2M Vicenza and the Russian patient was finally diagnosed with VWD type 2B New York/Malmoe. Our results suggest a significant contribution of this particular molecular mechanism to the manifestation of VWD. The location of the gene conversions, their extension and their occurrence as homozygous, compound heterozygous or heterozygous mutations determines the resulting phenotype.

Topics: Cells, Cultured; DNA Mutational Analysis; Ethnicity; Gene Conversion; Germany; Greece; Haplotypes; Humans; India; Mutation, Missense; Phenotype; Platelet Aggregation; Ristocetin; Russia; von Willebrand Diseases; von Willebrand Factor

We have previously described a monoclonal antibody (mAb), 1C1E7, against von Willebrand factor (VWF), that increases ristocetin-induced platelet aggregation (RIPA) and induces a preferential binding of the high-molecular-weight multimers of VWF to platelet GPIb. Further investigations using a rotational viscometer at a shear rate of 4000 s(-1) could now demonstrate that shear-induced platelet aggregation (SIPA) is significantly increased with 1C1E7 and that this could be completely inhibited by the anti-GPIb mAb 6D1. In contrast, platelet adhesion to a collagen surface at a shear rate of 2600 s(-1), using a rectangular perfusion chamber, was significantly inhibited in the presence of 1C1E7. When citrated whole blood was incubated with 1C1E7, a spontaneous binding of VWF to the platelet GPIb could be demonstrated by flow cytometric analysis. Parallel to this, a decrease of the highest molecular weight multimers of VWF in the plasma was found. Platelets with bound VWF on their surface were able to form macroaggregates but were no longer able to adhere. These phenomena are very similar to the alterations described in von Willebrand's disease type 2B. The epitope of this mAb could be localized to the N-terminal part of the subunit; therefore a distant conformational change in the A1 domain of VWF is suggested.

Topics: Antibodies, Monoclonal; Epitopes; Flow Cytometry; Humans; In Vitro Techniques; Platelet Adhesiveness; Platelet Aggregation; Protein Subunits; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Blood Platelet Disorders; Dimerization; Hemostasis; History, 20th Century; History, 21st Century; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We report on a new mutation (4337T-->C) in exon 28 of the von Willebrand factor (VWF) gene, resulting in a substitution of L with P at residue 1446 (L1446P) of pre-pro-VWF. The defect is transmitted as a dominant trait and induces a reduced VWF synthesis, an abnormal VWF multimer pattern and a deficient VWF-platelet glycoprotein Ib interaction. The proband had low plasma and platelet VWF antigen levels, a reduced VWF collagen-binding capacity, and a disproportionately low VWF ristocetin cofactor activity, associated with the absence of ristocetin-induced platelet aggregation. Multimer analysis showed that the smaller multimers were slightly low, whereas the larger ones were significantly reduced or absent, with a clear cutoff between the two patterns. Similar hemostatic findings were observed in the proband's sister and nephew. Desmopressin administration restored VWF levels to near normal, but this was not so for VWF ristocetin cofactor activity or ristocetin-induced platelet aggregation. VWF multimers improved after desmopressin, moreover, with the larger forms restored and the smaller ones still relatively more represented. Recombinant P1446 VWF synthesis was reduced at heterozygous level, and its multimer pattern was similar to that observed in plasma VWF. These findings confirm the role of L1446P mutation in determining the von Willebrand disease (VWD) phenotype observed in our patients. Given the lack of large and intermediate VWF multimers, and the fact that the VWF-platelet interaction defect appears to be partially independent of multimer pattern, the VWD associated with L1446P mutation may belong to the type 2A/2M VWD variant.

Topics: Adult; Blood Platelets; Collagen; Deamino Arginine Vasopressin; Hemostasis; Hemostatics; Heterozygote; Humans; Male; Mutation; Phenotype; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or botrocetin-induced binding of VWF to platelets in association with a decreased binding to botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to botrocetin and GPIb.

Topics: Adult; Animals; Binding Sites; Blood Platelets; COS Cells; Crotalid Venoms; Electrophoresis, Polyacrylamide Gel; Humans; Male; Mutation; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Protein Folding; Protein Structure, Tertiary; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Patients with von Willebrand's disease may have normal levels of von Willebrand factor (VWF) antigen. It is therefore important to measure not only the antigen concentration but also the VWF activity. The most widely used method for measurement of VWF activity is the ristocetin cofactor assay (VWF:RCo), which is still crucial for the laboratory diagnosis of von Willebrand's disease (VWD). However, VWF:RCo has low precision, poor inter-laboratory reproducibility and requires an aggregometer. Many routine laboratories are not equipped with aggregometers but have flow cytometers instead.. In this study a simple, precise and rapid flow cytometric assay was developed for the determination of von Willebrand factor activity, utilizing formalin-fixed platelets, fluorescein isothiocyanate-conjugated chicken anti-VWF antibodies (Fab-fragments) and phycoerythrine-conjugated anti-GPIIb/IIIa antibodies.. In samples from healthy controls and from patients with von Willebrand disease type 1, the flow cytometry assay showed good correlation with the VWF:RCo assay (r2 = 0.69) and the VWF antigen assays (r2 = 0.83), which was better than the correlation between the VWF:RCo assay and VWF antigen assays (r2 = 0.72). The flow cytometry method had good within-assay and total precision, C.V. 4.2%, and C.V. 7.5%, at a mean concentration of 0.40 IU/mL, respectively. Results obtained with the flow cytometric method on samples from two patients with von Willebrand disease 2B were lower than those obtained with the antigen method in accordance with the diagnosis.. The accuracy and precision of the von Willebrand activity assay may be improved if a flow cytometer is utilized for measurement of the impact of ristocetin on binding of VWF to formalin-fixed platelets instead of measuring agglutination utilizing an aggregometer. In addition, our flow cytometric method assay enables measurement of von Willebrand factor activity at many more hospitals than was previously possible with the traditional ristocetin cofactor platelet aggregometry assay, and this trend is likely to increase in the future when routine hematological instruments are equipped with built-in flow cytometers.

Topics: Antibodies; Blood Platelets; Calibration; Flow Cytometry; Hematology; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Menorrhagia is a common clinical problem and is unexplained in more than 50% of women. Although studies suggest that von Willebrand's Disease (VWD) is found in a substantial number of women with unexplained menorrhagia, the prevalence of platelet defects in women with menorrhagia is unknown. To determine the prevalence of platelet and other hemostatic defects, we evaluated women ages 17-55 diagnosed with unexplained menorrhagia. Seventy-four women (52 white, 16 black, six other) were studied. Bleeding time was prolonged in 23 women (31.5%). Maximal percent platelet aggregation was decreased with one or more agonists in 35 (47.3%) women. The most commonly found platelet function defects were reduced aggregation responses to ristocetin in 22 women and to epinephrine in 16 women. Sixteen of 22 women with reduced ristocetin aggregation had von Willebrand ristocetin cofactor (VWF:RCo) and von Willebrand factor antigen (VWF:Ag) > 60%. Platelet ATP release was decreased with one or more agonists in 43 (58.1%) women. Of the black women studied, 11/16 (69%) had abnormal platelet aggregation studies compared with 20/52 white women (39%) (P = 0.06). Black women with menorrhagia had a higher prevalence of decreased platelet aggregation in response to ristocetin and epinephrine than did white women (P = 0.0075, P = 0.02). Ten women (13.5%) had VWF:RCo and/or VWF:Ag < 60%. Using race and blood group specific ranges, 5 (6.8%) women had decreased VWF:RCo, VWF:Ag and/or collagen binding (VWF:CB). Mild factor XI deficiency was found in two women and one woman with mild factor V deficiency and one hemophilia A carrier were identified. We conclude that the prevalence of platelet function defects and other inherited bleeding disorders is substantial in a multiracial US population of women with unexplained menorrhagia.

Topics: Adolescent; Adult; Blood Coagulation Factors; Blood Coagulation Tests; Blood Platelet Disorders; Epinephrine; Female; Humans; Menorrhagia; Middle Aged; Platelet Aggregation; Platelet Function Tests; Prevalence; Racial Groups; Ristocetin; von Willebrand Diseases

Tests based on three different principles are reported to measure the activity of von Willebrand factor (VWF): ristocetin cofactor (VWF:RCo), collagen binding (VWF:CB), and the so-called "activity ELISA" (VWF:MoAb). We measured these and other diagnostic parameters in a population of 123 randomly selected female study controls, age 18-45 years. Type O subjects had significantly lower levels than non-O subjects in each test. Race differences were seen in all tests except VWF:RCo, with Caucasians having significantly lower levels than African-Americans. ABO differences accounted for 19% of the total variance in VWF:Ag (P < 0.0001) and race for 7% (P < 0.0001), for a total of 26%. Both effects were mediated through VWF:Ag and were independent. VWF:Ag level was the primary determinant of VWF function, accounting for approximately 60% of the variance in VWF:RCo and VWF:CB and 54% of the variance in factor VIII. The ratio VWF:RCo/VWF:Ag differed significantly by race within blood group. The median ratios were 0.97 for type O Caucasians vs. 0.79 for type O African-Americans and 0.94 for non-O Caucasians vs. 0.76 for non-O African-Americans. The ratio VWF:CB/VWF:Ag did not vary. This suggests racial differences in the interaction of VWF with GP1b but not with subendothelium. Alternatively, VWF:RCo may be regulated to maintain a relatively constant plasma level in the presence of excessive VWF:Ag. This heterogeneity within the normal population is partially responsible for the difficulty in defining diagnostic limits for von Willebrand disease.

Topics: ABO Blood-Group System; Adolescent; Adult; Black People; Blood Grouping and Crossmatching; Blood Proteins; Collagen; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Glycoproteins; Humans; Immunoglobulins; Middle Aged; Normal Distribution; Racial Groups; Ristocetin; von Willebrand Diseases; von Willebrand Factor; White People

Interaction between platelet glycoprotein (GP)Ibalpha and von Willebrand factor (VWF) has critical roles in both physiological hemostasis and thrombosis. Platelet-type von Willebrand disease (plt-VWD) is a congenital bleeding disorder characterized by gain-of-function mutations of GPIbalpha. To date, two mutations in GPIbalpha, G233V and M239V, have been reported in four unrelated families with plt-VWD.. The present study aimed to determine whether G233S of GPIbalpha, a new mutation observed in plt-VWD patients, causes the plt-VWD phenotype and to examine whether conversions to other residues at this position affect VWF binding.. The propositus was a 3-year-old Japanese male. He displayed bleeding symptoms and moderate thrombocytopenia. His brother was similarly affected. Platelets from both patients were analyzed by ristocetin- or shear-induced platelet aggregation. DNA sequencing was performed to analyze the GPIbalpha sequence. We examined the 125I-labeled VWF binding using a series of recombinant GPIbalpha fragments with different residues at position 233 (G233S, G233A, G233K, and G233D) together with naturally occurring mutations previously reported in patients (G233V and M239V).. Platelet function analysis indicated that platelets from both patients had a typical plt-VWD phenotype. DNA sequencing analysis showed a heterozygous mutation of Gly to Ser at residue 233 of GPIbalpha in both patients. The 125I-labeled VWF binding to mutant compared with the wild type displayed three patterns, gain-of-function (G233S, G233V, and M239V), equivalent function (G233A), and loss-of-function (G233K and G233D).. The G233S is a molecular basis of plt-VWD, and residue 233 plays critical roles in regulating VWF binding.

Topics: Bleeding Time; Blood Platelets; Blood Proteins; Cell Line; Child, Preschool; Dose-Response Relationship, Drug; Family Health; Genetic Vectors; Genotype; Glycine; Glycoproteins; Hemorrhage; Heterozygote; Humans; Immunoglobulins; Japan; Male; Mutation; Phenotype; Platelet Aggregation; Point Mutation; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Ristocetin; Serine; Thrombocytopenia; von Willebrand Diseases; von Willebrand Factor

Four hundred and ninety seven patients were referred to our center for platelet aggregation studies because of spontaneous mucocutaneous bleeds. All these patients had normal complete blood count, platelet count and peripheral smears except in ten patients of Bernard Soulier Syndrome where platelet count was marginally reduced in the presence of giant platelets. Two hundred and eighty patients were found to have normal platelet aggregation to ADP, collagen, ristocetin and arachidonic acid. Out of the remaining 217 patients, 62 patients were diagnosed to have Glanzmanns thrombasthenia, 10 Bernard Soulier Syndrome, 6 storage pool deficiencies, 7 cyclooxygenase deficiencies and 72 von Willebrand disease. In all the patients with GT and BSS, diagnosis was confirmed with flow cytometry using multiple monoclonal antibodies to GPIIb-IIIa and GPIb-IX. There were sixty patients where initial platelet aggregation studies showed reduced (<30%) aggregation to either ADP, collagen, ristocetin or arachidonic acid in its various combination, however in 12 such patients (20%) the platelet aggregation studies were normal on repetition. All our platelet aggregation studies were done only after assuring that the patient is not taking any medicine for at least 7-10 days which may affect the platelet function tests. The present study shows that single atypically abnormal platelet aggregation studies should always be repeated. Finally in 48/217 patients (22%) some aggregation abnormality to one or more of the agonists persisted, although we could not categorize these patients into any clear-cut platelet disorder. None of these 48 patients platelet associated immunoglobulin was increased by flow cytometry. It is possible that large number of patients from that disorder will finally prove to be some form of platelet secretory defect. In north India similar group of defect in a large number of patients have been reported as isolated PF3 abnormality or thrombasthenic thrombopathy.

Topics: Adenosine Diphosphate; Arachidonic Acid; Bernard-Soulier Syndrome; Blood Coagulation Disorders; Blood Platelets; Collagen; Flow Cytometry; Humans; Platelet Aggregation; Prostaglandin-Endoperoxide Synthases; Reference Values; Ristocetin; Thrombasthenia; von Willebrand Diseases

Topics: Antigens; Black People; England; Ethnicity; Factor VIII; Female; Genetic Variation; Humans; Male; Nigeria; Reference Values; Ristocetin; von Willebrand Diseases; von Willebrand Factor; White People

Topics: Blood Platelets; Collagen; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Collagen; Diagnosis, Differential; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Type Vicenza variant of von Willebrand disease (VWD) is characterized by a low plasma von Willebrand factor (VWF) level and supranormal VWF multimers. Two candidate mutations, G2470A and G3864A at exons 17 and 27, respectively, of the VWF gene were recently reported to be present in this disorder. Four additional families, originating from northeast Italy, with both mutations of type Vicenza VWD are now described. Like the original type Vicenza subjects, they showed a mild bleeding tendency and a significant decrease in plasma VWF antigen level and ristocetin cofactor activity but normal platelet VWF content. Unlike the original patients, ristocetin-induced platelet aggregation was found to be normal. Larger than normal VWF multimers were also demonstrated in the plasma. Desmopressin (DDAVP) administration increased factor VIII (FVIII) and VWF plasma levels, with the appearance of even larger multimers. However, these forms, and all VWF oligomers, disappeared rapidly from the circulation. The half-life of VWF antigen release and of elimination was significantly shorter than that in healthy counterparts, so that at 4 hours after DDAVP administration, VWF antigen levels were close to baseline. Similar behavior was demonstrated by VWF ristocetin cofactor activity and FVIII. According to these findings, it is presumed that the low plasma VWF levels of type Vicenza VWD are mainly attributed to reduced survival of the VWF molecule, which, on the other hand, is normally synthesized. In addition, because normal VWF-platelet GPIb interaction was observed before or after DDAVP administration, it is proposed that type Vicenza VWD not be considered a 2M subtype.

Topics: Blood Platelets; Deamino Arginine Vasopressin; DNA Mutational Analysis; Drug Stability; Factor VIII; Half-Life; Humans; Italy; Kinetics; Macromolecular Substances; Mutation; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

This study compares the utility of two functional assays for von Willebrand factor (VWF), the ristocetin cofactor assay (VWF:RCo) and the collagen-binding assay (VWF:CBA). We analysed a group of 32 patients with type 2 von Willebrand disease (VWD) (25 patients with type 2M, six with type 2A and one with type 2B) and 22 normal control subjects. VWF:RCo/VWF antigen (VWF:Ag) ratios and VWF:CBA/VWF:Ag ratios were compared between the patient and control groups. In the six patients with type 2A VWD, both VWF:RCo/VWF:Ag ratios and VWF:CBA/VWF:Ag ratios were discordant (< or = 0.7). In the 25 type 2M VWD patients, the VWF:CBA/VWF:Ag ratios were concordant (> 0.7), but the VWF:RCo/VWF:CBA ratios were discordant (< or = 0.7) (P = 0.001) compared with control subjects. Thus, VWF:RCo/VWF:Ag ratios were discordant in both type 2M and 2A VWD patient groups indicating a functional abnormality. However, VWF:CBA/VWF:Ag ratios were discordant in the type 2A VWD group but not in the type 2M VWD group. Our study showed that VWF:CBA is sensitive to functional variants associated with the loss of high-molecular-weight multimers, i.e. type 2A and 2B in VWD, but the assay was unable to discriminate defective platelet-binding VWD variants with normal multimeric patterns such as type 2M VWD. It was concluded that the VWF:CBA assay should be used in association with rather than as a replacement for the VWF:RCo assay.

Topics: Case-Control Studies; Collagen Type III; Humans; Molecular Weight; Platelet Aggregation; Predictive Value of Tests; Protein Binding; Ristocetin; Statistics, Nonparametric; von Willebrand Diseases; von Willebrand Factor

For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay systems can be used for diagnosis and subtyping of vWD, but CBA is more sensitive than either of the two RCo methods. The analysis of vWF multimers in the different fractions obtained by affinity chromatography on heparin Sepharose showed that the activity measured both with RCo assay and CBA correlated with the degree of multimerization. Our results suggest that measurement of the functional activity of vWF by the RCo procedure can be replaced by the more reliable CBA, reflecting the physiological

Topics: Agglutination Tests; Chromatography, Affinity; Collagen; Dimerization; Enzyme-Linked Immunosorbent Assay; Factor VIII; Humans; Molecular Probe Techniques; Protein Binding; Ristocetin; Structure-Activity Relationship; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (vWD) is the most common hereditary bleeding disorder due to either a qualitative or a quantitative defect in von Willebrand factor (vWF). vWF is a multimeric plasma protein that plays an important role in (1) primary hemostasis, by sustaining indirect platelet adhesion especially at high shear rates, and in (2) secondary hemostasis, by protecting factor VIIIc (FVIIIc) from degradation. A correct diagnosis of vWD is based on the accurate identification of one of the six different subtypes (type 1, 2A, 2B, 2M, 2N, 3). To do this, different laboratory tests are available. One aspect of the identification is the discrimination between type 1 and type 2 (2A, 2B, and 2M) vWD. In type 1 vWD, both vWF levels (vWF:Ag) and vWF activity (vWF:RCof) are decreased; in type 2, the vWF:Ag level is normal or decreased and vWF:RCof is decreased. Thus, ratios of vWF:Ag to vWF:RiCof above 1 allow identifation of type 2 vWD patients. The currently used vWF:RCof test is an agglutination test in which patients' plasma is added to washed fixed control platelets in the presence of ristocetin and the extent of agglutination is measured. This test suffers from high interlaboratory and intralaboratory variability. We have recently shown that the same vWF:RCof can also be measured in an enzyme-linked immunosorbent assay (ELISA) with a low interassay and intraassay variability and can be used to identify patients suffering from vWD. We here show that our test allows the discrimination between type 1 and type 2 vWD patients.

Topics: Agglutination Tests; Case-Control Studies; Collagen; Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; Humans; Molecular Probe Techniques; Protein Binding; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Type 2M von Willebrand disease (VWD) refers to variants with decreased platelet-dependent function that is not associated with the loss of high molecular weight (HMW) von Willebrand factor (VWF) multimers. This category includes the so-called "phenotype B" responsible for inexistent ristocetin-induced but normal botrocetin-induced binding of VWF to platelet glycoprotein lb. The missense mutation G1324S was identified in the first patient reported to display "phenotype B". We report here on the identification in four members of a French family of a missense mutation also affecting this glycine residue but changing it into an alanine residue. These individuals are heterozygous for this mutation and two of them display an additional quantitative VWF deficiency resulting from a stop codon at position 2470. After transient transfection in Cos-7 cells, the mutated recombinant protein harbouring the G1324A substitution was shown to exhibit normal multimers and inexistent ristocetin-induced but normal botrocetin-induced binding to GPIb, confirming the classification of this new mutation as a type 2M VWD mutation.

Topics: Adult; Amino Acid Substitution; Animals; Biopolymers; Chlorocebus aethiops; Codon; COS Cells; DNA Mutational Analysis; Exons; Female; France; Hemorrhage; Heterozygote; Humans; Male; Mutation, Missense; Pedigree; Phenotype; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoproteins; Point Mutation; Polymerase Chain Reaction; Protein Binding; Receptors, Cell Surface; Recombinant Fusion Proteins; Ristocetin; Transfection; von Willebrand Diseases; von Willebrand Factor

We describe a von Willebrand disease (VWD) variant characterized by low plasma and platelet von Willebrand factor (VWF), impaired ristocetin-induced VWF binding to platelet glycoprotein Ib (GPIb), and abnormal VWF multimer pattern not associated with the absence of large forms. A C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene was found; this mutation introduces a cysteine at the codon for Arg 611 of mature VWF. In addition to the decreased factor VIII (FVIII) and VWF levels, ristocetin-induced platelet aggregation (RIPA) was almost absent, and VWF ristocetin cofactor activity (VWF:RCo) was significantly more decreased than VWF antigen. The patients (mother and son) also showed a defect in VWF collagen-binding activity. Plasma VWF multimers were decreased, with no limit in the size of large forms, and the normal discontinuous multimer organization was replaced by a diffuse smear, especially detectable in the large forms. This picture was emphasized by 1-deamino-8-D -arginine vasopressin (DDAVP) infusion, so that the abnormal VWF multimers appeared to have a molecular weight higher than those present in, or released by, human umbilical vein endothelial cells. DDAVP also increased FVIII and VWF levels but did not normalize the GPIb-dependent VWF functions expressed as RIPA and VWF:RCo. We include this variant in type 2M VWD, focusing on the abnormality in GPIb-dependent VWF function. We advance that this defect depends on the mutation in the GPIb binding domain of VWF rather than the abnormal VWF multimer pattern.

Topics: Adult; Anti-Bacterial Agents; Cells, Cultured; Collagen; Deamino Arginine Vasopressin; Endothelium, Vascular; Factor VIII; Family Health; Female; Hemostatics; Humans; Male; Middle Aged; Molecular Weight; Platelet Aggregation; Point Mutation; Polymers; Ristocetin; Umbilical Veins; von Willebrand Diseases; von Willebrand Factor

The study identified 10 patients from 6 families with prolonged bleeding time, decreased von Willebrand factor (vWF) ristocetin cofactor activity (RCoF) to vWF:Ag (antigen) ratio, and reduced ristocetin-induced platelet agglutination as well as ristocetin- or botrocetin-induced binding of plasma vWF to platelet glycoprotein Ib (GpIb). In addition, all patients showed a decrease of intermediate-molecular-weight (intermediate-MW) and high-molecular-weight (HMW) multimers of vWF. In the heterozygous state, a cysteine-to-threonine (C --> T) transversion was detected at nucleotide 4193 of the VWF gene of all patients and lead to the arginine (R)522C substitution in the A1 loop of vWF mature subunit (R1315C in the preprovWF). By in vitro mutagenesis of full-length complementary DNA (cDNA) of vWF and transient expression in COS-7 cells, the mutated C552 recombinant vWF (C552rvWF) was found to exhibit decreased expression, abnormal folding, and lack of intermediate-MW and HMW multimers. In addition, direct binding of botrocetin to C552rvWF, as well as ristocetin- and botrocetin-induced binding of C552rvWF to GpIb, was markedly decreased. Although being localized in an area of the A1 loop of vWF where most of the type 2B mutations that induce a gain-of-function have been identified, the R552C mutation induces a 2A-like phenotype with a decrease of intermediate-MW and HMW multimers as well as a loss-of-function of vWF in the presence of either ristocetin or botrocetin. (Blood. 2001;97:952-959)

Topics: Adult; Amino Acid Substitution; Child; Crotalid Venoms; DNA Mutational Analysis; Female; Genes; Humans; Introns; Male; Middle Aged; Mutation, Missense; Point Mutation; Polymorphism, Genetic; Protein Folding; Protein Structure, Tertiary; Ristocetin; Structure-Activity Relationship; von Willebrand Diseases; von Willebrand Factor

The capability of von Willebrand factor (VWF) to bind platelet glycoprotein Ib (GPIb) and promote platelet plug formation is currently evaluated in vitro using the ristocetin co-factor activity (VWF:RCo) assay. The replacement of this cumbersome and not always reproducible test with the collagen binding activity of VWF (VWF:CBA) has been attempted with controversial results. To evaluate the capacity of VWF:CBA to identify classic and variant von Willebrand disease (VWD) compared with VWF:RCo, we studied 10 type 2A and 12 type 2B VWD patients, together with 30 type 1 VWD patients with reduced platelet VWF content. In both 2A and 2B VWD, VWF:CBA and VWF:RCo were decreased, but that of VWF:CBA was more consistent. The difference was more evident when values were expressed as a ratio, obtained by normalizing VWF:CBA and VWF:RCo with the VWF antigen value; the ratio for VWF:CBA was always below 0.2, while that for VWF:RCo was greater than 0.4, and in no patient was the VWF:CBA value higher than VWF:RCo. In contrast, in type 1 VWD, the decrease in VWF:CBA was similar to that seen in VWF:RCo with the ratios always within the normal range. To better investigate the relationship between VWF:CBA and VWF:RCo, and the representation of large/intermediate VWF multimers, to which both tests are sensitive, 1-deamino-cys-8-D-arginine-vasopressin (DDAVP) was infused in type 2A and 2B VWD patients. The differences between the two tests were even more evident after DDAVP, and in type 2A, even though large multimers were persistently decreased, VWF:RCo was normalized, while VWF:CBA remained defective. These findings clearly indicate that VWF:CBA detects the absence of large and intermediate VWF multimers better than VWF:RCo. Hence, we suggest adding VWF:CBA to the panel of tests employed in the diagnosis of VWD. Moreover, owing to the difficulty in performing VWF:RCo and its low reproducibility, we suggest that, when necessary, VWF:CBA may be substituted for VWF:RCo.

Topics: Blood Coagulation Tests; Collagen; Deamino Arginine Vasopressin; Humans; Mutation; Predictive Value of Tests; Protein Binding; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Diagnosis of von Willebrand disease (vWD) is based on a panel of laboratory tests that measure the amount and function of von Willebrand factor (vWF). In population studies, vWF is higher in African Americans than Caucasians. Bleeding time, factor VIII activity (FVIII), vWF antigen (vWF:Ag), "vWF activity" ELISA (vWF:Act), ristocetin cofactor (vWF:RCof), and ristocetin-induced platelet aggregation (RIPA) were measured on 123 women with menorrhagia and 123 randomly selected control women; 70 cases and 76 controls were African American. Among controls, African Americans had significantly higher levels of vWF:Ag (mean 120 vs. 102 U/dl, P = 0.017). Among all subjects, African Americans had higher levels of vWF:Ag (mean 123 vs. 103, P = 0.001), vWF:Act (mean 101 vs. 89, P = 0.006), and FVIII (mean 118 vs. 104, P = 0.008). VWF:RCof did not differ between races (93 vs. 94 U/dl). RIPA was reduced in African Americans (P < 0.0001). In both races, women with type O blood differed significantly from those with other ABO types in vWF:Ag, vWF:Act, FVIII, and vWF:RCof. Based on criteria of two or more tests below race- and ABO-specific reference ranges, 6.5% of menorrhagia cases and 0.8% of controls were classified as having vWD, or its phenocopy. Among Caucasians, no controls and 7 cases (15.6%) were classified as affected, and in African Americans, 1 control (1.3%) and 1 case (1.4%) were so classified. Racial differences in vWF further complicate the issues surrounding diagnosis of vWD. The finding of increased vWF:Ag not accompanied by increased vWF:RCof has implications for understanding the structure-function relationships of vWF. Published 2001 Wiley-Liss, Inc.

Topics: ABO Blood-Group System; Black People; Case-Control Studies; Factor VIII; Female; Humans; Menorrhagia; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor; White People

Acquired von Willebrand syndrome (AVWS) has been associated mainly with monoclonal gammopathy of uncertain significance (MGUS), clonal lymphoproliferative or myeloproliferative disorders and autoimmunity. In the present work we studied 6 patients with AVWS: four with MGUS IgG (lambda or kappa), one with small lymphocytic lymphoma and one with agnogenic myeloid metaplasia (AMM). All the patients underwent a pharmacokinetic analysis at presentation in order to study potential differences in recovery, clearance (CL) or terminal half-life (THL) following administration of von Willebrand factor (VWF) concentrate. In all the patients with AVWS an increase in clearance and a decrease in THL was observed as compared to these parameters in patients with hereditary type 3 von Willebrand disease (VWD). No difference in recovery was observed among the groups. The increase in clearance and the decrease in THL were significantly more pronounced in the group of MGUS patients (57.93 +/- 25.6 ml/h/kg, and 1.39 +/- 0.5 h, respectively) as compared to these parameters in the AMM (8.06 ml/h/kg, and 6.96 h, respectively) or the lymphoma (4.76 ml/h/kg, and 6.76 h. respectively) patients (p = 0.03 for clearance and 0.001 for THL). These data indicate that the pharmacokinetic analysis can be a useful tool to distinguish between MGUS-related and other causes of AVWS, and to plan an appropriate treatment accordingly.

Topics: Aged; Bleeding Time; Factor VIII; Female; Half-Life; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Metabolic Clearance Rate; Middle Aged; Monoclonal Gammopathy of Undetermined Significance; Primary Myelofibrosis; Ristocetin; von Willebrand Diseases; von Willebrand Factor

To maintain hemostasis under shear conditions, there must be an interaction between the platelet glycoprotein (GP) Ib-IX receptor and the plasma ligand von Willebrand factor (vWf). In platelet-type von Willebrand disease (Pt-vWD), hemostasis is compromised. Two mutations in the GPIbalpha polypeptide chain have been identified in these patients-a glycine-233 to valine change and a methionine-239 to valine change. For this investigation, these mutant proteins have been expressed in a Chinese hamster ovary cell model system. Ligand-binding studies were performed at various concentrations of ristocetin, and adhesion assays were performed under flow conditions. The Pt-vWD mutations resulted in a gain-of-function receptor. vWf binding was increased at all concentrations of ristocetin examined, and adhesion on a vWf matrix was enhanced in terms of cell tethering, slower rolling velocity, and decreased detachment with increasing shear rate. Two other mutations were also introduced into the GPIbalpha chain. One mutation, encompassing both the Pt-vWD mutations, created an increase in the hydrophobicity of this region. The second mutation, involving a valine-234 to glycine change, decreased the hydrophobicity of this region. Both mutations also resulted in a gain-of-function receptor, with the double mutation producing a hyperreactive receptor for vWf. These data further support the hypothesis that ligand binding is regulated by conformational changes in the amino-terminal region of GPIbalpha, thereby influencing the stability of the GPIbalpha-vWf interaction.

Topics: Animals; Cell Adhesion; Cell Aggregation; CHO Cells; Cricetinae; Hemostasis; Mutation; Phenotype; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The aim of our study was to characterise heparin-binding properties of mutated von Willebrand factor (VWF) in 24 patients plasmas with type 2 von Willebrand disease (VWD). and in 15 recombinant VWF (rVWF) with the corresponding mutations. Binding of mutated rVWF or plasma VWF was compared to that of WT-rVWF or normal pool plasma VWF. Four mutations, at positions C509, V551, R552 and R611 lead to significantly decreased binding to heparin in both plasma and rVWF. Interestingly, whereas these four residues are distant in the primary structure of VWF-A1domain, they are close to each other in its three-dimensional structure. Structural analysis suggested how folding problems and destabilisation due to these mutations could induce reorganisation of surface regions involved in heparin binding. In contrast, no heparin-binding defect was found associated with different type 2 VWF mutants, at positions G561, E596, I662, R543, R545, V553, R578 or L697.

Topics: Amino Acid Substitution; Animals; Antibodies, Monoclonal; Binding Sites; Chlorocebus aethiops; Codon; COS Cells; Cystine; Heparin; Humans; Hydrogen Bonding; Models, Molecular; Mutation, Missense; Platelet Glycoprotein GPIb-IX Complex; Point Mutation; Protein Binding; Protein Conformation; Protein Denaturation; Protein Folding; Protein Interaction Mapping; Recombinant Fusion Proteins; Ristocetin; Structure-Activity Relationship; Surface Properties; Transfection; von Willebrand Diseases; von Willebrand Factor

Topics: Anticoagulants; Blood Preservation; Blood Specimen Collection; Citric Acid; Collagen; False Positive Reactions; Molecular Weight; Protein Binding; Refrigeration; Ristocetin; Specimen Handling; Temperature; Transportation; von Willebrand Diseases; von Willebrand Factor

Inhibitors against von Willebrand factor (vWF) developed in two unrelated multitransfused patients (patients 1 and 2) with severe (type 3) von Willebrand disease (vWD) were analyzed. Both inhibitors were identified as antibodies of the IgG class by ELISA using immobilized purified vWF and either serum or purified Ig from the patients. Typing, mapping and functional studies of both antibodies revealed significantly distinct properties. Patient 1 antibody contained all subclasses of IgG (1, 2, 3 and 4) whereas antibody from patient 2 was a mixture of only IgG1 and 4. By ELISA using a series of immobilized purified proteolytic fragments of vWF, patient 1 antibody mainly bound to fragment SpIII and, to a lower extent, to fragments SpII and SpI; it poorly bound to P34 and the 39/34 kDa fragment. In contrast, patient 2 antibody only bound to fragments corresponding to the N-terminal portion of vWF but failed to bind to SpII. Functional studies were performed by testing the capacity of each antibody to inhibit vWF binding to its various ligands. Both antibodies blocked vWF binding to Factor VIII (FVIII), fibrillar type III collagen, bitiscetin and the subsequent induced binding to GPIb. Patient 1 antibody also blocked vWF binding to platelet GPIb when induced by ristocetin. However it failed to block vWF binding to GPIb when induced by botrocetin as well as the binding of botrocetin itself to vWF. Our data thus suggest that this inhibitor does not recognize the GPIb-binding site on vWF but the sites of vWF involved in its interaction with ristocetin. In contrast, we observed that patient 2 antibody blocked vWF binding to platelet GPIb induced by either agonist as well as vWF binding to botrocetin. Finally, the effect of the antibodies was tested on vWF binding to GPIIb/IIIa. As expected from the mapping experiments, only IgG from patient 1 blocked the interaction while IgG from patient 2 had no effect. In conclusion, we have shown that two multitransfused patients with type 3 vWD have developed alloantibodies with similar properties to those of polyclonal antibodies but with distinct effects on the functions of vWF.

Topics: Adult; Binding Sites, Antibody; Blood Platelets; Child; Collagen; Crotalid Venoms; Epitope Mapping; Factor VIII; Female; Hemagglutinins; Humans; Immunoglobulin G; Inhibitory Concentration 50; Iodine Radioisotopes; Isoantibodies; Male; Peptides; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Recombinant Proteins; Ristocetin; Snake Venoms; Transfusion Reaction; Viper Venoms; von Willebrand Diseases; von Willebrand Factor

Type 1 von Willebrand disease is characterized by a decreased plasma concentration of functionally normal von Willebrand factor (vWF) whereas type 2M is characterised by an abnormal vWF displaying decreased affinity for platelets. In these two types of patients, the multimeric structure of vWF is normal. We report here the identification, in two unrelated families from the UK and Algeria, of an in-frame 3 bp deletion, at the heterozygous state, resulting in the deletion of a lysine residue within a four lysine repeat at position 642-645 of the mature vWF subunit (del K 1405-1408 in pre-pro vWF). The patients who have a discrepancy between vWF antigen level and vWF ristocetin cofactor activity exhibited decreased ristocetin-induced binding but only a slight decrease in the percentage of high molecular weight (HMW) multimers in plasma. Recombinant vWF harbouring this deletion did not bind to platelet GPIb in the presence of ristocetin or botrocetin although the protein is multimerized. Consequently, this lysine deletion was considered as a type 2M vWD mutation.

Topics: Animals; Binding, Competitive; Blood Platelets; COS Cells; Crotalid Venoms; DNA Mutational Analysis; Family Health; Female; Hemagglutinins; Humans; Lysine; Male; Pedigree; Phenotype; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoproteins; Protein Binding; Protein Structure, Tertiary; Protein Subunits; Recombinant Proteins; Ristocetin; Sequence Deletion; Transfection; von Willebrand Diseases; von Willebrand Factor

Definitive diagnosis of type 1 von Willebrand Disease (VWD) remains a problem. Provisional consensus guidelines for the diagnosis of definite and possible type 1 VWD were prepared by the Scientific Subcommittee on von Willebrand factor (VWF) of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) during the 1996 annual meeting for the specific purpose of further evaluation in retrospective and prospective studies by a Working Party on Diagnostic Criteria (1996 Annual Report of the SSC/ISTH Subcommittee on VWF). In the first phase of this study, we compared 2 definitions of type 1 VWD. each with 3 criteria: significant bleeding history, laboratory investigations, and family history. Using the ISTH consensus guidelines for type 1 VWD definition, significantly fewer patients were diagnosed with definite type 1 disease as compared to our "in house" Hospital for Sick Children (HSC) criteria (4 vs. 31). While we recognize that the provisional ISTH consensus guidelines were not intended for clinical use, we believe that the results of our studies are of interest and will assist in any future refinements to the ISTH guidelines. In the second phase of this study, we investigated the utility of 2 new tests, a laboratory screening test and a functional test, for VWD in our well characterized, pediatric-based population. The Platelet Function Analyzer (PFA-100) provides an in vitro measure of primary hemostasis under conditions of high shear, using disposable cartridges containing collagen and either epinephrine or ADP. All tested subjects with types 2 or 3 VWD had prolonged PFA-100 closure times (CTs) with both cartridge types (n = 17) and prolonged bleeding times (n = 14). In subjects with definite type 1 VWD, 20/24 (83%) had prolonged CTs with the collagen/ADP cartridge (19/24 (79%) with collagen/epinephrine), compared with 7/26 (27%) with prolonged bleeding times. In subjects with definite types 1, 2, or 3 VWD, collagen/ADP CTs were abnormal in 37/41 subjects, giving an overall sensitivity of 90%. With this high sensitivity, the PFA-100 is a better screening test for VWD than the bleeding time. We also tested a VWF collagen-binding assay (VWF:CBA) as a functional test for VWF, in comparison with the more routinely-used ristocetin cofactor assay (VWF:RC0). The VWF:CBA is based on an ELISA technique, which has the potential to be more reproducible than the VWF:RC0. We found that the VWF:CBA detected 43

Topics: Adolescent; Bleeding Time; Blood Coagulation Tests; Child; Child, Preschool; Collagen; Dimerization; Electrophoresis, Polyacrylamide Gel; Family Health; Female; Humans; Infant; Male; Molecular Weight; Platelet Function Tests; Protein Binding; Reagent Kits, Diagnostic; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

Topics: Adult; Anesthesia, Epidural; Anesthesia, Obstetrical; Anti-Bacterial Agents; Contraindications; Deamino Arginine Vasopressin; Female; Hemostatics; Humans; Pregnancy; Pregnancy Complications, Hematologic; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Amino Acid Substitution; Biopolymers; Female; Humans; Nelfinavir; Platelet Aggregation; Point Mutation; Postoperative Care; Preoperative Care; Protease Inhibitors; Protein Structure, Tertiary; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T --> A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A --> T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

Topics: Base Sequence; Binding Sites; Blood Platelets; Collagen; Crotalid Venoms; DNA; Female; Hemagglutinins; Humans; Male; Models, Molecular; Mutation; Pedigree; Platelet Glycoprotein GPIb-IX Complex; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Desmopressin was administered intranasally to seven patients with von Willebrand disease (type 1: 4 patients, type 2A: 3 patients) to assess the response and safety. von Willebrand factor antigen ranged from 8% to 60% before treatment and increased significantly after intranasal DDAVP administration (the median relative increase: two- to threefold). Factor VIII levels also increased substantially over baseline levels after intranasal administration. Before treatment ristocetin cofactor activity was 32 +/- 12% in patients with type 1 vWD and 9 +/- 5% in patients with type 2A vWD. After intranasal administration, the levels of ristocetin cofactor activity increased to 56 +/- 21% and 29 +/- 9%, respectively. The bleeding time was normalized in 86% of the patients. The abnormality of vWF multimers in type 1 vWD returned more or less to normal after intranasal DDAVP administration whereas that in type 2A vWD did not. The intranasal administration of DDAVP is safe and effective for minor bleeding episodes and is adaptable for home use in patients with type 1 and type 2A vWD.

Topics: Administration, Intranasal; Bleeding Time; Coagulants; Crotalid Venoms; Deamino Arginine Vasopressin; Female; Humans; Male; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Platelet-type von Willebrand disease (vWD) is a congenital bleeding disorder characterized by heightened ristocetin-induced platelet aggregation caused by abnormally high affinity between the platelet membrane glycoprotein (GP) Ib/IX complex and von Willebrand factor (vWF). Two distinct point mutations, Gly233 to Val and Met239 to Val, have been reported in GPIb alpha. We have constructed a recombinant GPIb alpha fragment containing the latter mutation, Met239 to Val (M239V) and characterized the mutant molecule using two methods, ie, interaction between soluble vWF and immobilized M239V and inhibition of platelet aggregation by purified soluble M239V. Spontaneous binding (ie, binding without any inducers) was observed between 125I-vWF and immobilized M239V but not between 125I-vWF and immobilized wild-type (WT) GPIb alpha. The addition of low concentrations of ristocetin (0.2 mg/mL) induced specific 125I-vWF binding to immobilized M239V, but not to WT GPIb alpha. At high concentrations of ristocetin (1.2 mg/mL), both WT GPIb alpha and M239V specifically bound to 125I-vWF. Thus, M239V reproduced the unique functional abnormality of the GPIb/IX complex in platelet-type vWD. Moreover, the purified soluble M239V inhibited platelet aggregation induced by low concentration of ristocetin (0.3 mg/mL) in platelet-rich plasma from a patient having Met239 to Val mutation, whereas purified WT did not. These results provide direct evidences that the reported point mutation is the responsible molecular basis of this disorder.

Topics: Analysis of Variance; Humans; Methionine; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIb-IX Complex; Point Mutation; Recombinant Proteins; Ristocetin; Transfection; Valine; von Willebrand Diseases

Type 2B von Willebrand disease (vWD) is typically characterized by enhanced ristocetin-induced platelet aggregation (RIPA) caused by increased von Willebrand factor (vWF) affinity for platelets. Furthermore, absence of larger vWF multimers in plasma is characteristic of the originally described type IIB patients, now considered a subgroup of type 2B. We describe here three affected members of a family presenting with prolonged bleeding time, thrombocytopenia, markedly enhanced RIPA and spontaneous platelet aggregation, but normal plasma vWF antigen and ristocetin cofactor activity. Larger plasma vWF multimers, albeit decreased, were present in relatively greater proportion than in other type IIB patients. Genetic studies performed in two of these patients resulted in the identification of a previously unreported A-->G transition at nucleotide 4175 in the sequence of the pre-pro-vWF cDNA, corresponding to the substitution Ile546-->Val in the mature vWF subunit. This mutation appears to be responsible for an unusual type 2B phenotype, with greater enhancement of the vWF-platelet interaction than in typical cases but partial preservation of the larger vWF multimers in plasma.

Topics: Adult; Anti-Bacterial Agents; Child; Female; Humans; Isoleucine; Male; Middle Aged; Phenotype; Platelet Aggregation; Point Mutation; Restriction Mapping; Ristocetin; Sequence Analysis, DNA; Valine; von Willebrand Diseases; von Willebrand Factor

von Willebrand factor (vWF) is a multimeric glycoprotein that forms an adhesive link following vascular injury between the vessel wall and its primary ligand on the platelet surface, glycoprotein Ib (GpIb). Type 2b von Willebrand disease (vWD) is a qualitative form of vWD resulting from enhanced binding of vWF to platelets. Molecular characterization of the vWF gene in patients with type 2b vWD has resulted in identification of a panel of mutations associated with this disorder, all clustered within the GpIb binding domain in exon 28 of the vWF gene. We have expressed six of the most common type 2b vWD mutations in recombinant vWF and show that each mutation produces a similar increase in vWF binding to platelets in the absence or presence of ristocetin. Furthermore, expression of more than one type 2b vWD mutation in the same molecule (cis) or in different molecules within the same multimer (trans) failed to produce an increase in vWF platelet binding compared with any of the individually expressed mutations. Taken together, these data support the hypothesis that the vWF GpIb binding domain can adopt either a discrete "on" or "off" conformation, with most type 2b vWD mutations resulting in vWF locked in the on conformation. This model may have relevance to other adhesive proteins containing type A domains.

Topics: Animals; Base Sequence; Blood Platelets; Cell Line, Transformed; Chlorocebus aethiops; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Platelet Membrane Glycoproteins; Point Mutation; Protein Binding; Receptors, Cell Surface; Recombinant Fusion Proteins; Ristocetin; Transfection; von Willebrand Diseases; von Willebrand Factor

Type 2B von Willebrand disease is characterized by an abnormal von Willebrand factor molecule with increased affinity for the platelet glycoprotein (GP) Ib-IX receptor. A diagnostic feature of type 2B von Willebrand disease is a characteristic loss of von Willebrand factor high molecular weight multimers. In vitro, the soluble interaction of normal von Willebrand factor with platelets can be initiated with exogenous modulators, the most common being the antibiotic ristocetin. The variant molecules resulting in type 2B von Willebrand disease can sustain binding to platelets at subnormal levels of ristocetin. We characterized the von Willebrand factor gene of an individual with type 2B von Willebrand disease and identified a nucleotide transition resulting in an Arg543-->Trp amino-acid substitution within the GP Ib-IX binding domain of von Willebrand factor. In this study we demonstrate that a recombinant plasmid capable of expressing the isolated GP Ib-IX binding domain of von Willebrand factor, and containing the Arg543-->Trp amino-acid substitution, secretes a dimeric molecule that supports platelet agglutination using subnormal levels of ristocetin. These results demonstrate that the mutation at position 543 increases the affinity between the variant molecule and platelet GP Ib-IX as an intrinsic feature of the isolated von Willebrand factor domain. Thus, structural perturbations within the GP Ib-IX binding domain that are independent of the von Willebrand factor multimeric structure can sufficiently increase the affinity of von Willebrand factor to sustain platelet aggregation, using subnormal levels of ristocetin.

Topics: Adult; Animals; Base Sequence; CHO Cells; Cricetinae; DNA; Exons; Female; Humans; Macromolecular Substances; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments; Platelet Aggregation; Recombinant Proteins; Ristocetin; Sequence Analysis, DNA; Transfection; von Willebrand Diseases; von Willebrand Factor

Nine patients (10 infusions) with a confirmed diagnosis of type 3 VWD were infused with von Willebrand factor (human), a preparation of von Willebrand factor (VWF) with a very low factor VIII content. Each patient was infused with one dose of approximately 50 or 100 iu ristocetin cofactor activity (VWF:RiCoF) per kg body weight. Bleeding times were performed during the 24 h period after infusion. Plasma samples were obtained over the 96 h period after infusion and were analysed for factor VIII coagulant activity (FVIIIC), VWF:RiCoF, von Willebrand factor antigen (VWF:Ag), and multimers. The FVIIIC data were analysed by non-linear least-squares analysis assuming constant FVIIIC 'synthesis' and exponential decay. The VWF data were fitted for exponential decay. The average decay rates for FVIIIC, VWF:RiCoF and VWF:Ag were 0.041, 0.061 and 0.056 respectively. The average calculated 'synthesis' rate for FVIIIC was 6.4 u/dl/h. The synthesis of FVIIIC was slightly faster and the decay slightly slower following the infusion of 100 iu VWF:RiCoF/kg than of 50 iu VWF:RiCoF/kg. Correction of the bleeding time was strongly dose dependent. At 4 h post infusion the median bleeding time was 9 min following a dose of 50 iu VWF:RiCoF/kg versus 3 min with a dose of 100 iu VWF:RiCoF/kg. There was no decrease in the bleeding time until the level of VWF:Ag or VWF:RiCoF reached > 100 u/dl.

Topics: Adolescent; Adult; Aged; Bleeding Time; Child; Dose-Response Relationship, Drug; Factor VIII; Female; Humans; Male; Middle Aged; Ristocetin; von Willebrand Diseases; von Willebrand Factor

This report examines the genetic basis of a variant form of moderately severe von Willebrand disease (vWD) characterized by low plasma von Willebrand factor antigen (vWF:Ag) levels and normal multimerization, typical of type 1 vWD, but disproportionately-low agonist-mediated platelet-binding activity. We identified an in-frame deletion in vWF exon 28 in three generations of affected family members, who are heterozygous for this mutation. The deletion of nucleotides 4,173-4,205 results in the loss of amino acids Arg629-Gln639 in the Cys509-Cys695 loop of the A1 domain in mature vWF. The secreted mutant vWF showed a normal multimeric profile but did not bind to platelets in the presence of optimal concentrations of either ristocetin or botrocetin. The mutant vWF also failed to interact with heparin, and with vWF monoclonal antibody AvW3, which blocks the binding of vWF to GPlb. In addition, mutant vWF showed reduced secretion from transfected cells concomitant with increased intracellular levels. These results confirm that the deletion is the genetic defect responsible for the reduced interaction of vWF with platelets. We have designated this new variant type 2M:Milwaukee-1 vWD. Our analysis suggests that the potential frequency of this phenotype in individuals diagnosed with type 1 vWD is about 0.5%.

Topics: Alleles; Amino Acid Sequence; Base Sequence; Blood Platelets; Crotalid Venoms; DNA Mutational Analysis; Female; Heparin; Heterozygote; Humans; Male; Molecular Sequence Data; Pedigree; Phenotype; Platelet Membrane Glycoproteins; Protein Binding; Protein Structure, Tertiary; Receptors, Cell Surface; Recombinant Fusion Proteins; Ristocetin; Sequence Alignment; Sequence Deletion; Sequence Homology; von Willebrand Diseases; von Willebrand Factor

Acquired von Willebrand disease (vWD) has been described in a few patients with multiple myeloma. The present study characterizes an inhibitor of von Willebrand factor (vWF) isolated from a patient with multiple myeloma (IgG-kappa). Multimeric analysis of vWF from this patient's plasma showed a reduction in multimers of all sizes. The inhibitor (IgG) detected only the vWF subunit from plasma of normal individuals. It reacted with intact vWF subunit and a 39/34kDa dispase-digested fragment of vWF (residues; Leu480/Val481-Gly718), but did not react with platelet membrane glycoproteins (GPs) or adhesive proteins. The binding of vWF to GPIb mediated by ristocetin and by botrocetin was inhibited by the patient's IgG with an IC50s of 0.3 mg/ml and 0.48 mg/ml, respectively. The platelet aggregation induced by ristocetin or botrocetin was also inhibited by the IgG. These results indicate that this inhibitor may recognize the binding region of vWF to GPIb. Therefore, the antibody to vWF appears to represent the likely pathophysiological mechanism responsible for the acquired vWD in this patient.

Topics: Autoantibodies; Binding Sites; Blotting, Western; Crotalid Venoms; Humans; Immunoglobulin G; Macromolecular Substances; Male; Middle Aged; Multiple Myeloma; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Deamino Arginine Vasopressin; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases

The concentration of von Willebrand factor (vWf) in patients' plasma can be determined by measuring the ristocetin cofactor activity (vWf R:Co). However, this vWf R:Co assay is time consuming, which limits its routine use. Several commercial vWf R:Co tests, based on agglutination of lyophilized fixed platelets, are available. We evaluated the slide tests and aggregometer assays from Behring and Organon Teknika and compared them with the classic vWf R:Co aggregometer method. The within-run and between-run precisions of the two slide tests were better than those of the aggregometer methods. The correlation studies between the four commercial assays and the classic aggregation method were based on 23 plasma samples (range: 15-450% vWf R:Co). The correlation coefficients, which ranged from 0.923 to 0.950, did not differ significantly (P > 0.1). All four commercial assays gave significantly lower vWf R:Co values than the classic aggregation method (P < 0.01). We conclude that commercially available fixed platelets can be used for the rapid measurement of vWf R:Co with a slide test. The use of the aggregometer is time consuming and may result in a lower precision.

Topics: Humans; Platelet Aggregation; Reagent Kits, Diagnostic; Ristocetin; Sensitivity and Specificity; von Willebrand Diseases; von Willebrand Factor

Type 2B von Willebrand disease (vWD) is characterized by an increased affinity of von Willebrand factor (vWF) for binding to platelet glycoprotein Ib (GpIb). Most type 2B candidate mutations are clustered in the 509-695 disulphide loop but three of them (H505D, L697V and A698V) are outside this loop. We confirm here that the A698V mutation is a type 2B mutation by its expression in Cos-7 cells. As the L697V and A698V type 2B mutations both induce the presence of a valine residue in the 694-708 sequence, we created and expressed different mutated recombinant vWFs (rvWFs), in substituting the other leucine and alanine residues of this sequence (at positions 694, 701 and 706) into valine resides. V694rvWF and V706rvWF displayed decreased ristocetin-induced GpIb binding showing that it is not always the presence of a valine residue that may explain the increased affinity of type 2B vWF for GpIb. We also compared the interaction with platelets of V697rvWF and V698rvWF to those obtained with rvWFs reproducing two prevalent type 2B mutations located in the loop (R543W and V553M). We show that the two mutations located in the loop are more reactive than the two mutations identified outside the loop.

Topics: Base Sequence; DNA Primers; Female; Humans; In Situ Hybridization; Male; Middle Aged; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Pedigree; Platelet Glycoprotein GPIb-IX Complex; Polymerase Chain Reaction; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Type IIB von Willebrand disease is characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers from plasma and enhanced platelet agglutination of platelet-rich-plasma in the presence of low concentrations of ristocetin. We identified, in two related patients, a C-->G transversion resulting in the substitution of Valine for Leucine at position 697 of the mature subunit of vWF. We reproduced this mutation in vWF cDNA and expressed the recombinant protein in Cos-7 cells. The subunit composition and multimeric structure of mutated protein (rvWFLeu697Val) were similar to the wild-type recombinant (WTrvWF). Ristocetin-induced binding of rvWFLeu697Val to platelets was markedly increased in the presence of low doses of ristocetin and slightly increased with botrocetin as compared with that for WTrvWF, whereas collagen binding was not affected by the mutation. These data show that the Leu 697-->Val substitution is not a rare polymorphism but is responsible for the subtype IIB characteristic abnormalities identified in the two affected patients; however, it is not located in the area of vWF (amino acid 540 to amino acid 578) where most of the other type IIB mutations have already been reported.

Topics: Aged; Base Sequence; Blood Platelets; Crotalid Venoms; Female; Humans; Molecular Sequence Data; Mutation; Phenotype; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Type IIB von Willebrand disease (vWD) is characterized by a selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma due to their abnormally enhanced reactivity with platelets. Several missense mutations in the platelet glycoprotein Ib (GPIb) binding domain of vWF were recently characterized that cause type IIB vWD. The effect of type IIB mutation Arg(545)Cys on vWF binding to platelet GPIb was studied using recombinant wild type (rvWFWT) and mutant rvWFR545C expressed in COS-7 cells. In the absence of ristocetin, 50% of rvWFR545C bound spontaneously to platelet GPIb and the binding increased to 70% in the presence of 0.2 mg/ml ristocetin; rvWFWT did not bind significantly under either condition. Botrocetin-induced binding of rvWFR545C was only slightly increased compared to rvWFWT. These data demonstrate that the Arg(545)Cys mutation increases the affinity of vWF for GPIb, resulting in the characteristics gain-of-function type IIB vWD phenotype.

Topics: Arginine; Base Sequence; Blood Platelets; Cysteine; Humans; Molecular Sequence Data; Mutation; Platelet Membrane Glycoproteins; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We report a 28-year-old-Japanese male who had a skin tumor derived from variant type xeroderma pigmentosum (XP), combined with factor XI (FXI) deficiency and type IIB von Willebrand's disease (vWd). The patient had abnormal bleeding history on tooth extraction. FXI clotting activity (FXI:C) and antigen (FXI:Ag) were remarkably decreased (< 0.01 U/ml, < 0.02 U/ml, respectively). Factor VIII (FVIII) clotting activity, von Willebrand factor antigen (vWf:Ag), and ristocetin cofactor (RCoF) were 0.43 U/ml, 45%, and 57%, respectively. Ristocetin-induced platelet agglutination (RIPA) revealed hyper-aggregation compared with a normal control. Multimeric composition of vWf in plasma showed a reduction in high molecular weight forms. The family study revealed two other subjects with homozygous hereditary FXI deficiency and vWd, and five subjects with heterozygous FXI deficiency. The relationship between FXI deficiency and vWd is discussed and previously reported cases are reviewed.

Topics: Adult; Asian People; Blood Coagulation; Factor VIII; Factor XI Deficiency; Family Health; Female; Heterozygote; Humans; Japan; Male; Pedigree; Ristocetin; von Willebrand Diseases; von Willebrand Factor; Xeroderma Pigmentosum

Thrombocytopenia after desmopressin (DDAVP) infusion is usually observed in patients with type IIB von Willebrand disease (vWD). No other subtypes of vWD with thrombocytopenia after DDAVP have been reported so far. We describe here the occurrence of thrombocytopenia after DDAVP in a 39 year old male and his son with phenotypic characteristics of type I vWD, "platelet discordant subtype." After DDAVP, the abnormal ristocetin cofactor/von Willebrand factor antigen ratio in plasma was not corrected and the bleeding time remained markedly prolonged. Platelet count dropped 30 min after DDAVP (from 279 to 96 x 10(3)/microL in the propositus and from 298 to 116 x 10(3)/microL in his affected son) and returned to normal at 60 min. Platelet clumping was evident on peripheral blood smears obtained after infusion. These cases indicate that after DDAVP thrombocytopenia can occur in vWD other than type IIB.

Topics: Adult; Blood Platelets; Child; Deamino Arginine Vasopressin; Electrophoresis, Polyacrylamide Gel; Female; Humans; In Vitro Techniques; Infusions, Intravenous; Kinetics; Male; Ristocetin; Thrombocytopenia; von Willebrand Diseases; von Willebrand Factor

Type IIB von Willebrand disease is characterized by increased affinity of mutant von Willebrand factor (vWF) for platelet glycoprotein Ib. Eight different missense mutations that cause this phenotype have been reported within the disulfide loop defined by Cys-509 and Cys-695 of the mature vWF subunit; this disulfide loop is required for normal binding of vWF to platelet glycoprotein Ib. A new mutation was identified in a patient with type IIB von Willebrand disease (vWD) characterized by a lifelong bleeding disorder, mild thrombocytopenia, normal levels of factor VIII, vWF antigen, and ristocetin cofactor activity but increased ristocetin-induced platelet agglutination at low concentrations of ristocetin. Exon 28 of the patient's vWF gene was amplified, cloned, and sequenced. At nucleotide 3802 (numbering the cDNA from translation initiation), a C to G transversion was identified, which changes the encoded amino acid sequence from His-505 to Asp. The corresponding mutant recombinant vWF was expressed in transiently transfected COS cells. Relative to wild type vWF, the mutant vWF exhibited markedly increased binding to platelets at low concentrations of ristocetin, confirming the association between the His-505-->Asp substitution and the type IIB vWD phenotype. The His-505-->Asp mutation lies outside the disulfide loop affected by other type IIB vWD mutations and implicates a new segment of vWF in the regulation of platelet glycoprotein Ib binding.

Topics: Alleles; Amino Acid Sequence; Aspartic Acid; Base Sequence; Binding Sites; Blood Platelets; Crotalid Venoms; DNA; Electrophoresis, Agar Gel; Female; Hemagglutinins; Histidine; Humans; Kinetics; Middle Aged; Molecular Sequence Data; Oligodeoxyribonucleotides; Plasmids; Platelet Membrane Glycoproteins; Point Mutation; Polymerase Chain Reaction; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We report two cases of von Willebrand (type I) disease with vWF antigen levels of 21 and 25%, vWF ristocetin cofactor concentrations of 32 and 30%, and coagulation FVIII levels of 10 and 50% respectively. Template bleeding times were 20 and 11 minutes respectively. Both of these women exhibited a progressive and marked improvement of the vWF/FVIII complex during pregnancy. In the first, all parameters had completely normalized in the 37th week when a cesarean section was performed without hemorrhagic complications. The second case had borderline normal levels of the vWF/FVIII complex in the 37th week and underwent normal delivery in the 39th week. The improvement persisted for at least one week post-partum and in the first case values were still higher 10th weeks after delivery than they had been prior to the pregnancy. These observations and similar cases in the recent literature suggest that obstetrical operations and normal delivery are not associated with major blood losses in cases of moderately severe vWD of type I. These patients probably will not need vWF/FVIII concentrates for adequate hemostasis.

Topics: Adult; Cesarean Section; Factor VIII; Female; Humans; Pregnancy; Pregnancy Complications, Hematologic; Reference Values; Remission, Spontaneous; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The platelet GP Ib-IX receptor supports platelet adhesion and activation by binding to vWf in the exposed subendothelial matrix. An abnormal GP Ib-IX complex exists in platelet-type or pseudo-von Willebrand disease and has a characteristic increased affinity for soluble vWf resulting in impaired hemostatic function due to the removal of larger vWf multimers from the circulation. Genetic studies within an afflicted family have demonstrated that the disease is linked to a Gly233-->Val amino acid substitution within the alpha-subunit of the oligomeric GP Ib-IX complex (Miller, J.L., D. Cunningham, V.A. Lyle, and C. L. Finch. 1991. Proc. Natl. Acad. Sci. USA. 88:4761-4765). To evaluate the functional consequences of this mutation, we constructed a recombinant analogue of the alpha-subunit of GP Ib containing Val233. Experiments comparing molecules with either Gly233 or Val233 revealed that the Val substitution generates a molecule with increased affinity for vWf. The recombinant fragment reproduces the functional abnormality of the GP Ib-IX complex in platelet-type von Willebrand disease, thus establishing the molecular basis of the bleeding disorder within this family. Moreover, it becomes apparent that structural elements responsible for the regulation of hemostasis through modulation of vWf affinity for platelets reside within the alpha-subunit of the GP Ib-IX complex.

Topics: Amino Acid Sequence; Animals; Blood Platelets; CHO Cells; Cricetinae; Crotalid Venoms; Genetic Variation; Glycine; Hemagglutinins; Humans; Kinetics; Macromolecular Substances; Phenotype; Platelet Membrane Glycoproteins; Recombinant Proteins; Ristocetin; Transfection; Valine; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574----Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non-ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.

Topics: Animals; Base Sequence; Blood Platelets; Cloning, Molecular; DNA; Endothelium, Vascular; Female; Humans; Leukocytes; Male; Molecular Sequence Data; Mutation; Oligonucleotides, Antisense; Pedigree; Platelet Membrane Glycoproteins; Polymerase Chain Reaction; Protein Binding; Receptors, Cell Surface; Recombinant Proteins; Ristocetin; RNA; Transfection; von Willebrand Diseases; von Willebrand Factor

von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.

Topics: Amino Acid Sequence; Base Sequence; Crotalid Venoms; Humans; Molecular Sequence Data; Oligodeoxyribonucleotides; Platelet Membrane Glycoproteins; Point Mutation; Protein Binding; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Type IIB von Willebrand disease is an autosomal dominant bleeding disorder characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma, presumably due to their abnormally increased reactivity with platelets. We and others have recently identified a panel of missense mutations clustered in the platelet glycoprotein Ib binding domain of vWF from patients with type IIB von Willebrand disease. We now report functional analysis of one of the most frequent type IIB missense mutations, Arg-543----Trp (vWF R543W). vWF from a human umbilical vein endothelial cell culture heterozygous for the vWF R543W mutation showed markedly increased binding of large vWF multimers to platelets in the presence of a low dose of ristocetin compared to vWF from a normal control culture. Recombinant vWF containing the vWF R543W mutation expressed in COS-7 cells also demonstrated increased binding of large vWF multimers. Mixed multimers obtained by cotransfection of mutant and wild-type cDNAs showed partial dominance of the vWF R543W mutation. Thus these data demonstrate that the vWF R543W mutation alone is sufficient to confer increased binding of large vWF multimers to platelets in a dominant fashion and that no other factors relating to vWF posttranslational processing or secretion in endothelial cells are required for this effect.

Topics: Binding Sites; Blood Platelets; Endothelium, Vascular; Humans; Macromolecular Substances; Mutation; Platelet Aggregation; Platelet Membrane Glycoproteins; Recombinant Proteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Acquired von Willebrand's disease in a 40-year-old woman affected with essential thrombocythemia (ET) is reported. The profile of plasma von Willebrand factor (vWF) revealed decreased ristocetin cofactor activity and diminished large multimers of vWF in spite of a normal vWF antigen level. There was no evidence of circulating inhibitor against the factor VIII complex. The vWF abnormality improved by controlling the platelet count following treatment for ET with interferon-alpha 2b and ranimustine. The possible mechanism of the development of AvWD in ET is briefly discussed.

Topics: Adult; Female; Humans; Interferon alpha-2; Interferon-alpha; Platelet Aggregation; Recombinant Proteins; Ristocetin; Thrombocythemia, Essential; von Willebrand Diseases; von Willebrand Factor

By providing some examples of variations in the levels of von Willebrand factor antigen (vWF:Ag), ristocetin cofactor, and Factor VIII during one month, the authors wish to emphasize the difficulty of diagnosing mild forms of von Willebrand's disease (vWD), especially type I. In three of 15 normal female volunteers the vWF:Ag levels, on some sampling occasions, were so low (0.25-0.30 IU/mL, normal greater than 0.50 IU/mL) that the diagnosis of vWD type I might be made while on other occasions normal levels were obtained. The coefficients of variation (CV) for vWF:Ag in these three women were 12%, 25%, and 43%. However, CVs of similar magnitude were also observed for "non-diseased" males and females. The ratio F VIII/vWF:Ag also varied greatly. In the three women with suspected vWD it was 36%, 15%, and 34%. A representative level for the entire cycle of vWF:Ag and ristocetin cofactor seems to have been obtained in the follicular phase and therefore it is suggested that in order to make the diagnosis of vWD type I, at least in females, blood samples should be taken in this phase.

Topics: Adult; Factor VIII; Female; Humans; Male; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We have identified a patient with von Willebrand's disease (vWD) resembling type IIB vWD, with increased ristocetin induced platelet aggregation (RIPA), the absence of the large multimers of von Willebrand factor (vWF) in plasma, and the presence of the large multimers in platelets in whom a family study indicated a probable double heterozygous inheritance pattern. The propositus was a 12-year-old boy with frequent epistaxis and bruising. Abnormal hemostatic findings included a prolonged bleeding time (BT), decreased levels of factor VIII coagulant activity (VIIIC), von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (RCof), and an increased RIPA. In the presence of ristocetin, binding of the patient's plasma vWF to normal platelets was increased but binding of normal vWF to his platelets was normal. SDS-agarose gel (1.5%) electrophoresis revealed that plasma vWF lacked the large multimers, and 3.0% gel electrophoresis revealed that the multimers had a 5-band pattern similar to normal. The above findings were consistent with type IIB vWD, but 1-deamino[8-D-arginine]-vasopressin (DDAVP) infusion resulted in a shortened BT and the transient appearance of large multimers without a decrease in the platelet count. Family studies revealed that his mother has mild bleeding symptoms, decreased VIIIC, vWF:Ag, and RCof levels and normal to slightly reduced RIPA with a multimer pattern consistent with type I vWD. In contrast, the father, sister, and paternal grandfather were asymptomatic, with a slightly decreased VIIIC level but a normal BT and vWF:Ag and RCof levels. Their RIPA and vWF binding to normal platelets were increased, but unlike the propositus their plasma contained large multimers. We concluded that the propositus is a type IIB-like variant differing from previously reported IIB variants in two ways: 1) his response to DDAVP and 2) a possible double heterozygous mode of inheritance rather than the usual dominant route.

Topics: Blood Platelets; Child; Deamino Arginine Vasopressin; Electrophoresis, Agar Gel; Heterozygote; Humans; Infusions, Intravenous; Male; Pedigree; Platelet Aggregation; Ristocetin; Sodium Dodecyl Sulfate; von Willebrand Diseases; von Willebrand Factor

Topics: Antibodies, Monoclonal; Blood Platelets; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The differentiation of type IIb von Willebrand's disease from other variants of von Willebrand's disease, especially platelet-type (pseudo-) von Willebrand's disease, poses a significant clinical problem because, although they are similar in the clinical and diagnostic laboratory settings, the therapy of type IIb von Willebrand's disease is different from the therapy of platelet-type von Willebrand's disease. This discrimination has required cumbersome assays using fresh platelet-rich plasma that often yielded equivocal results. Because it was shown by other researchers that type IIb von Willebrand factor binds to normal platelets with increased avidity at low concentrations of ristocetin, it was reasoned that von Willebrand factor from patients with type IIb von Willebrand's disease would also bind to formalin-fixed washed platelets at low concentrations of ristocetin. Using the radiolabeled "neutral" monoclonal antibody AVW1 to label plasma von Willebrand factor, the binding of von Willebrand factor to formalin-fixed washed platelets was studied as a function of ristocetin concentration. These studies demonstrated that the 125I-AVW1 von Willebrand factor from 13 patients with type IIb von Willebrand's disease binds to formalin-fixed washed platelets at significantly lower concentrations of ristocetin than plasma von Willebrand factor from 18 normal individuals, 3 patients with platelet-type von Willebrand's disease and 8 patients with other variant forms of von Willebrand's disease. This radiolabeled "neutral" monoclonal antibody technique provides a rapid, simple method for the differentiation on frozen plasma samples of type IIb von Willebrand's disease from platelet-type and other variants of von Willebrand's disease.

Topics: Antibodies, Monoclonal; Blood Platelets; Humans; Reference Values; Ristocetin; Time Factors; von Willebrand Diseases; von Willebrand Factor

Thrombocytopenia has been reported in patients with type IIB von Willebrand's disease (vWd) during pregnancy. In the present work we report the behaviour of platelet count and von Willebrand factor (vWf) multimers in a pregnant type IIB vWd patient who presented with mild thrombocytopenia in the steady state. The evolution of pregnancy was associated with a progressive decrease of platelet count which showed its lowest value two days before delivery (20 x 10(9)/l). The tendency of the platelet count to decrease was suddenly reversed a few days later (77 x 10(9)/l). At the same time, the vWf multimeric pattern showed a strict but inverse correlation with the platelet count. In fact, a progressive increase in low and intermediate sized vWf multimers, which proceeded until platelets reached their minimum level, was noted. A few days after delivery, concomitant with the prompt platelet increase, low and intermediate multimers became decreased. During pregnancy, the patient's platelet showed additional increased responsiveness to ristocetin but did not demonstrate spontaneous platelet aggregation (SPA). On the contrary, the patient's plasma, collected both during and after pregnancy, caused normal platelets to aggregate spontaneously. SPA appeared completely blocked by an anti-GPIIb-IIIa monoclonal antibody (MAb), which recognized the binding site for fibrinogen, vWf and fibronectin. In contrast, a MAb against ristocetin-induced vWf binding site on GPIb did not affect SPA. These findings suggest that the common stimulus or stimuli, responsible for the pregnancy-induced decrease of platelet count and improvement of vWf multimeric pattern in type IIB vWd is strictly related to pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Antibodies, Monoclonal; Blood Coagulation Tests; Female; Fibrinogen; Fibronectins; Humans; Platelet Aggregation; Platelet Count; Platelet Membrane Glycoproteins; Pregnancy; Pregnancy Complications, Hematologic; Ristocetin; Thrombocytopenia; von Willebrand Diseases; von Willebrand Factor

Platelet von Willebrand factor (vWF) has been suggested to play an important role in the hemostatic process. Clinical and experimental data indicate that bleeding time (BT) and platelet-vessel wall interaction cannot be normalized unless the defect of platelet vWF is also corrected. We have examined the effect of normal platelet concentrate transfusion 1 hour after cryoprecipitate infusion in five type III von Willebrand disease (vWD) patients. The cryoprecipitate infusion attained normal circulating levels of ristocetin cofactor, vWF antigen, and factor VIII activity. In two patients, cryoprecipitate infusion did not modify the BT (greater than 30 minutes), whereas in the remaining three patients BT was only partially corrected (from greater than 30 to 12, 18, and 21 minutes). However, the immediate platelet transfusion completely corrected the BT in four cases, and in one case it shortened the BT to 8.30 minutes (n = 3 to 8 minutes). In the perfusion study, cryoprecipitate infusion only resulted in a slight increase in platelet deposition (surface coverage range: 2.4% to 11.3%), whereas the platelet concentrate transfusion elicited a more marked improvement (range: 8.2% to 26.4%; P less than .02 v post-cryoprecipitate). These results suggest an important in vivo role of the platelet vWF in supporting platelet-vessel wall interaction. They also give support to the occasional addition of normal platelet transfusion to the cryoprecipitate infusion for the control of serious bleeding episodes resistant to cryoprecipitate in severe vWD patients.

Topics: Bleeding Time; Blood Platelets; Blood Transfusion; Endothelium, Vascular; Factor VIII; Fibrinogen; Hemostasis; Humans; Platelet Transfusion; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Platelets from patients with platelet-type von Willebrand disease (vWD) were used as immunogens for the production of murine monoclonal antibodies (MoAbs). One such MoAb, C-34, inhibited ristocetin-induced aggregation of patient or normal platelets, but not aggregation induced by other aggregating agents. As demonstrated by crossed-immunoelectrophoresis, C-34 recognized an epitope within the GPIb/IX complex. In indirect immunofluorescence studies on fresh platelets, the ratio of any of four different anti-GPIb MoAbs to one another was near unity (0.88-1.14) both for normals and for patients. In contrast, the ratio of the binding of C-34 to such a MoAb (AP-1) was 0.31 +/- 0.02 (means +/- SE) for normal platelets and significantly increased to 0.54 +/- 0.01 for patient platelets (P less than 0.001). In NP-40 lysates of 3H-labelled platelets, saturating concentrations of C-34 produced much fainter bands than did AS-2 or other anti-GPIb MoAbs in the GPIb and GPIX regions. In contrast to the other anti-GPIb MoAbs, C-34 did not bind to the purified 125I-labelled glycocalicin fragment of GPIb, to the glycocalicin derivative identified by crossed-immunoelectrophoresis, or to the amino-terminal approximately 40 kDa portion of GPIb alpha cleaved from 3H-labelled platelets by trypsin. C-34 appears to be the first MoAb that is quantitatively informative in identifying the abnormal platelets in platelet-type vWD. The observed differences between the patient and normal platelets may reflect an abnormality in the primary structure of the GPIb/IX complex. Alternatively, patient platelets may have an abnormality of other structures near this region that impose less of a steric hindrance upon binding of antibody to the C-34 epitope.

Topics: Animals; Antibodies, Monoclonal; Blood Platelets; Epitopes; Humans; Immunoelectrophoresis, Two-Dimensional; Mice; Mice, Inbred BALB C; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; von Willebrand Diseases

The authors have identified six Southeast Asian patients ranging in age from 14 to 21 years with hemoglobin E-beta(0) thalassemia and a coagulopathy involving von Willebrand factor (vWF). These patients had normal or only slightly decreased plasma clotting factor levels. The activated partial thromboplastin time was prolonged in four of the patients. The abnormal feature common to all patients was a qualitative loss of high molecular weight multimers of vWF by crossed immunoelectrophoresis (vWF:CIE). Plasma vWF antigen concentration (vWF:Ag) and ristocetin cofactor activity (vWF:RCo) also were decreased and bleeding time prolonged in three patients. Epistaxis was present in two. No family history of increased bleeding tendency was present in any patient. Coagulation parameters and vWF:CIE were normal in two first-degree relatives without this hemoglobinopathy. vWF abnormalities and clinical manifestations were greatest in those patients with the most severe anemia and hepatosplenomegaly. These six patients appear to have an acquired abnormality of vWF, although they lack the clinical characteristics of acquired von Willebrand disease. While the etiology of this abnormality is unclear, the authors speculate that proteolysis of vWF secondary to extramedullary hematopoiesis or loss through high cardiac output shear stress in these anemic patients may be involved.

Topics: Adolescent; Adult; Antigens; Bleeding Time; Blood Coagulation Tests; Factor VIII; Female; Hemoglobin E; Hemoglobins, Abnormal; Humans; Immunoelectrophoresis; Laos; Male; Minnesota; Molecular Weight; Ristocetin; Thalassemia; von Willebrand Diseases; von Willebrand Factor

A prenatal diagnosis for fetal disease was performed at 20 weeks gestation in a severely affected patient with type IIA von Willebrand disease. In the fetal cord blood sample obtained under ultrasound guidance, the level of von Willebrand ristocetin cofactor activity was similar to that of von Willebrand factor antigen, and all the multimers were present. These results were compared to those obtained in 51 normal fetuses of similar gestational age (19-21 weeks). Normal fetuses showed slightly lower levels of von Willebrand factor than normal adults and in addition to all adult multimers, the presence of unusual large forms. This data compared with the case, allowed the exclusion of the diagnosis of type IIA von Willebrand disease in our patient's fetus. This was confirmed at birth in the cord blood.

Topics: Adult; Factor VIII; Female; Fetal Blood; Humans; Pregnancy; Pregnancy Trimester, Second; Prenatal Diagnosis; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We describe a man and his daughter from a large New Zealand family with the extremely rare variant von Willebrand's (VW) disease type IID. These two patients had a severe bleeding history following minor surgery and displayed easy bruising. However, routine laboratory screening tests and factor VIII studies were essentially normal except for slightly reduced ristocetin cofactor activity and prolonged skin bleeding times. Although lacking higher molecular weight forms of VW antigen in common with VW type II's the patients' multimer patterns were clearly different from types IIA and IIB. Instead of showing the characteristic 'triplet' pattern in each multimer band the patients gave a single prominent band with faint satellite bands different in mobility to those in normals. Von Willebrand factor from the patients' platelets gave a similar abnormal pattern. DDAVP failed to correct the bleeding time in either patient and multimer analysis confirmed that there was no increase in the higher molecular weight VWF antigen.

Topics: Adolescent; Adult; Bleeding Time; Blood Protein Electrophoresis; Factor VIII; Female; Humans; Male; Molecular Weight; Pedigree; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Von Willebrand's disease is categorized into types and subtypes based on multimeric analysis of plasma von Willebrand's factor. Such categorization is of value because both the mode of inheritance and the choice of therapeutic material differ between subtypes. The Type IIB variant is characterized by hypersensitivity in vitro to ristocetin and thrombocytopenia after administration of desmopressin (DDAVP). Hypersensitivity to ristocetin has also been described in Type I variants but without thrombocytopenia after DDAVP. This report describes a new Type II variant characterized by the converse situation, absence of hypersensitivity to ristocetin in vitro but transient thrombocytopenia after intravenous administration of DDAVP.

Topics: Deamino Arginine Vasopressin; Drug Hypersensitivity; Female; Humans; Infusions, Intravenous; Male; Pedigree; Platelet Aggregation; Platelet Count; Ristocetin; Thrombocytopenia; von Willebrand Diseases; von Willebrand Factor

We have studied the interaction of the congenitally abnormal type IIA and IIB von Willebrand factor (vWF) molecules, both lacking the larger multimeric forms, with the two vWF binding sites on platelets, the glycoprotein (GP) Ib-IX and GP IIb-IIIa complexes. Variant as well as normal (N) vWF were purified from plasma. Estimates for binding of subunit molecules per platelet at saturation (Bmax) and dissociation constant in moles/liter (Kd), respectively, were obtained from binding isotherms of 125I-labeled vWF, with the following results. In the presence of ristocetin (binding to GP Ib-IX): N, 25,693 and 0.5 x 10(-8); IIA, both parameters not measurable; IIB, 17,708 and 0.87 x 10(-8). After thrombin stimulation (binding to GP IIb-IIIa): N, 17,059 and 1.12 x 10(-8); IIA, 23,751 and 4.87 x 10(-8); IIB, 19,890 and 2.52 x 10(-8). Distinct experiments based on measuring the ability of the variant species (from the same patients and one additional IIB patient) to inhibit the binding of normal 125I-vWF to platelets gave results in agreement with those reported above. Other studies showed that only IIB vWF bound to platelets in the absence of any mediating substance (Kd = 5.21 x 10(-8) mol/liter and Bmax = 9,599 subunits per platelet) and induced aggregation at a concentration of 10 micrograms/ml (3.6 x 10(-8) M). Thus, IIB vWF binds to GP Ib-IX with high affinity and induces platelet aggregation, whether with or without ristocetin, in spite of the absence of larger multimers. In contrast, the binding of IIA vWF to GP Ib-IX occurs with very decreased affinity, and this defective function may result from specific structural abnormalities rather than just being a reflection of the absence of larger multimeric forms. Both IIA and IIB vWF exhibit decreased affinity for GP IIb-IIIa. In this case, the extent of the defect correlates with the absence of larger multimers.

Topics: Blood Platelets; Humans; In Vitro Techniques; Macromolecular Substances; Platelet Activation; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; Sialic Acids; Thrombin; von Willebrand Diseases; von Willebrand Factor

Commercial concentrates of factor VIII (FVIII) were analyzed in order to 1) determine the effects of viral inactivation on von Willebrand factor (vWF); 2) evaluate the vWF content of the new, immunopurified concentrates; and 3) assess their potential for correcting the long bleeding time of von Willebrand disease (vWD). Included in our study were products that had been treated to inactivate viruses; older, untreated products; and the new, immunopurified concentrates. We measured von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity (RCoF), and vWF multimeric and subunit composition. A newly developed radioimmunoassay (RIA) was used to quantitate vWF:Ag. The vWF:Ag content varied from 0.083 micrograms/IU FVIII:C for Hemofil M to 32.2 micrograms/IU FVIII:C for Humate-P, whereas pooled normal human plasma (NHP) contained 6.3 micrograms/IU FVIII:C. The RCoF varied from 0.0007 to 2.09 U/IU FVIII:C, with the immunopurified concentrates having the lowest values and Humate-P the highest. The ratio of RCoF to vWF:Ag ranged from 11 to 96 U/mg, as compared to a ratio of 160 for NHP. All of the concentrates lacked the largest vWF multimers, and all had abnormal triplet patterns. Modest differences between some untreated concentrates and their treated counterparts were noted. As expected, the immunopurified concentrates had much lower levels of all vWF activities than the conventionally prepared products. Our data suggest that none of the concentrates have as great a capacity as NHP to correct the prolonged bleeding time of von Willebrand disease.

Topics: Antigens; Chemical Phenomena; Chemistry; Factor VIII; Humans; Immunologic Techniques; Radioimmunoassay; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A factor VIII (FVIII) concentrate, virus-inactivated by the solvent/detergent procedure, was studied in vitro. In contrast with most high-purity, virus-inactivated FVIII concentrates, it contains not only high levels of von Willebrand factor (vWF) antigen and ristocetin cofactor activity but also high molecular weight forms of von Willebrand factor. Furthermore, it is able to promote platelet adhesion on collagen in a perfusion system. In vivo studies performed in patients with different types of von Willebrand's disease provided evidence that this concentrate corrects Duke's bleeding time and prevents or stops haemorrhages. Thus, the particular advantages of this FVIII/vWF preparation are safety, low content of contamination proteins, and efficacy in von Willebrand's disease.

Topics: Antigens; Bleeding Time; Detergents; Drug Contamination; Factor VIII; Humans; Molecular Weight; Platelet Adhesiveness; Ristocetin; Solvents; Viruses; von Willebrand Diseases; von Willebrand Factor

In this study a new variant of type II von Willebrand disease is identified by multimeric analyses of increasing resolving power. Prior to multimeric analysis, the patient was misdiagnosed as carrying an undefined abnormality in platelet function because of his normal von Willebrand factor antigen (vWF:Ag) and low borderline ristocetin cofactor (Ricof) levels. Absence of the largest multimers from the patient's plasma and platelets was shown in a low-resolution system, but all the multimers were present in his relatives. An abnormality in the complex multimeric structure was demonstrated in both plasma and platelets with high-resolution agarose gels. The plasma of the proband and of several family members shows a broader central band with a minor, faster moving satellite band differing from the typical "triplet pattern" observed with normal plasma. Platelets show a "doublet" that runs with a mobility different from the "doublet" in normals. Therefore the proband may be either a homozygote or double heterozygote for this new abnormality. Treatment with desmopressin (DDAVP) on several occasions corrected the prolonged bleeding time of the patient only transiently. Factor VIII increased significantly, but vWF:Ag and Ricof responded poorly. We conclude that this vWF abnormality is different from those observed in the other variants (II A-G) previously described. Therefore the proposed designation for this new variant is type II H.

Topics: Adult; Antigens; Bleeding Time; Blood Platelets; Deamino Arginine Vasopressin; Factor VIII; Genetic Variation; Humans; Macromolecular Substances; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

To characterize the heterogeneity of severe (type III) von Willebrand disease (vWD), plasma and platelet von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (Ricof) were measured in 28 obligatory heterozygotes (ie, parents or children of probands from 15 different kindreds with severe vWD). On the average, heterozygotes had low levels of vWF in both platelets and plasma. There was, however, considerable heterogeneity, with four distinct patterns. Eleven heterozygotes had concordant reduction of vWF:Ag and Ricof in both plasma and platelets; five had low levels of vWF:Ag and Ricof in plasma contrasting with normal levels in platelets; eight had a peculiar pattern, the reverse of the above (ie, low levels in platelets and normal levels in plasma); and in one, both vWF measurements were normal in plasma and platelets. These patterns were genetically determined: they were consistent in four couples of consanguineous heterozygotes and in two couples carrying the same gene deletion. Only the remaining three heterozygotes had no clearly identifiable pattern. Other findings of this study were that although most of the heterozygotes had normal bleeding times, the 7 of 28 who had prolonged bleeding times had concordantly low levels of vWF measurements in both plasma and platelets. In conclusion, this large series of obligatory heterozygotes provides evidence for phenotypic and genotypic heterogeneity of severe vWD.

Topics: Bleeding Time; Blood Platelets; Heterozygote; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The method of sample collection and time interval from venipuncture to test performance is considered to be important in platelet function testing. Platelet function studies can be performed in whole blood with the use of an impedance lumi-aggregometer, and an examination of the influence of these variables on the outcome of such a study is important. Twelve healthy donors had blood drawn with the use of conventional approach for platelet function testing and a Vacutainer method. Platelet function studies were performed at three time points over a three-hour period. No significant differences in results were observed between the methods of collection and the time to test performance. It is concluded that for platelet function screening in whole blood, with the use of an impedance lumi-aggregometer, a Vacutainer sample maintained at room temperature and tested within three hours is satisfactory.

Topics: Adenosine Triphosphate; Adult; Blood Specimen Collection; Female; Humans; Male; Platelet Aggregation; Platelet Function Tests; Ristocetin; Thrombin; Time Factors; von Willebrand Diseases

Platelet function testing in von Willebrand disease is generally performed in platelet rich plasma using an optical system. The characteristic abnormality is an abnormal response to the agglutinating agent, ristocetin. Platelet aggregation can be also studied in whole blood using either an impedance aggregometer or a particle counter. Fifteen patients with von Willebrand disease and fifteen normal subjects were studied using both whole blood methods. In the impedance system, normal subjects responded to ristocetin (1 mg/ml) with short lag phases (less than 70 sec) and normal maximum aggregation greater than 5 omega). Only three patients with von Willebrand disease responded with normal maximal aggregation but each of these had a prolonged lag phase, and this may be a useful diagnostic parameter. In the particle counter, discrimination between normals and some von Willebrand disease patients was possible but an overlap between normals and von Willebrand patients is evident. It is concluded that the platelets in patients with von Willebrand disease exhibit the same abnormalities in whole blood as in platelet rich plasma and that the combination of impedance aggregometry and a factor VIII procoagulant assay is a time-efficient and sensitive method to screen for von Willebrand disease.

Topics: Humans; Osmolar Concentration; Platelet Aggregation; Reaction Time; Reference Values; Ristocetin; von Willebrand Diseases

Topics: Blood Platelets; Flow Cytometry; Galactans; Humans; Platelet Membrane Glycoproteins; Protein Binding; Receptors, Cell Surface; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A heat-treated factor VIII (F VIII) concentrate (Haemate P) has been administered to patients with various types of von Willebrand's disease (vWD). The 4 activities of F VIII/vWF as well as change in the multimeric structure of vWF were then studied. In 4 patients with type I vWF who were given a Ristocetin cofactor (Rcof) dose of 42-78 U/kg, there was a clear reduction of the bleeding time and an increase of F VIII: C, F VIII: Ag, Rcof and vWF: Ag for several hours. The recovery of Rcof. after 1 h was 50-75%. Although the multimeric composition of vWF in these patients was similar to that of normal plasma, the density of each multimer band was very low. After infusion, however, the density of all multimer bands increased for several hours, to decrease again after 24 h. In 4 patients with type II A vWD who received a dose of Rcof of 55-76 U/kg, the 4 activities of F VIII/vWF increased similarly as was the case in type I. All patients had only 3-4 smaller multimer bands. New larger and intermediate multimers appeared for several hours after infusion of the preparation. Two patients with type III vWD who received doses of Rcof of 52 and 65 U/kg showed also a similar increase in the 4 activities of F VIII/vWF after infusion. All the multimers lacking in these patients appeared for several hours after infusion.

Topics: Adolescent; Adult; Bleeding Time; Child; Child, Preschool; Factor VIII; Female; Hemostasis; Hot Temperature; Humans; Male; Middle Aged; Ristocetin; von Willebrand Diseases

Collagen cofactor (CCo), an activity of von Willebrand factor (vWF) which increases the rate of adhesion of human fixed washed platelets (FWP) to collagen, was measured in plasma from normal individuals and individuals with von Willebrand's disease (vWD). CCo in vWD plasma was compared to vWF antigen (vWF:Ag), ristocetin cofactor (RCo), factor VIII (VIII) coagulant activity (VIII:C) and the quantitative bleeding time. There was close correlation between CCo and VIII:C (r = 0.909), vWF:Ag (r = 0.975), and RCo (r = 0.936). However, there was no correlation between CCo and the quantitative bleeding time. Plasma CCo in type IIA vWD was markedly lower than vWF:Ag and the ratio of CCo/vWF:Ag was 0.08, which was less than a mean value of 0.92 in type I vWD. CCo activity in normal plasma was completely inhibited by monoclonal antibody CLB-RAg 201, an antibody that inhibits the binding of vWF to collagen, suggesting that the binding of vWF to collagen is required for the expression of CCo. Furthermore, the partial inhibition of CCo by monoclonal antibody CLB-RAg 35 that inhibits the binding of vWF to platelet in the presence of ristocetin, suggests that CCo is partly mediated through platelet membrane glycoprotein Ib. Large multimers of vWF:Ag in normal plasma were preferentially absorbed by collagen. These studies demonstrate that CCo is another functional activity of vWF and the measurement of CCo may be useful for the detection of new variant forms of vWD.

Topics: Antibodies, Monoclonal; Antigens; Bleeding Time; Blood Preservation; Collagen; Factor VIII; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A familial bleeding disorder characterized by an association of Type IIB von Willebrand's disease (vWD) with a complex thrombocytopenic thrombocytopathy is described in two patients from the same generation. Findings typical of type IIB vWD included enhanced ristocetin-induced binding of patient von Willebrand factor (vWF) to platelets of patients and normal individuals in association with the absence of larger multimers from plasma. Abnormalities in platelet function included deficient platelet aggregation to ADP, collagen, epinephrine, and arachidonic acid; and defective release of 14C-serotonin, vWF, and platelet factor 4 (PF4) in response to thrombin, collagen, or ADP. Platelet factor 4 and platelet vWF were decreased when measured per mg of total platelet protein. In addition, the binding of normal vWF to patient platelets stimulated with thrombin was decreased. Platelet size was increased with a very heterogeneous distribution width. Electron microscopic evaluation showed giant platelets with dense and alpha bodies present. The platelet count was borderline or slightly decreased in the resting state and declined to frankly thrombocytopenic levels at the time of acute bleeding episodes; this state was associated with the presence of platelet aggregates in blood smears.

Topics: Antibodies, Monoclonal; Blood Platelets; Female; Humans; Kinetics; Male; Pedigree; Platelet Aggregation; Reference Values; Ristocetin; Thrombocytopenia; von Willebrand Diseases; von Willebrand Factor

The clinical and laboratory features of a patient with a recently recognized new variant of von Willebrand's disease are presented. The importance of this variant is that it is associated with a clinically significant bleeding diathesis but with a normal skin bleeding time, PTTK, factor VIIIc and platelet aggregation with 1 mg/ml ristocetin. The distinctive laboratory features are increased platelet sensitivity to low concentrations of ristocetin, and the presence of all plasma von Willebrand factor multimers, but in reduced amounts. The need for thorough investigation of patients with significant bleeding history despite apparently normal screening tests is emphasized.

Topics: Adult; Humans; In Vitro Techniques; Male; Platelet Aggregation; Protein Conformation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Circulating inhibitors against von Willebrand factor (vWF) that show the properties of heterologous IgG antibodies have been described in a few patients with severe von Willebrand disease (vWD). The present study provides further characterization of inhibitors from two patients with severe vWD. Inhibitors in both, like polyclonal rabbit antibody, detected all sizes of multimers and the complex structure of each multimer from platelets and plasma of normal individuals as well as from plasma of patients with IIA, IIB, and IIC vWD. Both inhibitors and the rabbit antibody reacted mainly with the intact 225-Kd vWF subunit and the 189-H and 140-Kd fragments in contrast to monoclonal antibodies specific for vWF fragments that detected a higher relative proportion of 176-Kd fragment. Furthermore, all these antibodies recognized fragment III, although one inhibitor and rabbit polyclonal antibody reacted poorly and the other inhibitor did not react at all with reduced fragment II of vWF digested with Staphylococcus aureus V-8 protease. These data suggest that although human inhibitors from severe vWD patients may behave, to some extent, as polyclonal heterologous antibodies against native vWF, the former show striking differences in their target specificity as well as a much broader specificity than that described for human factor VIII inhibitors.

Topics: Adult; Antibodies, Heterophile; Binding, Competitive; Female; Humans; Immunoglobulin G; Macromolecular Substances; Molecular Weight; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Multiple assays have been described which quantify von Willebrand Factor (vWF) in plasma or other specimens. An assay using a modification of the microtitre method(1) is described. Washed platelets fixed in formalin and frozen in dimethylsulfoxide are substrate for the reaction. Platelets, ristocetin and the specimen to be assayed are combined in the wells of a microtitre plate. The reaction is mixed for defined times and agglutination of platelets read visually. By adjusting the concentration of platelets, concentration of ristocetin and time, the sensitivity can be adjusted over a wide range.

Topics: Humans; Methods; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

In a case of severe IgG kappa myeloma with cryoglobulinaemia usual concentrations of epinephrine, collagen, ADP, arachidonic acid, thrombin and ristocetin caused no aggregation of platelets in platelet rich plasma. However, in contrast to other agents ristocetin induced platelet aggregation in higher concentrations. The investigations showed, that the aggregating activity was inhibited by binding of ristocetin to the abnormal protein. Following saturation of the monoclonal protein, the surplus ristocetin caused normal aggregation. This indicates that platelets actually preserved their responsiveness to ristocetin. Possible causes of the phenomenon are discussed.

Topics: Aged; Cryoglobulinemia; Female; Hemorrhagic Disorders; Humans; Immunoglobulin G; Immunoglobulin kappa-Chains; Multiple Myeloma; Platelet Aggregation; Protein Binding; Ristocetin; von Willebrand Diseases

Thrombocytopenia may accompany variant (type IIB) von Willebrand's disease (vWD) and is thought to result from binding of the abnormal von Willebrand factor (vWF) to the patient's platelets with subsequent platelet aggregate formation and clearance. We have studied a patient with type IIB vWD who became thrombocytopenic during two pregnancies. During the third trimester of pregnancy, her platelet counts dropped to 20,000 to 30,000/microL, and an increase in the intermediate-sized vWF multimers was seen on agarose gel electrophoresis. During this time her platelet-rich plasma showed spontaneous platelet aggregation, and her plasma caused spontaneous aggregation of normal washed platelets. Antibody to platelet glycoprotein Ib completely blocked the spontaneous platelet aggregation, while antibody to platelet glycoprotein IIb/IIIa did not block the response at the concentrations used. Inhibitors of platelet function that elevate platelet cyclic AMP also blocked the response, but aspirin had no effect on the spontaneous platelet aggregation. The patient illustrates that the platelet counts in one individual can vary greatly in type IIB vWD and that the thrombocytopenia that occurs can appear under physiologic conditions that stimulate the endogenous production of the patient's abnormal vWF. The mechanisms leading to spontaneous platelet aggregation and thrombocytopenia appear to be similar to those described for other patients with type IIB vWD.

Topics: Adult; Aspirin; Blood Coagulation Tests; Bucladesine; Epoprostenol; Female; Humans; Platelet Aggregation; Pregnancy; Pregnancy Complications, Hematologic; Ristocetin; Thrombocytopenia; von Willebrand Diseases

A patient with acquired von Willebrand disease associated with multiple myeloma (IgG-lambda) is described. Mixture of his plasma or IgG fraction with washed control platelets resulted in the inhibition of aggregation with ristocetin, but mixture of control plasma or IgG fraction with washed patient platelets showed no inhibition of ristocetin-induced aggregation. Although his vWF: Ag, RCo, and factor VIII coagulant activity were all normal, inactivation of RCo was induced in normal plasma by incubation with patient plasma. Crossed immunoelectrophoretic analysis showed that vWF:Ag was composed of much more anodic component. A marked increase of Factor VIII and a rapid return of RCo to the baseline after 1-deamino-8-arginine vasopressin (DDAVP) infusion were observed. A transient increase in vWF:Ag after the infusion of DDAVP showed with less anodic forms and in the relative proportion as in normal. Treatment of the underlying disease also led to a correction of the bleeding time, improvement of platelet adhesion and ristocetin-induced aggregation, and normalization of crossed immunoelectrophoresis of vWF:Ag. The present study showed that myeloma-associated IgG interacted specifically with the antigenic sites on the von Willebrand portion of the Factor VIII complex.

Topics: Adult; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Blood Coagulation Tests; Blood Platelets; Factor VIII; Glycoproteins; Humans; Male; Melphalan; Myeloma Proteins; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Three monoclonal antibodies produced against vWF:Ag by conventional hybridoma technique did not inhibit factor VIII coagulant activity (F. VIII:C) but did inhibit VIII ristocetin cofactor activity. The antibodies were used in an indirect competitive ELISA for quantifying von Willebrand's antigen (vWF:Ag) and compared with values obtained by the Laurell technique using commercial antibody by means of a ratio: ELISA/Laurell. For one monoclonal BD2-CC9, vWF:Ag values obtained in the two assays were in good agreement for normal and hemophilia A plasmas (normal, n = 19, ratio = 1.13 +/- .17, hemophilia A, n = 10, ratio = 0.91 +/- .15). However, type II vWD patients had a disproportionately low value of vWF:Ag with the ELISA. Use of the ratio normalized the difference among individual plasma values and allowed a significant separation of type II vWD plasma (n = 9, ratio = 0.46 +/- .19) from normal plasma (p = .0001) and type I vWD plasma (n = 8, ratio = 1.52 +/- .34) from type II vWD plasma (p = .0003) using BD2-CC9. Although the sample size was small, the greater degree of discrimination among the vWD plasmas tested with BD2-CC9 (compared with the other two antibodies [CA3-AE4, CC6-BG10]) suggests that this antibody may recognize conformational epitopes that reflect the degree of multimeric polymerization of the vWF molecule rather than simply recognize a decreased number of antigenic sites in a basic subunit. BD2-CC9 may be valuable in investigating the various types of vWD and/or the process of polymerization of this complex protein.

Topics: Antibodies, Monoclonal; Antibody Specificity; Antigens; Enzyme-Linked Immunosorbent Assay; Factor VIII; Female; Humans; Male; Partial Thromboplastin Time; Platelet Aggregation; Protein Conformation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF-induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.

Topics: Adenosine Triphosphate; Asialoglycoproteins; Binding Sites; Blood Platelets; Edetic Acid; Humans; Platelet Aggregation; Ristocetin; Thromboxane B2; von Willebrand Diseases; von Willebrand Factor

Cryoprecipitate has proved to correct the hemostatic defects in von Willebrand's disease (vWD) and platelet-type vWD. However, recent studies have revealed that transmission of the AIDS retrovirus (HIV) occurs through exposure to blood products including cryoprecipitate. Treatment with heat-treated factor VIII/von Willebrand factor (vWf) concentrates may have certain advantages over treatment with nonheated products, if these preparations are efficacious in these disorders. We found that a commercially available factor VIII/vWf concentrate, Haemate P, contained the high-molecular-weight multimers of vWf and had a ratio of ristocetin cofactor (RCof) to vWf antigen (vWf:Ag) close to unity. In addition, its capacity to directly induce aggregation of platelet-type vWD platelets in vitro was similar to that for cryoprecipitate. When infused into a patient with platelet-type vWD, Haemate P shortened the prolonged bleeding time and caused spontaneous platelet aggregation in vitro with a mild diminution of platelet count. These results indicate that some of the heat-treated factor VIII/vWf concentrates may provide a safer, yet still effective, treatment for platelet-type vWD.

Topics: Factor VIII; Female; Hot Temperature; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions. Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients. The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added. Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma. Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes. All maturation stages of morphologically recognizable megakaryocytes were aggregated. The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated). Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin. A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions. The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population. We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics. This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.

Topics: Animals; Cattle; Cell Aggregation; Cell Separation; Cell Survival; DNA; Guinea Pigs; Megakaryocytes; Plasma; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Electrophoresis, Agar Gel; Genetic Variation; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Chemical Phenomena; Chemistry; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases

The authors present a study of a human myeloma-produced monoclonal protein (IgG-k) directed against von Willebrand factor that caused an acquired von Willebrand's disease (vWD)-like syndrome. The illness was characterized by upper gastrointestinal bleeding, prolonged bleeding time, decreased platelet adhesiveness, lack of platelet aggregation in response to ristocetin, and a qualitatively abnormal Factor VIII related antigen (vWF) by two-dimensional immunoelectropheresis. Patient plasma or IgG fraction mixed with normal platelet-rich plasma completely inhibited aggregation with ristocetin, but patient platelets resuspended in normal plasma aggregated normally with ristocetin. VWF was markedly elevated and the two-dimensional immunoelectropheresis of vWF revealed a vWD type II-like pattern with an absence of the higher molecular weight forms of the vWF. Marked inhibitory activity was observed in the ristocetin cofactor assay but disappeared at the highest dilutions of patient plasma used in the assay. Infusion of cryoprecipitate following plasmapheresis led to a correction of the bleeding time, improvement in platelet adhesiveness, transient disappearance of inhibitory activity in the Factor VIII ristocetin cofactor assay, and no significant normalization of two-dimensional immunoelectropheresis of vWF. This case demonstrated a myeloma-associated monoclonal antibody that interacted specifically with that part of the Factor VIII molecule necessary for Factor VIII ristocetin cofactor activity, normal platelet adhesiveness, and bleeding time.

Topics: Aged; Antibodies, Monoclonal; Blood Coagulation Tests; Factor VIII; Female; Fibrin Fibrinogen Degradation Products; Humans; Myeloma Proteins; Plasmapheresis; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A variant of von Willebrand disease (vWD) has been identified in a 19-year-old woman with a severe bleeding syndrome. She had a very prolonged bleeding time (over 20 min), 24 U/dl factor VIII coagulant activity (F.VIII:C), 16 U/dl von Willebrand factor antigen (vWF:Ag), no ristocetin cofactor activity, and an anodal mobility of vWF:Ag on crossed immunoelectrophoresis (CIE). vWF:Ag was markedly reduced in her platelet lysate. In plasma and platelets, SDS-agarose electrophoresis consistently demonstrated the absence of large multimers, a relatively increased concentration of the fastest-moving multimer, and gross abnormalities of the internal structure of each vWF multimeric unit. Five members from the maternal side of the family had a double vWF:Ag peak by CIE and a relative increase of the fastest-moving vWF multimer by SDS-agarose electrophoresis; no quantitative or qualitative vWF defects were found in the paternal side of the family. The pattern of the findings in the propositus and her family is similar to those of type IIC vWD. However, there are some unique characteristics suggesting phenotypic variability in this subtype, such as low level of platelet vWF:Ag and the absence of increase of vWF after DDAVP administration.

Topics: Adult; Antigens; Bleeding Time; Deamino Arginine Vasopressin; Electrophoresis; Factor VIII; Female; Humans; Immunoelectrophoresis, Two-Dimensional; Macromolecular Substances; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet-rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.

Topics: Adult; Endothelium; Female; Humans; In Vitro Techniques; Macromolecular Substances; Male; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

In eight members of one family, platelets in platelet-rich plasma aggregated at much lower ristocetin concentrations than normal. Ivy bleeding time was variously prolonged, and von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity, and factor VIII coagulant activity were decreased. Most of the affected members had had slight to rather severe bleeding symptoms. Platelet-type von Willebrand's disease (vWD) could be ruled out. All multimers of vWF:Ag were found in plasma as well as platelets. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) to the propositus did not cause thrombocytopenia, and platelet-poor plasma obtained immediately after did not aggregate normal platelets. The molecular defect in this family, inherited as an autosomal dominant, resembles the one in type IIB because of the response to ristocetin but differs from IIB because all vWF:Ag multimers are present in plasma and the response to DDAVP is atypical. We conclude that this family has a new subtype of vWD and propose that structural as well as functional criteria should be used for a proper classification of vWD.

Topics: Blood Platelets; Deamino Arginine Vasopressin; Humans; Immunoelectrophoresis, Two-Dimensional; Macromolecular Substances; Pedigree; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Two patients from two separate families were diagnosed as having type IIB von Willebrand disease, because they had lifelong bleeding tendencies, prolonged bleeding times, no large von Willebrand factor multimers, and low levels of ristocetin cofactor in plasma with heightened ristocetin-induced platelet aggregation. There was no history of bleeding, and no laboratory abnormalities were found in the parents and sibship of either propositi, in contrast with the autosomal dominant pattern of inheritance usually observed in type IIB von Willebrand disease. Abnormalities of ristocetin-induced von Willebrand factor-platelet interactions were less severe than in a patient from a previously reported family with type IIB von Willebrand disease studied in parallel. The peculiar features of these cases provide additional evidence of the existence of heterogeneity within this variant.

Topics: Blood Platelets; Child; Female; Humans; Male; Pedigree; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Adult; Animals; Antibodies, Monoclonal; Epitopes; Factor VIII; Humans; Male; Platelet Adhesiveness; Platelet Aggregation; Rabbits; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The three main probes for functional vWF activity--ristocetin, botrocetin, and the PAggF test--and similarities and differences in their elicited vWF activities have been reviewed. Emphasis has been placed on the technologies dependent on these probes, with a brief description of a series of relatively simple and sensitive tests developed in this laboratory. These tests include the development of the PAF test for vWF in certain animal plasmas; the development and use of fixed lyophilized platelets that retain receptor activity for vWF; the purification of botrocetin (venom coagglutinin) freed of thrombinlike enzymes and its use in vWF assays; the development of macroscopic platelet aggregation tests for screening and assay of vWF; and the application of the macroscopic test for rapid screening and quantitation of human plasmas for acquired inhibitors of vWF utilizing each of the three probes. Historically, the similarities of the ristocetin and botrocetin probes were first observed. For normal human plasmas and for patients with classic vWD, both homozygous or heterozygous, similar values for vWF were obtained with these two probes. Similar platelet binding of vWF in the presence of the two probes was likewise noted. However, further studies of these two probes revealed striking differences. Especially important for study of animal plasmas generally as well as a canine model of vWD was the observation that the vWF in all animal plasmas tested with botrocetin was highly reactive, whereas with ristocetin nearly all plasmas were resistant. Similarly, all animal platelets tested for vWF-dependent aggregation with the two probes were highly reactive with botrocetin, but inactive with ristocetin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies; Antigen-Antibody Complex; Cattle; Crotalid Venoms; Disease Models, Animal; Dogs; Epitopes; Hemagglutinins; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A murine monoclonal antibody has been produced (RFF-VIII:R/2) that binds specifically to human factor VIII-related antigen (VIII:RAg) in plasma and in vascular endothelial cells but has no reactivity with factor VIII procoagulant antigen (VIII:cAg). This antibody is a potent inhibitor of von Willebrand factor activity (VIII:vWF) in that it can totally neutralize ristocetin-induced aggregation of platelet rich plasma and inhibit platelet adhesion at high flow rates. RFF-VIII:R/2 can be used in a one-stage, fluid phase immunoradiometric assay that can detect VIII:RAg at concentrations of 0.001 u/ml. This method has been used to analyse plasma from patients with von Willebrand's disease (vWD). Results obtained in these patients showed a high degree of correlation between the monoclonally-defined epitope and VIII:vWF levels measured by ristocetin-induced aggregation of washed platelets. This correlation was maintained in those patients with the 'variant' types of vWD who exhibit highly disparate VIII:vWF and VIII:RAg levels when the latter is determined using polyclonal antisera. It appears that this monoclonal antibody recognizes a site on the VIII:RAg molecule which is associated with its interaction with the platelet membrane. Immunoradiometric assays using RFF-VIII:R/2 offer a simplified, reproducible means of detecting functionally-active VIII:RAg as an alternative or supplement to techniques involving platelet interactions.

Topics: Animals; Antibodies, Monoclonal; Antigens; Blood Coagulation Factors; Epitopes; Factor VIII; Humans; Mice; Platelet Aggregation; Radioimmunoassay; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Two unrelated patients with "pseudo" ("platelet-type")-von Willebrand's disease (vWD) are described demonstrating thrombocytopenia with a prolonged bleeding time, ristocetin-induced platelet aggregation at low stimulus concentrations, decreased levels of ristocetin-cofactor activity (vWF:RCo), slight cryoprecipitate-induced platelet aggregation in the absence of ristocetin, and a lack of the high molecular weight factor VIII-von Willebrand factor (FVIII/vWF) multimers in plasma. Isolated washed patient platelets bound more FVIII/vWF at high (1 and 0.75 mg/ml) and low (0.5 and 0.25 mg/ml) ristocetin concentrations than control platelets. Fresh or paraformaldehyde-fixed washed platelets from these patients also bound more specific monoclonal antibody to glycoprotein Ib (25,000 binding sites per cell) than normal platelets (15,000 +/- 3,300 binding sites per cell). Results obtained in control patients with thrombocytopenia and increased platelet size (May-Hegglin anomaly and vWD type IIB) excluded a nonspecific increase of glycoprotein Ib in the platelets of the patients with pseudo-vWD. These data indicate that in pseudo-vWD, the primary abnormality lies in the platelet and is related to a quantitative and/or qualitative anomaly of platelet membrane glycoprotein Ib.

Topics: Factor VIII; Glycoproteins; Humans; Kinetics; Membrane Proteins; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We have investigated and characterized the abnormalities in four unrelated patients with von Willebrand's disease (vWd) who have (a) enhanced ristocetin-induced platelet aggregation (RIPA) at low ristocetin concentrations, (b) absence of the largest plasma von Willebrand factor (vWf) multimers, and (c) thrombocytopenia. The platelet-rich plasma of these patients aggregates spontaneously without the addition of any agonists. When isolated normal platelets are resuspended in patient plasma spontaneous aggregation occurs; however, the patients' plasmas did not induce platelet aggregation of normal washed formalinized platelets. When the patients' platelets are suspended in normal plasma, spontaneous aggregation is not observed. The spontaneous platelet aggregation (SPA) is associated with dense granule secretion as measured by ATP release and alpha granule release as measured by beta-thromboglobulin and platelet factor 4 release. The SPA is totally inhibited by 5 mM EDTA, prostaglandin I2, and dibutryl cyclic AMP, while it is only partially inhibited by 1 mM EDTA, acetylsalicylic acid, or apyrase. A monoclonal antibody directed against glycoprotein Ib (GPIb) and/or a monoclonal antibody against the glycoprotein IIb/IIIa (GPIIb/IIIa) complex totally inhibits the SPA. The vWf was isolated from the plasma of one of these patients. The purified vWf induced platelet aggregation of normal platelets resuspended in either normal or severe vWd plasma, but the vWf did not induce platelet aggregation of normal platelets resuspended in afibrinognemic plasma. Sialic acid and galactose quantification of the patient's vWf revealed approximately a 50% reduction compared with normal vWf. These studies indicate that a form of vWd exists, which is characterized by SPA that is induced by the abnormal plasma vWf. The SPA is dependent on the presence of plasma fibrinogen, and the availability of the GPIb and the GPIIb/IIIa complex. In this variant form of vWd the abnormal vWf causes enhanced RIPA, SPA, and thrombocytopenia.

Topics: Antigens; Factor VIII; Fibrinogen; Galactose; Glycoproteins; Humans; Membrane Proteins; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; Sialic Acids; Thrombocytopenia; von Willebrand Diseases; von Willebrand Factor

Topics: Blood Platelets; Child; Factor VIII; Humans; Hypersensitivity; Immunoelectrophoresis; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases

It has been reported that botrocetin, a Bothrops venom factor, induces platelet aggregation dependent on von Willebrand factor (vWF), and that platelet aggregation induced by Polybrene, a synthetic polycation, is enhanced by vWF. This report describes the platelet aggregability on stimulation with botrocetin and Polybrene in four patients with platelet-type von Willebrand disease (vWD) who showed increased platelet aggregation with low concentrations of ristocetin as the result of a platelet abnormality. Enhanced platelet aggregability with botrocetin was observed in platelet-rich plasma (PRP) from the patients. Platelet aggregation induced by botrocetin in a mixture of normal washed platelets and patient plasma was either decreased or normal, being dependent on the amount of plasma vWF. In contrast with ristocetin and botrocetin, Polybrene did not cause increased aggregation of patient PRP. Polybrene aggregated normal washed platelets less extensively in the presence of patient plasma than normal plasma. These studies demonstrated that botrocetin induced heightened interaction between platelets and vWF, but Polybrene did not, in platelet-type vWD, and that the enhanced responsiveness of patient platelets to botrocetin is related to an intrinsic platelet abnormality.

Topics: Crotalid Venoms; Hexadimethrine Bromide; Humans; Platelet Aggregation; Polyamines; Ristocetin; von Willebrand Diseases

Type IIB von Willebrand's disease (vWD) is a distinct form of this disorder in which the largest multimers of the von Willebrand factor (vWF) are lacking in plasma but present in platelets. When the vasopressin analogue, 1-deamino-8-D-arginine vasopressin (DDAVP), is given to patients with type IIB vWD, an abnormal vWF is released to plasma. This vWF causes thrombocytopenia in vivo and platelet aggregation in vitro. Aggregation occurs in the plasma milieu and thus at physiological fibrinogen concentration. In this study we demonstrate that IIB post-DDAVP vWF aggregated only metabolically active platelets. The platelet aggregation was completely inhibited by EDTA and PGE1, and either inhibited or greatly weakened by ASA, demonstrating the role of divalent cations and thromboxane A2 formation. In spite of inhibiting platelet aggregation, EDTA, PGE1 and ASA did not prevent platelet binding of IIB post-DDAVP vWF. An antiserum against GP Ib made normal platelets less responsive to the IIB vWF although neither platelet aggregation nor vWF binding were completely prevented. The aggregation was fibrinogen-dependent and platelets from patients with Glanzmann's thrombasthenia were unresponsive. The studies provide evidence that IIB post-DDAVP vWF is bound to unstimulated platelets and that the interaction between vWF and platelets in type IIB vWD is different from ristocetin-induced as well as thrombin- and epinephrine-induced binding to platelets of normal vWF.

Topics: Blood Platelets; Deamino Arginine Vasopressin; Fibrinogen; Humans; Platelet Aggregation; Ristocetin; Thrombasthenia; Thrombocytopenia; von Willebrand Diseases; von Willebrand Factor

A monoclonal antibody directed against the von Willebrand factor moiety (vWF) of factor VIII-von Willebrand factor (FVIII-vWF), which blocks ristocetin-induced platelet aggregation as well as the binding of FVIII-vWF to platelets in the presence of ristocetin, inhibited platelet adherence to human artery subendothelium when present in normal flowing blood. This monoclonal antibody, CLB-RAg 35, inhibited platelet adherence as a function of the shear rate. At wall shear rates below 500 s-1, platelet adherence was not affected, but at higher shear rates platelet adherence was gradually inhibited, reaching an average of 11% of the normal value at 2,500 s-1. Indirect immunofluorescence established the reactivity of CLB-RAg 35 with vWF present in artery subendothelium. Pretreatment of normal vessel walls with this antibody inhibited adherence of platelets in blood from a patient with severe homozygous von Willebrand's disease and in blood from normal individuals. The inhibition was shear-rate dependent and significant at high shear rates (2,500 s-1). By adding increasing amounts of purified FVIII-vWF to normal blood, the inhibition was gradually overcome. These data indicate that vWF present in the vessel wall contributes appreciably to platelet adherence. At high wall shear rates, platelet adherence is mediated virtually completely by both plasma FVIII-vWF and vWF in the vessel wall. At low wall shear rates (below 500 s-1), platelet adherence occurs independent of FVIII-vWF in plasma and vWF in the vessel wall.

Topics: Antibodies, Monoclonal; Binding, Competitive; Blood Coagulation Factors; Endothelium; Factor VIII; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Perfusion; Platelet Adhesiveness; Ristocetin; Umbilical Arteries; von Willebrand Diseases; von Willebrand Factor

In vitro investigations have demonstrated a high F VIII:Rcof potency and a high F VIII:Rcof/F VIII R:Ag ratio of two heat-treated F VIII concentrates. We therefore studied the in vivo effectiveness of these preparations (F VIII HSR, Behringwerke Marburg and F VIII HTR, Travenol) in five patients with von Willebrand's disease (vWd). In the steady state in vivo recoveries of F VIII:Rcof ranged from 73-153% after transfusion of F VIII HSR and from 11.5-17% after F VIII HTR respectively. The gain of F VIII-complex after F VIII HS was comparable to cryopecipitate (KryobulinR SP, Immuno AG Wien). All three products shortened the bleeding-time. Three of our five patients underwent surgery (Billroth I, papillotomy, laparatomy, open heart surgery) under F VIII HS cover without bleeding complications. The dose applied ranged from 20 to 40 U/kg at 8 or 12 h intervals for a period of approx. 14 days. Serum-transaminase elevations were observed in two of four patients after F VIII HT treatment. Although the risk of hepatitis of heat-treated F VIII concentrates remains to be determined, these products proved to be effective in vWd. The major advantages of these preparations are stability, rapid solubility, a low content of contaminating proteins, and a rapid, general availability.

Topics: Chemical and Drug Induced Liver Injury; Dose-Response Relationship, Drug; Factor VIII; Gluconates; Hemorrhage; Hot Temperature; Humans; Isotonic Solutions; Magnesium Chloride; Platelet Aggregation; Potassium Chloride; Prospective Studies; Ristocetin; Sodium Acetate; Sodium Chloride; von Willebrand Diseases

Four unrelated patients with a bleeding diathesis (bleeding time longer than 30 min), some spontaneous platelet aggregation, thrombocytopenia and large platelets, had decreased levels of factor VIII-von Willebrand factor (FVIII-VWF) related properties and impaired platelet adherence to human artery subendothelium. The largest multimers of plasma FVIII-VWF were absent and enhanced ristocetin induced platelet aggregation in platelet-rich plasma was observed. 'Pseudo-von Willebrand's disease' was excluded, because FVIII-VWF from normal subjects did not initiate platelet aggregation. Perfusion studies with reconstituted blood, consisting of respectively patient platelets in normal plasma or normal platelets in patient plasma, yielded evidence for an intrinsic platelet abnormality in two out of the four patients and a plasma defect in the other two patients. Similar experiments with platelets and plasma from four patients with von Willebrand's disease (VWD) subtype I and four patients with VWD subtype IIa showed that the impaired platelet adherence in blood from these patients was caused by a plasma defect. These experiments indicate that patients with laboratory findings in many respects similar to those originally reported for patients with VWD subtype IIb may represent a heterogeneous group of individuals. The data derived from our study imply that perfusion experiments with reconstituted blood offer a new approach in characterization of these patients, which may be more relevant for the treatment of the patients than characterization by ristocetin induced adsorption of FVIII-VWF to platelets.

Topics: Adsorption; Blood; Blood Platelets; Diagnosis, Differential; Factor VIII; Humans; Perfusion; Platelet Adhesiveness; Platelet Aggregation; Platelet Count; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The response to infusions of cryoprecipitate and factor VIII concentrate was studied in a patient with platelet-type von Willebrand disease (vWD) who showed lack of the large multimers of von Willebrand factor in plasma, increased platelet aggregation with low concentrations of ristocetin, and in vitro platelet aggregation by normal plasma. The cryoprecipitate and factor VIII concentrate to be infused induced platelet aggregation when added to patient platelet-rich plasma at concentrations higher than 0.86 U/ml and 3 U/ml of factor VIII-related antigen (VIIIR:Ag), respectively. Administration of cryoprecipitate (41.9 U VIIIR:Ag/kg body weight) was followed by a shortening of the bleeding time, and hemostasis was achieved during tooth extractions. Factor VIII concentrate (70.2 U VIIIR:Ag/kg) failed to correct the prolonged bleeding time and proved ineffective to control the gum bleeding. No significant diminution of the platelet count was observed following any infusion. These results indicate that cryoprecipitate is hemostatically effective and safe when infused in such a dosage, but factor VIII concentrate is not effective in platelet-type vWD in analogy to what is observed in various types of vWD.

Topics: Antigens; Bleeding Time; Blood Coagulation; Child; Cryoglobulins; Factor VIII; Female; Humans; Immunoelectrophoresis, Two-Dimensional; Ristocetin; Tooth Extraction; von Willebrand Diseases; von Willebrand Factor

Factor VIII von Willebrand may be detected by a Ristocetin-dependent platelet agglutination reaction. The authors have measured plasma levels of Factor VIII-related Ristocetin cofactor (VIIIR:RCoF) using formalin-fixed platelets tested by an aggregometric method or by a new, commercially available glass slide test. Plasmas were obtained from patients with von Willebrand's disease and Cushing's syndrome, and from normal controls. Von Willebrand factor (vWf) levels varied between 0-500% of the normal. A good correlation (r = 0.95) was found comparing the results obtained by the two methods over the entire range. If all patients were considered, a satisfactory correlation was obtained between VIIIR:RCoF and VIII-related antigen (VIIIR:Ag), both by the aggregometric method (r = 0.79) and by the glass slide method (r = 0.88). On the contrary, if values below 25% of the normal are taken into account, only a mild correlation was found between VIIIR: RCoF (aggregometric method) and VIIIR:Ag and no correlation between VIIIR:RCoF (glass slide method) and VIIIR:Ag. No correlation, regardless of the method used, was found between VIIIR:RCoF and VIIIR:Ag for values above 120%.

Topics: Antigens; Blood Coagulation Tests; Cushing Syndrome; Factor VIII; Female; Humans; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Type IIB von Willebrand disease is characterized by enhanced ristocetin-induced platelet aggregation and absence of large von Willebrand factor multimers from plasma. An alteration of the von Willebrand factor molecule resulting in increased reactivity with platelets appears to be the basis for these abnormalities. We have now identified a new variant of type IIB von Willebrand disease in a family in which the four affected members also have chronic thrombocytopenia, in vivo platelet aggregate formation, and spontaneous platelet aggregation in vitro. In spite of repeatedly prolonged bleeding times and persistent thrombocytopenia, their bleeding diathesis is only moderate.

Topics: Adult; Blood Coagulation Tests; Blood Platelets; Electrophoresis, Agar Gel; Factor VIII; Female; Humans; Immunoelectrophoresis, Two-Dimensional; Male; Pedigree; Platelet Aggregation; Ristocetin; Thrombocytopenia; von Willebrand Diseases

The binding of von Willebrand factor (vWf) to stimulated platelets in the plasma milieu was performed using a radiolabeled monoclonal antibody to vWf. Plasma proteins specifically inhibited the thrombin- and ADP/epinephrine-induced vWf binding to activated platelets but did not inhibit the ristocetin-induced vWf binding. When normal plasma was heat defibrinated, monoclonal-labeled vWf was bound to platelets following thrombin or ADP/epinephrine stimulation. Furthermore, monoclonal-labeled vWf from afibrinogenemic plasma bound normally to platelets. The binding of vWf to stimulated platelets in either heat-defibrinated normal plasma or afibrinogenemic plasma was specifically inhibited by the addition of normal plasma fibrinogen in a concentration-dependent manner. At levels of fibrinogen less than 1 mg/ml, however, vWf binding could be demonstrated. The inhibition by fibrinogen of vWf binding to platelets was competitive and overcome by increased concentrations of vWf. These studies show that thrombin-induced and ADP/epinephrine-induced vWf binding to platelets does not occur in the plasma milieu, although at reduced levels of fibrinogen, vWf binding to stimulated platelets can be demonstrated.

Topics: Adenosine Diphosphate; Afibrinogenemia; Antibodies, Monoclonal; Blood Coagulation Factors; Blood Platelets; Blood Proteins; Epinephrine; Fibrinogen; Glycoproteins; Humans; Platelet Membrane Glycoproteins; Ristocetin; Thrombin; von Willebrand Diseases; von Willebrand Factor

The interaction of platelets and von Willebrand factor (vWF) in platelet-type von Willebrand's disease (vWD) was characterized using formalin-fixed platelets from the patients. Formalin-fixed patient platelets were agglutinated directly by human vWF in normal plasma and type IIB vWD plasma, but not in type IIA vWD plasma. In the presence of a small amount of normal vWF, ristocetin-induced agglutination of patient platelets was enhanced with low concentrations of ristocetin. Wheat germ agglutinin and EDTA inhibited vWF-induced agglutination, although EDTA had no effect on ristocetin (plus vWF)-induced agglutination. These results demonstrate that vWF-induced agglutination of platelet-type vWD platelets is independent of active platelet metabolism but requires divalent cations, and suggest that platelet membrane glycoprotein I (GPI) would be involved in this agglutination.

Topics: Blood Coagulation Factors; Edetic Acid; Glycoproteins; Humans; In Vitro Techniques; Membrane Proteins; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We studied four patients who showed aggregation of platelets in platelet-rich plasma at lower concentrations of ristocetin than those required for normal platelet-rich plasma and who demonstrated an increased capacity of the platelets to bind normal von Willebrand factor. The four patients were from two Japanese families. Platelets from one family aggregated spontaneously in vitro, and platelets from both families aggregated upon the addition of normal plasma and cryoprecipitate, in the absence of ristocetin or other agonists. Analysis of the multimeric composition of von Willebrand factor by sodium dodecyl sulfate-agarose gel electrophoresis revealed a decrease in large multimers or a decrease in both large and intermediate multimers in plasma, but normal multimers in platelets. 1-Deamino-[8-D-arginine]-vasopressin caused by an immediate appearance of larger multimers in plasma, followed by the rapid disappearance of these multimers from circulating plasma. Analysis of platelet membrane glycoproteins from the patients showed that there were two distinct bands in the glycoprotein I region; one migrated in a slower region and the other in a faster region than normal glycoprotein Ib. We suggest that the platelet receptor abnormality in these patients is related to this abnormality of glycoprotein Ib.

Topics: Deamino Arginine Vasopressin; Electrophoresis, Agar Gel; Glycoproteins; Humans; Membrane Proteins; Platelet Aggregation; Ristocetin; Sodium Dodecyl Sulfate; von Willebrand Diseases; von Willebrand Factor

Topics: Blood Coagulation Factors; Counterimmunoelectrophoresis; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Two family members (daughter and mother) with a bleeding disorder showed prolonged bleeding time and activated partial thromboplastin time associated with decreased plasma levels of factor VIII procoagulant activity, factor VIII-related antigen, and factor VIII-ristocetin cofactor activity. The ristocetin-induced platelet agglutination (RIPA) was enhanced, ADP-, collagen- and Ca ionophore-induced platelet aggregation were also increased at low concentrations of these compounds. In the mother, spontaneous platelet aggregation (SPA) was also observed. Contrary to type II B von Willebrand's disease (vWd), pseudo-vWd and platelet-type vWd, the patients did not show any increased binding of factor VIII/vWf to platelets in the presence of ristocetin. The RIPA in normal controls were inhibited by addition of antifactor VIII antiserum to the platelet-rich plasma, but not in cases 1 and 2. In this atypical vWd, therefore, the hyperreactivity of platelet aggregation may be due to an intrinsic abnormality of the platelets, but not to any enhanced interaction between plasma factor VIII and the platelets.

Topics: Adult; Child, Preschool; Factor VIII; Female; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A new type II variant form of von Willebrand's disease has been recognized in a mother and daughter who have bleeding manifestations typical of von Willebrand's disease. Laboratory findings include consistently prolonged bleeding times, with normal levels of factor VIII procoagulant and antigen, but decreased ristocetin cofactor activity. Electrophoresis in SDS 1.5% agarose gel and reaction with 125I-labeled anti-factor VIII-related antigen rabbit IgG, followed by autoradiography, revealed that both plasma and platelets lack the large multimers of factor VIII-related antigen. In 2.5% gel, the propositus plasma lacked the normal "triplet" pattern. In 3.0% gel, a 5-band pattern was observed in normal, type IIA, and type IIB plasma, whereas type IIC plasma revealed a 2-band pattern. The patient's plasma revealed a 4-band pattern distinctly different from normal or other type II variants. We suggest that this new variant be labeled type IID, until a more appropriate nomenclature is developed.

Topics: Adult; Antigens; Electrophoresis, Agar Gel; Factor VIII; Female; Genetic Variation; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The effect of intravenous 1-deamino (8-D-arginine)vasopressin (DDAVP) administration on platelet interaction with human artery subendothelium was investigated with flowing blood from five normal individuals and 12 patients with von Willebrand's disease (vWD). Three of the patients were diagnosed as vWD subtype I, four as subtype IIa, and five as subtype IIb. DDAVP administration to normals enhanced platelet adherence, in parallel with increasing plasma levels of factor VIII-related antigen ( FVIIIR :Ag) and ristocetin cofactor activity ( FVIIIR :RCF). Platelet aggregate formation was transiently increased within 90 minutes. Platelet adherence in patient blood before DDAVP infusion was subnormal. In patients with subtype I, administration of DDAVP normalized the bleeding time, enhanced the platelet adherence, and transiently improved the platelet aggregate formation. The platelet adherence was more corrected than would have been expected on the basis of the FVIIIR :Ag and FVIIIR :RCF levels. In patients with subtype IIa, infusion of DDAVP increased the FVIIIR :Ag levels approximately threefold, without affecting the FVIIIR :RCF levels, and in only two of four patients was a transiently enhanced platelet adherence with a corresponding shortening of the bleeding time observed. In patients with subtype IIb, administration of DDAVP increased the FVIIIR :Ag levels about threefold and the FVIIIR :RCF levels five to tenfold, but decreased the platelet adherence significantly. The bleeding time values were not normalized. A close association between the bleeding time values and corresponding platelet adherence values before and after DDAVP infusion was observed. Normalization of the bleeding time was paralleled with normalization of platelet adherence. We conclude that DDAVP improves the primary hemostasis by causing enhanced FVIII-vWF-mediated platelet adherence. DDAVP has little or no effect on the bleeding time in patients with subtype IIa and subtype IIb, because the platelet adherence is not normalized.

Topics: Arginine Vasopressin; Bleeding Time; Deamino Arginine Vasopressin; Factor VIII; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Platelet-type von Willebrand's disease is a recently described disorder of primary hemostasis, possessing attributes both of von Willebrand's disease and of a qualitative platelet abnormality. This article discusses the clinical features, laboratory aspects, pathophysiology, and treatment of platelet-type von Willebrand's disease.

Topics: Blood Coagulation Tests; Blood Platelet Disorders; Cell Membrane; Dose-Response Relationship, Drug; Factor VIII; Humans; Platelet Aggregation; Platelet Function Tests; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Antigens; Factor VIII; Hemophilia A; Humans; Methods; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Adult; Blood Coagulation Factors; Female; Hemophilia A; Humans; Male; Middle Aged; Platelet Count; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A monoclonal antibody to human antihemophilic factor (AHF, factor VIII) was derived from BALB/c mouse spleen cells fused with P3x63Ag8 mouse plasmacytoma cells. This antibody, harvested from culture medium or ascites fluid, reacted with purified AHF and with plasmas with normal subjects or classic hemophiliacs, as measured by enzyme-linked immunosorbent assay (ELISA), but not with plasmas from patients with severe von Willebrand's disease. The antibody possessed only IgG, heavy chains and kappa light chains. It blocked ristocetin-induced platelet agglutination and, to a lesser degree, platelet retention by glass bead columns, but it did not inhibit the procoagulant activity of AHF significantly. An amount of rabbit antiserum against AHF that provided equivalent inhibition of ristocetin-induced platelet agglutination inhibited glass bead retention much more effectively than the mouse monoclonal antibody. This difference was exaggerated in studies of the corresponding Fab fragments. These data suggest that the site or sites on the AHF complex molecule that are associated with ristocetin-induced platelet agglutination differ quantitatively or qualitatively from those associated with enhancement of platelet retention by glass beads. ELISA titers of immunoreactive AHF, using the monoclonal antibody, were closely correlated to those using rabbit antiserum against AHF in normal, hemophilic, and most von Willebrand's disease plasma.

Topics: Antibodies, Monoclonal; Blood Coagulation Factors; Factor VIII; Glass; Hemophilia A; Humans; Platelet Aggregation; Ristocetin; Spleen; von Willebrand Diseases; von Willebrand Factor

We have studied four patients suffering from acquired von Willebrand's disease. All patients had a severe bleeding diathesis with recurrent life-threatening haemorrhages. Three of the patients had a monoclonal gammopathy and one of these developed multiple myeloma. In three patients tested, a plasma inhibitor to ristocetin cofactor activity was detected. In each case this was localized to the IgG fraction of plasma. In addition, VIII:C activity was found to be associated with the IgG fraction of patients' plasma and altered mobility of VIII:C was detected on Laurell immunoelectrophoresis. Furthermore, plasma from all four patients and the IgG fraction therefrom resulted in a dissociation of normal VIII:C into two components separable by gel-filtration on Sepharose 6B. Finally the circulating half-life of the three factor VIII activities was found to be markedly reduced in the patients with acquired von Willebrand's disease. We conclude that in the patients studied the coagulation defect was related to the presence of a circulating inhibitor to the factor VIII complex and that this inhibitor was associated with the IgG fraction of plasma.

Topics: Aged; Antigens; Chromatography, Gel; Factor VIII; Female; Half-Life; Humans; Immunoglobulin G; Male; Middle Aged; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Arteriosclerosis; Blood Coagulation Tests; Blood Transfusion; Deamino Arginine Vasopressin; Factor VIII; Humans; Platelet Aggregation; Prenatal Diagnosis; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Platelet-type von Willebrand's disease is a recently described autosomal dominant bleeding disorder characterized by decreased ristocetin cofactor activity, lack of the higher molecular weight von Willebrand Factor (vWF) multimers on SDS agarose gel electrophoresis, increased platelet aggregation with low concentrations of ristocetin, and increased ristocetin-induced binding of normal vWF to patient platelets. In this report the authors describe a 17-month-old male with Platelet-type von Willebrand's disease, inherited from the paternal side of his family, who developed an inhibitor specific to Factor VIII:C. The patient's plasma inhibited normal plasma VIII:C and partially purified VIII:C; it did not appear directed against normal VIIIR:Ag or ristocetin cofactor. This antibody is therefore similar to inhibitors that develop in some transfused hemophilia A patients. Since low VIII:C, VIII:CAg, and VIII:C/VIIIR:Ag ratio were encountered in his mother, it is likely that this patient has inherited hemophilia A in addition to Platelet-type von Willebrand's disease.

Topics: Adult; Antigens; Blood Coagulation; Factor VIII; Female; Humans; Infant; Male; Middle Aged; Pedigree; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Three families with von Willebrand's disease (vWd) type I were investigated. A reliable identification of healthy and diseased individuals was achieved by number of bleeding symptoms, assays of bleeding time, FVIII:C (one stage and two stage), VIIIR:Ag (EIA) and ristocetin cofactor. The diagnoses-vWd or non-vWd were confirmed by laboratory indices based on predictive values of positive and negative tests, also including VIIIR:Ag (IRMA and RIA). The last mentioned two variables did not contribute to significantly better identification of vWd versus health. The best single test variable for this purpose was ristocetin cofactor. One vWd family had significantly higher levels of ristocetin cofactor and shorter bleeding time than the other two vWd families and is probably the typical example of a family transmitting classical severe vWd.

Topics: Adult; Aged; Antigens; Blood Coagulation Tests; Child; Factor VIII; Genes, Dominant; Genes, Recessive; Heterozygote; Humans; Middle Aged; Radioimmunoassay; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The assay of factor VIII, co-factor of Ristocetin (VIIIR:Co) is a relatively delicate procedure which is presently reserved to specialized laboratories. It requires the use of an aggregometer and a long and difficult preparation of human platelets. In parallel with this classical method, we have used a new, rapid, semi-quantitative slide test whose advantages are: simple technique and rapid answer (2 minutes), small volume of plasma required for the test (50 microliters) and the possibility of using citrated or heparinized plasma taken form a venous or capillary blood sample. Using this test, we have assayed factor VIII co-factor of ristocetin: in 31 hospitalized adults patients with no previous history of bleeding and no disturbance of haemostasis and in 18 patients with Willebrand's factor deficiency in comparison with the standard technique using aggregometry (Allain's method); - in 28 normal neonates, in comparison with the VIIIR:Ag factor assay (Laurell's technique), only because of the small sample volume available; - in 17 patients with various disease associated with an abnormality of the VIII complex in comparison with the assay of VIIIR:Ag and VIIIC. The results obtained in the normal adults show a satisfactory correlation between the two methods. The mean level of factor VIII:Co is 100 +/- 10 per cent (M +/- SD) with the semi-quantitative slide test and 109 +/- 20 per cent (M +/- SD) with the method taken as the reference. The correlation is also satisfactory for patients with a deficit of Willebrand's factor. The test performed on the neonates gives a mean value of 101 +/- 37 per cent (M +/- SD) with good correlation between the factor VIIIR:Ag and the factor VIIIR:Co.

Topics: Adult; Blood Coagulation Factors; Diagnosis, Differential; Factor VIII; Female; Hematologic Diseases; Humans; Infant, Newborn; Male; Pregnancy; Reference Values; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Antigens; Bleeding Time; Factor VIII; Female; Humans; Immunoelectrophoresis; Male; Platelet Function Tests; Ristocetin; von Willebrand Diseases

An autosomally transmitted bleeding diathesis sharing some, but not all, features previously described in von Willebrand's disease (vWd) was studied in five patients representing three generations of a single family. Bleeding times in the upper normal range in conjunction with low-normal platelet counts, normal factor VIII coagulant activity and VIII-related antigen, decreased VIII-ristocetin cofactor activity, selective decrease of the higher molecule weight factor VIII/von Willebrand factor (VIII/vWF) multimers, and increased ristocetin-induced platelet agglutination at low ristocetin concentrations were characteristic. Binding of patient VIII/vWF to washed normal platelets was within normal limits, whereas binding of normal VIII/vWF to patient platelets was significantly increased (p less than 0.001 at 0.6 mg/ml ristocetin). This disorder accordingly appears to involve an intrinsic platelet abnormality affecting platelet-VIII/vWF interactions. It is proposed that the concept of vWD be broadened to include patients with this abnormality, which may appropriately be called "Platelet-type von Willebrand's disease."

Topics: Antigens; Bleeding Time; Blood Platelets; Factor VII; Female; Humans; Male; Partial Thromboplastin Time; Platelet Aggregation; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Ristocetin; von Willebrand Diseases

Four members (from four generations) of a family with a mild bleeding disorder and intermittent thrombocytopenia had decreased plasma levels of properties related to factor VIII/von Willebrand factor (FVIII/VWF), an absence of high-molecular-weight forms of FVIII/VWF in the plasma (but normal multimeric structure in the platelets), and increased ristocetin-induced platelet aggregation, as in Type IIB von Willebrand's disease. However, unlike the abnormality in FVIII/VWF in Type IIB disease, the basic defect in this family was in their platelets, which absorbed FVIII/VWF high-molecular-weight multimers at lower concentrations of ristocetin than did normal platelets. In addition, either in platelet-rich plasma or suspended in buffer, their platelets were aggregated by unmodified normal human FVIII/VWF without ristocetin. Since the abnormalities of plasma FVIII/VWF in this family may be secondary to the platelet abnormalities, the term "pseudo-von Willbrand's disease" may be suitably descriptive of their disorder.

Topics: Adsorption; Adult; Aged; Antigens; Blood Coagulation Factors; Blood Platelets; Child, Preschool; Factor VIII; Female; Glycoproteins; Humans; Male; Middle Aged; Molecular Weight; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A form of von Willebrand's disease has been described with enhanced ristocetin-induced platelet aggregation and anodal migration of the factor VIII/von Willebrand factor protein (type IIb). We studied two families with this form of von Willebrand's disease and macrothrombocytopenia. We have found that these platelets bind more of the normal and intermediate-sized multimers of the factor VIII/von Willebrand factor than normal platelets. Analysis of the binding data show an increased affinity of these vWd platelets for the factor VIII/von Willebrand factor. These findings are consistent with an increased number of platelet receptors, which, either by their native topography or migration on the platelet surface, bind factor VIII/von Willebrand factor protein with greater affinity than normal platelets, platelets of other vWd patients, and large platelets of other etiologies.

Topics: Adolescent; Adult; Aged; Antibody Affinity; Blood Coagulation Factors; Blood Platelets; Child; Factor VIII; Female; Humans; Male; Middle Aged; Platelet Aggregation; Receptors, Immunologic; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Antibodies; Blood Coagulation Factors; Female; Humans; Middle Aged; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Blood Coagulation Factors; Diagnosis, Differential; Hemophilia A; Humans; Platelet Aggregation; Reagent Kits, Diagnostic; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Binding Sites; Blood Platelets; Blood Transfusion; Chemical Precipitation; Chromatography, Gel; Factor VIII; Freezing; Humans; Immunoelectrophoresis, Two-Dimensional; Iodine Radioisotopes; Ristocetin; Time Factors; von Willebrand Diseases

Topics: Factor VIII; Female; Humans; Male; Platelet Adhesiveness; Platelet Aggregation; Prothrombin Time; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Two cases of von Willebrand disease (vWD) associated with familial thrombocytopenia were reported. The proband (daughter) and her father showed thrombocytopenia with large platelets and decreased von Willebrand factor activity (VIIIR:WF). Factor VIII procoagulant activity (VIII:C) and factor VIII-related antigen (VIIIR:AG) were normal, but both patients revealed an increased ristocetin-induced platelet aggregation and a qualitative abnormality of the factor VIII protein, which was characterized by fast electrophoretic mobility of VIIIR:AG and an abnormal elution of factor VIII-related activities on Sepharose 2B. DDAVP was hemostatically effective even in this thrombocytopenic patient undergoing a dental extraction.

Topics: Adult; Blood Platelets; Child; Factor VIII; Female; Humans; Male; Pedigree; Platelet Aggregation; Ristocetin; Thrombocytopenia; von Willebrand Diseases

Topics: Adolescent; Adult; Blood Platelets; Child; Factor VIII; Female; Humans; Male; Ristocetin; von Willebrand Diseases

Topics: Factor VIII; Female; Humans; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A patient with a reduced response to the platelet-aggregating agent ristocetin and a prolonged bleeding time was found to have an abnormally high level of vWf as measured by Laurell immunoelectrophoresis. Characterization of the patient's vWf by disc-gel electrophoresis and crossed immunoelectrophoresis showed it to be indistinguishable from vWf isolated from normal plasma. The specific activity of the purified vWf in supporting RIPA was the same for both patient and normal vWf. The diminished RIPA was therefore not due to decreased levels of vWf or the presence of a modified form of the vWf. The patient's platelet-poor plasma inhibited the ristocetin-induced platelet aggregating activity of normal PRP, indicating the presence of an inhibitor. Fractionation of the plasma by ion-exchange chromatography showed the inhibitory activity to be in the patient's gamma globulin fraction. The gamma globulin was not directed against either the patient's vWf or against the patient's platelets but appeared to interfere with RIPA by binding ristocetin.

Topics: Aged; Female; Humans; Immunoglobulin G; Lymphoproliferative Disorders; Platelet Aggregation; Protein Binding; Ristocetin; von Willebrand Diseases

Topics: Blood Coagulation Factors; Cells, Cultured; Deamino Arginine Vasopressin; Dexamethasone; Endothelium; Epinephrine; Humans; Hydrocortisone; Ristocetin; Umbilical Veins; Vasopressins; von Willebrand Diseases; von Willebrand Factor

Topics: Humans; Platelet Aggregation; Platelet Function Tests; Ristocetin; von Willebrand Diseases

Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Female; Hemagglutination Tests; Humans; Male; Pedigree; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The form of von Willebrand's disease characterized by a qualitative abnormality of Factor VIII/von Willebrand factor (FVIII/vWF) in plasma has been designated as Type II. We have now identified 20 persons from five families whose qualitatively abnormal FVIII/vWF shows heightened responsiveness to ristocetin. We have classified this form of the disease as Type IIB and reclassified as Type IIA the form previously described as Type II, in which the interaction of the abnormal FVIII/vWF with platelets is decreased or absent in the presence of ristocetin. The enhanced reactivity of FVIII/vWF in Type IIB was evident in studies of ristocetin-induced platelet agglutination and of binding of FVIII/vWF to platelets in the presence of ristocetin. In both Type IIA and IIB, crossed immunoelectrophoresis of plasma FVIII/vWF demonstrated similar absence of the larger, less anodic forms. These findings suggest that ristocetin-mediated interactions between platelets and FVIII/vWF do not accurately reflect the "bleeding-time" (von Willebrand factor) defect in this newly described subtype of von Willebrand's disease.

Topics: Bleeding Time; Blood Coagulation Factors; Blood Platelets; Factor VIII; Female; Humans; Immunoelectrophoresis, Two-Dimensional; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Aged; Factor VIII; Humans; Male; Partial Thromboplastin Time; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Child, Preschool; Factor VIII; Female; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Two cases of von Willebrand disease (VWD) that revelaed an increased ristocetin-induced platelet aggregation (RIPA) and a qualitative abnormality of the factor VIII protein are reported. The threshold concentration of ristocetin giving a 30% increase in light transmission was 0.5 mg/ml in the proband and 0.4 mg/ml in her father (normal: 1.16 +/- SD 0.18 mg/ml) although both patients showed reduced plasma von Willebrand factor activity (VIIIR:WF). In both patients, the amount of factor VIII related antigen (VIIIR:AG) in their platelets were normal, but an increased binding affinity of platelets to plasma factor VIII was demonstrated. The qualitative abnormality of the factor VIII protein was characterized by an increasead anodal migration of VIIIR:AG in crossed immunoelectrophoresis (CIE), a delayed elution pattern as demonstrated by gel filtration on Sepharose 2B, and a decreased precipitation with concanavalin A (Con A). The response to DDAVP was also investigated.

Topics: Adult; Blood Platelets; Child, Preschool; Dose-Response Relationship, Drug; Factor VIII; Female; Humans; Male; Molecular Conformation; Platelet Aggregation; Ristocetin; Stimulation, Chemical; von Willebrand Diseases

The variability of laboratory findings in von Willebrand's disease (vWd) was evaluated by performing serial studies of bleeding time (BT), factor VIII coagulant activity (VIII:C), factor-VIII-related antigen (VIIIR:Ag) and ristocetin cofactor (VIIIR:Rcof) in 50 individuals from 25 families with this disorder. The types of results were characterized from 1 to 16 based on the possible combinations of findings using these four tests. The only patients observed to have consistently abnormal results of all four tests were three individuals with homozygous vWd. Individuals with autosomal dominant vWd were found to have a variety of results and all 16 possible types were observed. Although a consistent pattern was present within some families, others with equivalent history of bleeding demonstrated widely variable types of results. The results within some families, others with equivalent history of bleeding demonstrated widely variable types of results. The results of serial studies of the same tests in 10 normal individuals indicated relative stability, with nearly all values within the usual range of normal, but some independent variation of factor-VIII-related activities was observed. These studies indicate that: (1) the results of BT, VIII:C, VIIIR:Ag, and VIIIR:Rcof vary considerably from time to time in many individuals with vWd, (2) a classification of "variants" of vWd based solely on such studies may be inappropriate, particularly if the tests are not repeated, and (3) repeated testing may be required to establish the diagnosis of vWd in some individuals.

Topics: Bleeding Time; Blood Coagulation; Factor VIII; Genetic Variation; Humans; Ristocetin; von Willebrand Diseases

Topics: Antigens; Factor VIII; Humans; Methods; Ristocetin; von Willebrand Diseases

Topics: Antigens; Factor VIII; Humans; Radioimmunoassay; Ristocetin; von Willebrand Diseases

Acquired von Willebrand's syndrome with a regressive evolution is described in a 66 year old man with Waldenström's disease. An inhibitor electively directed against Ristocetin cofactor activity has been demonstrated, active in vitro after incubation at 37 degrees C. Serum fractionation showed that the inhibitor was independent of the monoclonal IgM and subsequent purification that it was IgG in nature. The results permit its classification as an auto-antibody.

Topics: Aged; Autoantibodies; Autoimmune Diseases; Factor VIII; Humans; Immunoglobulin G; In Vitro Techniques; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases; Waldenstrom Macroglobulinemia

Addition of Ristocetin to formalin-fixed platelets suspended in diluted PPP induces a marked increase in turbidity which is not caused by platelet shape change but by the formation of microaggregates. If diluted PPP of patients with severe von Willebrand's disease is used, only an initial increase in turbidity and no further decrease is observed without any form variation of the platelets but again with formation of many microaggregates. In normal PRP diluted with buffered EDTA, ADP induces an increase in turbidity without further changes in optical density. Simultaneously platelets immediately change their shape with formation of pseudopodia and sphering but at the same time also microaggregates appear. Shape change and microaggregate formation can also be observed after the addition of Collagen to undiluted PRP which is followed by the formation of large aggregates and a decrease in optical density. Increase in optical density in undiluted PRP is not a specific indicator of platelet shape changes. Microaggregates can alone or partially be responsible for these changes. For the evaluation of platelet shape changes but also for the estimation of microaggregate formation microscopic methods are preferred.

Topics: Adenosine Diphosphate; Blood Platelets; Cells, Cultured; Collagen; Culture Media; Edetic Acid; Formaldehyde; Humans; Nephelometry and Turbidimetry; Platelet Aggregation; Ristocetin; Stimulation, Chemical; von Willebrand Diseases

Topics: Binding Sites; Factor VIII; Female; Humans; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Agglutinins; Animals; Blood Coagulation Factors; Freeze Drying; Humans; Platelet Aggregation; Ristocetin; Snake Venoms; Time Factors; von Willebrand Diseases; von Willebrand Factor

Dogs with VWD provide useful models for the study of the factor VIII complex. However, the study of canine VIIIR:WF has been hampered by the lack of a routine plasma assay for canine RCF, an activity that is usually used as a measure of VIIIR:WF. This study shows that canine plasma can be assayed for RCF with a macroscopic tilt-tube method using formalin-fixed human platelets, ristocetin, and extra canine albumin (5.0 mg/ml) to prevent plasma precipitation. An assay was also developed for canine plasma PBCF, an activity that is closely related to RCF. In 45 normal canine plasmas, VIII:C was not correlated with RCF, PBCF, or VIIIR:AG. However, RCF, PBCF, and VIIIR:AG were well correlated with each other. In 26 canine VWD plasmas, VIII:C was frequently normal, whereas VIIIR:AG, RCF, and PBCF were almost always deficient. The patterns of VIIIR:AG, RCF, and PBCF deficiencies in the canine VWD plasmas suggested that some canine breeds have a "classic" form of VWD whereas other breeds have "variant" forms of disease.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Disease Models, Animal; Dog Diseases; Dogs; Hexadimethrine Bromide; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The quantitative assay of von Willebrand factor using ristocetin aggregation is simplified and improved by the use of frozen normal platelets. The main advantages of the method described are excellent reproducibility, maintenance of a stable optimal pH and the possibility of eliminating interfering factors, such as inhibitors, by dilution.

Topics: Blood; Blood Coagulation Factors; Blood Platelets; Freezing; Humans; Hydrogen-Ion Concentration; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Precipitating antibodies directed toward human F.VIII/WF were found in the plasma of four out of 17 multitransfused patients with severe, homozygous-like VWD. The familial incidence was illustrated by the development of these antibodies in three patients from the same kindred. Such antibodies, titrated with newly developed quantitative assays of anti-VIIIR:Ag and anti-VIIIR:RCo, were directed toward only these components of F.VIII/WF. VIII:C was neutralized in a time-independent manner in plasma and was not inactivated when separated from F.VIII/WF by solid-phase PE absorption. Plasma, serum, or immunoglobulin reacted in precipitation systems (immunodiffusion, EID, CIE) with human VIIIR:Ag, with some degree of cross-reactivity toward VIIIR:Ag from other mammalian plasmas. When used in IRMA, these antibodies demonstrated the same abnormalities as heterologous antisera in variant VWD: decreased binding affinity or nonparallelism of the dose-response curves. They are polyclonal IgG with both kappa and lambda light chains. It is suggested that in some patients with severe homozygous-like VWD, the synthesis of the component of F.VIII/WF carrying VIIIR:Ag and VIIIR:RCo is suppressed whereas VIII:C production is not completely abolished.

Topics: Antibody Formation; Blood Coagulation Factors; Child; Child, Preschool; Factor VIII; Female; Humans; Immunodiffusion; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Diagnosis of deficiencies of coagulation factor VIII can be difficult to establish in some cases. The use of the factor VIII-related antigen and the use of the ristocetin cofactor assays have increased the reliability of diagnosis of factor VIII deficiency in patients with hemophilia A or von Willebrand's disease, and in carriers of hemophilia A. The authors re-evaluated samples, from frozen storage, of blood from patients previously diagnosed as having von Willebrand's disease. This diagnosis was based on clinical history, family history, bleeding time, factor VIII procoagulant activity, and response to ristocetin in platelet-aggregation studies. Eleven cases were studied by the review of previously obtained data and the addition of the factor VIII-related antigen and ristocetin-cofactor assays. In two of eleven cases, the diagnosis was changed to possible hemophilia A carrier state.

Topics: Adolescent; Adult; Antigens; Blood Proteins; Child; Child, Preschool; Factor VIII; Female; Hemophilia A; Humans; Male; Methods; Middle Aged; Ristocetin; von Willebrand Diseases

Ristocetin was trace-labeled with [3H] by the reductive methylation method. It was shown to agglutinate human platelets in the presence of VIIIR:WF in a manner indistinguishable from unlabeled ristocetin. The binding of the labeled ristocetin to normal and enzyme-modified human platelets was studied both in the presence and absence of VIIIR:WF and at nonagglutinating and agglutinating concentrations of ristocetin. Virtually no difference in [3H]ristocetin binding was seen whether VIIIR:WF was present or not. Platelets treated with chymotrypsin, which destroys their ability to agglutinate to VIIIR:WF and ristocetin, did not bind less ristocetin than did control platelets. A pronounced, direct relationship was found between [3H]ristocetin bound by normal platelets and total ristocetin concentration. This implies that at the higher (agglutinating) concentrations of ristocetin either more binding sites are exposed or, more probably, aggregation of ristocetin occurs.

Topics: Alkylation; Binding Sites; Blood Platelets; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases

In classic von Willebrand's disease (vWd), assignment of the heterozygous genotype for genetic studies and diagnosis for clinical purposes (which are not exactly the same) are formidable problems. We have pointed out in the first report in this series that almost 50% of the members of two large kindred who transmitted this disease, and were therefore heterozygous, were scored as normal by the usual tests of hemostasis. This report describes how this large proportion can be significantly reduced by application of discriminant analysis. Using linear discriminants in three variables--coagulation factor VIII (VIII:C), factor-VII-related antigen (VIIIR:Ag), and the ristocetin cofactor related to factor VIII (VIIIR:WF)--we were able to classify as heterozygous more than 80% of the transmitters in the two large kindred. It was of particular interest that the four parents of two related vWd homozygotes could be scored as heterozygous by discriminant analysis even though all their laboratory tests were within the normal ranges.

Topics: Adult; Aged; Antigens; Factor VIII; Female; Genotype; Heterozygote; Humans; Male; Middle Aged; Pedigree; Ristocetin; Statistics as Topic; von Willebrand Diseases

Topics: Adolescent; Aged; Arginine Vasopressin; Blood Coagulation; Blood Donors; Child; Deamino Arginine Vasopressin; Factor VIII; Female; Hemophilia A; Humans; Male; Ristocetin; Tranexamic Acid; von Willebrand Diseases

Topics: Agglutination; Animals; Blood Platelets; Blood Proteins; Dogs; Factor VIII; Humans; Ristocetin; Serum Albumin, Bovine; von Willebrand Diseases

Topics: Bleeding Time; Factor VIII; Hemostasis; Humans; Molecular Weight; Platelet Aggregation; Ristocetin; von Willebrand Diseases

An inexpensive assay for von Willebrand factor using microtitration plates, formalin-fixed platelets, and ristocetin is described. With this technic, the assay for von Willebrand factor is simple enough to be considered for use in any diagnostic laboratory for patients with prolonged bleeding times.

Topics: Blood Coagulation Factors; Blood Coagulation Tests; Blood Platelets; Factor VIII; Female; Formaldehyde; Humans; Male; Methods; Ristocetin; von Willebrand Diseases; von Willebrand Factor

In a patient with a clinically serious Willebrand-Jürgen's syndrome an inhibitor appeared at the age of 1 1/2 years, which, contrary to inhibitors in haemophilia A, was directed against all properties of factor VIII molecule. In a quantitative test the height of the inhibitor level to factor VIIIag was determined. Administrations of plasma concentrate resulted in a titre increase which could not be suppressed even by an immunosuppressive therapy with cyclophosphamide. After a long break in the substitution a severe bleeding could be successfully treated and the substitution effect for factor VIIIc, factor VIIIag and Ristocetin co-factor could be identified for several days.

Topics: Antigens; Blood Transfusion; Child, Preschool; Factor VIII; Humans; Male; Plasma; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Animals; Antigens; Arteriosclerosis; Factor VIII; Hemostasis; Humans; Ristocetin; von Willebrand Diseases

Topics: Antigens; Blood Platelets; Factor VIII; Humans; Radioimmunoassay; Ristocetin; von Willebrand Diseases

31 patients with the diagnosis or presumed diagnosis of von Willebrand's disease were reinvestigated by means of Ristocetin cofactor-activity and factor VIII-associated antigen. In addition a family with a variant of von Willebrand's disease is described. Ristocetin cofactor-activity was found the most reliable test, its value for the diagnosis of mild forms and of variants of von Willebrand's disease is further established by these results.

Topics: Adult; Antigens; Blood Coagulation Tests; Child; Factor VIII; Female; Genetic Variation; Humans; Male; Ristocetin; von Willebrand Diseases

Topics: Adult; Blood Platelets; Dextrans; Factor VIII; Humans; Male; Ristocetin; von Willebrand Diseases

Topics: Animals; Factor VIII; Humans; Immune Sera; Immunoassay; International Cooperation; Platelet Adhesiveness; Platelet Aggregation; Rabbits; Radioimmunoassay; Ristocetin; von Willebrand Diseases

Topics: Drug Stability; Female; Freezing; Humans; Male; Platelet Aggregation; Ristocetin; Time Factors; von Willebrand Diseases

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Calcium Chloride; Dose-Response Relationship, Drug; Factor VIII; Humans; Hydrogen-Ion Concentration; Methods; Platelet Aggregation; Ristocetin; Sodium Chloride; Swine; von Willebrand Diseases; von Willebrand Factor

Results of functional and immunologic tests of factor VIII and of platelet function were followed in two patients with severe von Willebrand's disease who were given cryoprecipitate during preparation for surgery. Initial correction of factor VIII coagulant activity was found to persist from 48 to 72 hours. Correction of immunologically measured factor VIII persisted for 48 hours and correlated with the patients' platelet response to ristocetin aggregation. Bleeding times were corrected for only 6 to 8 hours and showed fairly good correlation with the ristocetin aggregation factor, as suggested by Weiss. The administration of commercial factor VIII concentrate corrected all parameters except the bleeding time and the ristocetin aggregation factor measurement.

Topics: Adult; Autoantigens; Blood Coagulation; Chemical Precipitation; Cryoglobulins; Dose-Response Relationship, Drug; Factor VIII; Female; Half-Life; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Adult; Antigens; Blood Coagulation Factors; Factor VIII; Female; Humans; Male; Physical Exertion; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Adolescent; Adult; Aged; Blood Platelets; Blood Transfusion; Chemical Phenomena; Chemistry; Factor VIII; Female; Hemophilia A; Humans; Infant, Newborn; Macromolecular Substances; Male; Middle Aged; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

The clinical picture, family history and laboratory findings of 100 patients with von Willebrand's disease (VWD) have been studied in Italy in a multicentre survey from two rural areas with a known high incidence of the disease and from two large cities (Rome and Milan). Bleeding time, procoagulant factor-VIII (VIIIAHF) assay, platelet retention and PRP-ristocetin aggregation were measured in each centre, and plasma samples were frozen and subsequently assayed for factor VIII-related antigen (VIIIAGN) and ristocetin co-factor (VIIIVWF) in one laboratory (Milan). On the basis of the inheritance pattern, clinical severity of the disease and laboratory findings, patients with VWD were separated into two groups. In 17 patients the absence of a family history of bleeding, a high incidence of parental consanguinity and involvement of both sexes suggested an autosomal recessive mode of inheritance; the unusual severity of the disease with markedly abnormal results of all six laboratory measurements (and frequent reduced levels of VIIIAGN and VIIIVWF in the unaffected parents) were consistent with the homozygous state. In 83 patients, the disease was familial (being transmitted as an autosomal dominant trait) and of moderate clinical severity. In these, VIIIVWF was usually much lower than VIIIAHF and VIIIAGN. The findings suggest that although some patients may present with simultaneous abnormalities of all six studied laboratory measurements, the majority show a variation in the spectrum of the abnormal results, without any obvious simple pattern.

Topics: Antigens; Blood Coagulation Tests; Factor VIII; Female; Genes, Recessive; Humans; Male; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Ristocetin and vancomycin are structurally similar glycopeptide antibiotics. Both vancomycin and ristocetin in high concentrations (3.0 mg/ml) cause the precipitation of fibrinogen, plasminogen, and IgG from platelet-poor plasma (PPP). In contrast to ristocetin, vanomycin (0.5-1.5 mg/ml) does not agglutinate platelets in normal platelet-rich plasma (PRP) or formalin-treated platelets in the presence of normal PPP. Preincubation of vancomycin (0.5-1.25 mg/ml) with normal PRP, von Willebrand platelets in normal PPP, or formalinized platelets results in inhibition of platelet agglutination induced by ristocetin (0.7-1.25 mg/ml) or ristocetin and normal PPP. This inhibition can be overcome by increasing the final concentration of ristocetin in the platelet suspension. Preincubation of formalin-treated platelets with the major fraction obtained by carboxymethyl-Sephadex C-50 chromatography of commercial vancomycin also results in inhibition of agglutination induced by ristocetin and normal PPP. Incubation with vancomycin (1.25 mg/ml) does not interfere with von Willebrand factor (vWF) or factor VIII coagulant activities in normal PPP or in Sepharose 4B void volume fractions of PPP. These results indicate that vancomycin interacts with normal, von Willebrand, and formalin-treated platelets and inhibits the binding of ristocetin (or ristocetin-vWF complexes).

Topics: Binding, Competitive; Drug Interactions; Factor VIII; Formaldehyde; Humans; Platelet Aggregation; Ristocetin; Vancomycin; von Willebrand Diseases

Aortic endothelial cells from normal pigs and pigs with von Willebrand disease have been established in long-term cultures. Both cultures appeared similar in terms of general growth characteristics, morphologic features and ultrastructure. Immunofluorescent staining of these cultures with chicken (or rabbit) antiporcine ristocetin-Willebrand factor sera (or IgG) resulted in extensive perinuclear staining of the cells in both cultures. Additionally, staining of semiconfluent cultures of normal cells for ristocetin-Willebrand factor revealed an extensive meshwork of distinct, immunologically identifiable ristocetin-Willebrand factor-containing filaments between cells. Immunoreactive material was considerably decreased and more diffuse between cells in semiconfluent cultures from affected pigs. Through immunocytochemical staining with peroxidase-coupled antiserum, the filaments (of indeterminate length) were found to have a diameter of approximately 300 A. Finally, washed porcine platelets interacted extensively with scrape-damaged cultures of affected endothelial cells. This interaction of platelets with damaged normal cultures was abolished by pretreatment of the cultures with rabbit antiporcine ristocetin-Willebrand factor IgG.

Topics: Animals; Aorta; Blood Platelets; Cell Division; Cells, Cultured; Endothelium; Microscopy, Electron; Ristocetin; Swine; von Willebrand Diseases; von Willebrand Factor

Commercially available antiserum to factor VIII was used in several tests to determine whether it might serve as a reference between research laboratories involved in investigation of the factor VIII complex and whether the antiserum might be useful in the screening of large populations of patients with hereditary disorders of factor VIII. In Ouchterlony plates, the antiserum gave a single line of identity with concentrated factor VIII, cryoprecipitate, and human plasma. The antiserum was capable of inhibiting the ristocetin response of normal platelets. Testing antigenic factor VIII by the Laurell technic with the commercial antiserum on plasmas from normal and stressed normal controls, patients with von Willebrand's disease, patients with hemophilia A, and obligate carriers of hemophilia A gave diagnostic and reproducible results.

Topics: Adult; Antigens; Factor VIII; Female; Hemophilia A; Humans; Immune Sera; Immunodiffusion; Male; Middle Aged; Physical Exertion; Platelet Aggregation; Ristocetin; von Willebrand Diseases

A plasmatic component required for the ristocetin-induced aggregation of platelets has been purified from normal human and porcine plasma by gel filtration (4% agarose) and anion-exchange chromatography (DEAE cellulose). No factor VIII coagulant activity was found associated with the purified human or porcine component. Urea sodium dodecylsulfate electrophoretic analysis of the purified component of both species indicated that the apparent molecular weight with intact disulfides is in excess of 500,000; after disulfide reduction with 2-mercaptoethanol, single components with an apparent subunit molecular weight of 230,000 were observed. Purified porcine ristocetin-Willebrand factor (RWF) co-sedimented in sucrose gradients with the factor present in normal plasma. Amino acid analysis of both human and porcine RWF indicated that all normal amino acids are present, whereas amino sugars were undetected. However, lipid analysis indicated 1% to 2% lipids present, including monoglycerides, di- and tri-glycerides, cholesterol, cholesterol esters, some free fatty acids, and a trace of phospholipid. A single line of identity was observed between normal human plasma and purified human RWF when immunodiffusion plates were run with purified rabbit anti-human RWF immunoglobulins. Antisera raised against human and porcine RWF's do not inhibit the factor VIII coagulant activity of the homologous plasma, nor is "spontaneously occurring" human factor VIII inhibitor neutralized by the isolated material of either species.

Topics: Amino Acids; Animals; Blood Coagulation Factors; Blood Coagulation Tests; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Humans; Immunodiffusion; Lipids; Methods; Platelet Aggregation; Proteins; Ristocetin; Swine; von Willebrand Diseases; von Willebrand Factor

Samples of apparently normal skin from one patient with von Willebrand's disease (vWD) and five patients without vWD were examined with fluorescein-tagged antiserum to the component of factor VIII required for aggregation of platelets by ristocetin (VIII-R.A.F.). No evidence of VIII-R.A.F. was found in the vWD skin, while bright granules were seen on and/or in the endothelial cells of dermal capillaries in all patients without vWD. VIII-R.A.F. granules were also found in the interstitial vasculature of all of twelve renal-biopsy specimens from patients without vWD. These observations support the concept that an abnormality of the vascular endothelium is involved in the pathogenesis of vWD.

Topics: Blood Platelets; Capillaries; Factor VIII; Female; Fluorescent Antibody Technique; Humans; Immune Sera; Kidney; Platelet Aggregation; Ristocetin; Skin; von Willebrand Diseases

A reduced level of factor XII has been observed in 9 of 39 patients with von Willebrand's disease. This apparent common variant of von Willebrand's disease has not previously been described, and its significance is not known.

Topics: Adult; Aged; Child; Diagnosis, Differential; Factor VIII; Factor XII; Female; Humans; Male; Middle Aged; Platelet Aggregation; Ristocetin; Terminology as Topic; von Willebrand Diseases

Plasmas from a pregnant patient with von Willebrand's disease and from a patient with von Willebrand's disease who is taking Premarin had elevated levels of Factor VIII-related antigen and Factor VIII activity without any improvement in their platelet retention abnormality. When these were added to the blood from nonpregnant patients with von Willebrand's disease no significant improvement in platelet retention followed. This suggests that the "retention factor" is independent of the antigenic Factor VIII-like material.

Topics: Blood Coagulation; Blood Transfusion; Estrogens, Conjugated (USP); Factor VIII; Female; Humans; Isoantigens; Platelet Adhesiveness; Platelet Aggregation; Pregnancy; Pregnancy Complications, Hematologic; Ristocetin; von Willebrand Diseases

Antiserum specific for that part of the factor VIII complex designated ristocetin aggregation factor (VIIIRAF) was prepared by immunizing rabbits with VIIIRAF derived from the plasma of a hemophilic patient with circulating antibody to factor VIII procoagulant activity (VIIIcoag). The rabbit antiserum prevented ristocetin-induced platelet aggregation and platelet retention by glass-bead columns. Although the antiserum also inactivated VIIIcoag in normal plasma, it did not inactivate VIIIcoag which had been dissociated from VIIIRAF by chromatography in 1 M NaCl, thus establishing the antigenic specificity of these two factors. When the VIIIRAF antibody was conjugated with fluorescein isothiocyanate and used to examine platelets from normal subjects and patients with von Willebrand's disease, the normal platelets showed a granular fluorescence similar to that observed with antifibrinogen serum whole von Willebrand platelets showed no fluorescent staining. The normal platelets retained the VIIIRAF granules during 18 hours incubation and 5 washings in artificial medium while the von Willebrand platelets failed to acquire granules after 18 hours in normal plasma. When the unstained platelets from patients with von Willebrand's disease were suspended in normal plasma and then aggregated by the addition of ristocetin, the aggregates not only stained brightly for VIIRAF, but fluorescent granules could be seen on individual platelets in the aggregates. These observations suggest that ristocetin causes the binding of VIIIRAF to platelets as well as platelet-to-platelet adhesion.

Topics: Blood Platelets; Factor VIII; Fluorescent Antibody Technique; Hemophilia A; Humans; Immune Sera; Platelet Aggregation; Receptors, Antigen, B-Cell; Ristocetin; von Willebrand Diseases

A girl with symptoms of von Willebrand's disease was found to have a slightly reduced or normal FVIII procoagulant activity, normal FVIII-related antigen (VIIIR:AG) and virtually absent von Willebrand's factor. The electrophoretic mobility of the VIIIR:AG in this patient's plasma and plasma fractions was increased and has been compared with that of two reported patients with FVIII variants. Her lysed platelets contained increased amounts of VIIIR:AG which had an increased anodal migration identical to her plasma VIIIR:AG. Experiments involving the selective absorption of a rabbit antiserum with the patient's plasma provide evidence that VIIIR:AG and von Willebrand's factor are immunologically distinct.

Topics: Adolescent; Antigens; Chemical Precipitation; Chromatography, Gel; Cryoglobulins; Factor VIII; Female; Genetic Variation; Humans; Immune Sera; Immunoelectrophoresis, Two-Dimensional; Immunosorbent Techniques; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Dogs with von Willebrand's disease (VWD) were infused with a highly purified human and canine factor VIII preparation. Control infusions were performed using isotonic saline solution. Following all of the concentrate infusions, the bleeding times were shortened to within the normal range for up to 4 h. Factor VIII activity rose immediately after infusion, rapidly returned to preinfusion levels, and then increased again at 18 h. The levels of factor VIII-related antigen (F VIII-RA) as measured with anticanine factor VIII also increased after infusion and then gradually declined. There was no further increase in F VIII-RA at 18 h, when the factor VIII activity showed the secondary increase characteristic of VWD. With antihuman factor VIII, two identifiable antigens were present in the plasma of the dogs given human factor VIII. The reduced platelet retention of these animals was not significantly altered following infusion, whereas ristocetin-induced platelet aggregation was corrected in one of the dogs given human factor VIII. These studies indicate that factor VIII procoagulalant and von Willebrand factor activities reside on a single molecule or form part of a molecular complex. In addition, the factor VIII preparations appeared to contain the factor VIII synthesis-stimulating factor and/or a precursor of factor VIII procoagulant activity.

Topics: Animals; Antigens; Blood Coagulation Factors; Dogs; Factor VIII; Female; Immunodiffusion; Infusions, Parenteral; Isotonic Solutions; Male; Platelet Aggregation; Prothrombin Time; Ristocetin; Sodium Chloride; Time Factors; von Willebrand Diseases; von Willebrand Factor

Platelet aggregation in citrated and heparinized plasma by ionophore A 23187 and Ristocetin was studied in normal subjects and in patients with von Willebrand's disease and congenital afibrinogenemia. Aggregation by ionophore was normal in all groups both in citrated and heparinized plasma. Aggregation by Ristocetin in citrated plasma was normal in congenital afibrinogenemia, in normal subjects and in types II and III of von Willebrand's disease. It was absent in classical von Willebrand's disease, type I. In heparinized plasma it was absent in all groups, except in some patients with von Willebrand's disease, type III. It is concluded that ionophore A 23187 behaves in a different way than Ristocetin and has no diagnostic implications.

Topics: Afibrinogenemia; Citrates; Female; Heparin; Humans; Ionophores; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases

The antibiotic ristocetin only aggregates platelets in the presence of plasma von Willebrand factor. Platelets from patients with Bernard-Soulier syndrome do not aggregate upon addition of ristocetin although, in contrast to von Willebrand's disease, plasma levels of factor VIII complex (factor VIII clotting activity, von Willebrand factor activity, and von Willebrand antigen) are normal. The membrane surface of normal platelets was modified and compared to the surface of platelets from a patient with Bernard-Soulier syndrome in an attempt to identify the receptor involved in von Willebrand factor-ristocetin-induced aggregation. After the incubation of washed normal platelets with a preparation of ristocetin previously shown to contain a proteolytic contaminant, the aggregation response is significantly decreased on addition or normal plasma. Analaysis by gel electrophoresis of such platelets when stained for carbohydrate revealed a decrease in the relative amounts of membrane glycopro-eins. Chymotrypsin-treated normal platelets had less membrane glycoproteins in addition to giving a reduced aggregation response in ristocetin-induced aggregation. Staining of gels for protein and carbohydrate indicated that there was an extensive change in the surface of Bernard-Soulier platelets, whereas those from patients with von Willebrand's disease appeared the same as normal. Platelets from patients were labeled by the lactoperoxidase iodination technique. Not only was the relative intensity of staining of platelet-specific proteins and glycoproteins changed in Bernard-Soulier platelets, but the iodination of the glycoproteins on the membrane surface relative to other membrane constituents was lower. In contrast, platelets from patients with von Willebrand's disease showed a normal exposure of membrane components. These data suggest therefore that membrane glycoproteins may play a functional role in ristocetin-induced aggregation.

Topics: Binding Sites; Blood Platelets; Cell Membrane; Glycoproteins; Humans; Peptide Hydrolases; Platelet Aggregation; Purpura, Thrombocytopenic; Ristocetin; Syndrome; von Willebrand Diseases; von Willebrand Factor

Crossed antigen-antibody electrophoresis was used to determine the electrophoretic properties of factor VIII/Von Willebrand (F. VIII/VW) protein in normal plasma using a specific rabbit antiserum against purified human factor VIII. The electrohoretic patterns in seven patients with severe Von Willebrand's disease in two different families were studied. In these patients the prolonged bleeding time was related to a functionally abnormal F. VIII/VW protein which was unable to induce platelet aggregation in presence of ristocetin. F. VIII/VW protein had an increased electrophoretic mobility in all the patients. The electrophoretic pattern was similar in different members of the same family but differed from one family to the other. Plasma samples collected after transfusion of normal cryoprecipitate to one member of the first family showed an increasing amount of F. VIII/VW protein, but even one hour after transfusion only one peak migration in an abnormal position was found. However, when mixed in vitro with normal plasma, the plasmas of the same patient showed two peaks, one in position of normal F. VIII/VW protein and one with increased electrophoretic mobility. These results suggest that normal F. VIII/VW protein transfused to recipients with Von Willebrand's disease synthesizing a functionally deficient factor undergoes a rapid alteration.

Topics: Antigen-Antibody Reactions; Antigens; Blood Coagulation Factors; Blood Coagulation Tests; Blood Protein Electrophoresis; Blood Transfusion; Factor VIII; Female; Humans; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Nine probands with von Willebrand's disease, and their family members, totalling 43 people, were examined. Twenty-seven had a history of bleeding; 29 had an increased factor VIII activity:factor VIII related antigen ratio; 24 had a decreased factor VIII related antigen; 23 had a prolonged bleeding time; 19 had a reduced platelet adhesiveness; 16 had a decreased factor VIII activity; and 14 had an abnormal ristocetin-induced platelet aggregation. Eight members with both normal beleeding time and normal factor VIII activity were found to have other abnormal tests: elevated ratio of factor VIII activity to factor VIII related antigen in seven; decreased factor VIII related antigen in four; and reduced platelet adhesiveness in one. Therefore, ratio of factor VIII activity to factor VIII related antigen and factor VIII related antigen are more sensitive and may be used for the detection of heterozygous carriers of von Willebrand's disease. Although patients with thrombocytopathy may have a prolonged bleeding time, decreased platelet adhesiveness and reduced platelet aggregation by ristocetin, their factor VIII activity, factor VIII related antigen and ratio of factor VIII activity to factor VIII related antigen are normal and their abnormal ristocetin test cannot be corrected by the addition of factor VIII concentrate. Hemophilic subjects and hemophilic carriers, who are deficient in factor VIII activity, usually have a normal bleeding time, normal platelet adhesiveness, and normal ristocetin test. In contrast to patients with von Willebrand's disease, their factor VIII related antigen is normal or slightly increased and their ratio of factor VIII activity to factor VIII related antigen is significantly reduced. We conclude that ratio of factor VIII activity to factor VIII related antigen and factor VIII related antigen are not only more sensitive but also more specific for the diagnosis of von Willebrand's disease.

Topics: Antigens; Blood Coagulation Tests; Blood Platelet Disorders; Diagnosis, Differential; Factor VIII; Female; Hemophilia A; Heterozygote; Humans; Male; Pedigree; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

A newly available dried concentrate of antihemophilic factor (Profilate, Abbott Laboratories) was compared with standard, blood-bank prepared cryoprecipitate in the control of bleeding in a patient with von Willebrand's disease. Profilate effectively raised plasma levels of factor VIII but produced only half the expected increase in plasma ristocetin aggregation factor (RAF), and this RAF did not bind readily to the platelets in the presence of ristocetin. Furthermore, the Profilate had little effect upon the bleeding time or the clinical hemorrhage. In contrast, the cryoprecipitate did increase plasma RAF to the expected level, and this RAF bound readily to the patient's platelets in the presence of ristocetin. Cryoprecipitate promptly controlled bleeding. We conclude that the RAF present in Profilate retains in vitro activity but is incapable of augmenting platelet function in vivo.

Topics: Adult; Blood Coagulation Tests; Cryoglobulins; Factor VIII; Humans; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases

The authors report a case of chronic eosinophilic leukemia. Ristocetin did not aggregate patient's platelets in the absence of bleeding disorder. A possible relationship between arterial thrombosis observed in patients with eosinophilic leukemia and platelet abnormality is evoked.

Topics: Arteries; Blood Platelet Disorders; Cytoplasmic Granules; Diagnosis, Differential; Eosinophils; Hematopoiesis; Heparin Antagonists; Humans; Male; Middle Aged; Myeloproliferative Disorders; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Thrombosis; von Willebrand Diseases

Topics: Animals; Animals, Newborn; Antigens; Blood Coagulation Factors; Factor VIII; Female; Immunoelectrophoresis; Male; Platelet Aggregation; Ristocetin; Swine; von Willebrand Diseases; von Willebrand Factor

This investigation was undertaken in order to determine the most suitable methods for diagnosing von Willebrand's disease (v. W. d.), with particular reference to "mild" cases. 50 healthy persons, 21 patients with "severe" v.W.d. (verified according to all hitherto-established criteria) and 39 persons suffering from "mild" v.W.d. were examined. Even though - as far as the latter group is concerned - some of the characteristic laboratory findings were absent, the fact that these persons are related to the patients with "severe" v.W.d. made verification of the diagnosis nevertheless possible. In the group with "mild" v.W.d. 85% had decreased functional factor VIII activity and 82% showed reduced platelet adhesiveness; a prolonged bleeding time according to Borchgrevink was recorded in 72% of the cases and 64% had pathological values for the Ristocetin-induced platelet aggregation were pathological in less than 50% of the group. There was a close relation between the diagnostic significance and reproducibility of the various methods; in regard to the group of patients with "mild" v.W.d., the methods yielding the greatest number of pathological findings (functional factor VIII and platelet adhesiveness) were the ones with the lowest variation coefficients, i.e. 3.1 and 2.1%, respectively. The consideration of a time-dependent rise in the aggregation capability of washed platelets with Ristocetin was of decisive importance in the reproducibility of the rather sophisticated technique of determining the Ristocetin cofactor. The addition of EDTA largely inhabited this effect. Even in the group of patients with "severe" v.W.d., part of the findings obtained with screening tests of the intrinsic coagulation system remained within the normal range. There was no statistical correlation between the individual laboratory findings in the group of healthy persons. In the group with "mild" v.W.d., there was a significant correlation between platelet adhesiveness and Ristocetin-induced platelet aggregation on the one hand, and platelet adhesiveness and the Ristocetin cofactor on the other. In addition, a verified correlation was found to exist between Ristocetin-induced platelet aggregation and Ristocetin cofactor and between functional factor VIII and factor VIII-related antigen. In the group of "severe" v.W.d., only platelet adhesiveness and functional factor VIII were not significantly correlated, whilst all other correlations were of statistical significance. Further p

Topics: Factor VIII; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Treatment of von Willebrand disease with two plasma antihemophilic factor (AHF) concentrates, cryoprecipitate and glycine-precipitated AHF, was compared. Both concentrates were equally effective in immediately raising the plasma levels of factor VIII, the factor VIII-related antigen, and the ristocetin-related von Willebrand factor (vWF) and in stimulating a secondary rise in plasma factor VIII. Given either concentrate, the vWF activity, the antigen, and factor VIII levels were normalized in a patient with von Willebrand disease. However, correction of the prolonged bleeding time and control of bleeding occurred only with the cryoprecipitate. The bleeding-time corrective factor and the ristocetin-related vWF or platelet-aggregating factor are dissociable, distinct activites.

Topics: Antigens; Blood Coagulation Disorders; Blood Coagulation Tests; Chemical Precipitation; Factor VIII; Glycine; Hemostasis; Humans; Male; Middle Aged; Ristocetin; von Willebrand Diseases

Topics: Adolescent; Adult; Antigen-Antibody Complex; Blood Transfusion; Child; Factor VIII; Female; Humans; Immunoglobulin G; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Factor VIII procoagulant activity (VIIIc), antigen (vWa), mobility of the antigen on two dimensional immunoelectrophoresis and platelet function were studied in 9 families with reduced ristocetin induced platelet aggregation rate (RIPA) and/or deficiency of plasma factor(s) required for ristocetin aggregation of washed normal platelets (vWf). the families could be subdivided into 4 groups. Group I showed dominant inheritance and reduced levels of VIIIc and vWa characteristic of typical von Willebrand's disease. All patients had reduced vWf and in 7 of 10 RIPA was reduced. Group II showed normal levels of VIIIc but reduced vWa. All showed reduced vWf but RIPA was reduced in one patient only. There was a good correlation between vWf and vWa and VIIIc in both groups. The bleeding time correlated with vWf in group I but not group II. Group III showed normal or nearly normal VIIIc and vWa but there was an increased mobility of vWa compared to normals and to groups I and II. RIPA was markedly reduced as was the vWf in one patient. Group IV is represented by one child with a strong family history of bleeding, who had reduced RIPA and defective platelet release reaction. The vWf in this child was normal and the ratio between VIIIc and vWa was similar to that seen in carriers of haemophilia. This spectrum of abnormalities of ristocetin aggregation justifies the use of the term 'von Willebrand's syndrome'.

Topics: Animals; Blood Coagulation Tests; Blood Platelets; Factor VIII; Female; Humans; Immunoelectrophoresis; Male; Platelet Aggregation; Rabbits; Ristocetin; von Willebrand Diseases

A case of acquired von Willebrand's syndrome (vWs) is described which appeared to be due to antibodies directed against factor VIII clotting activity (FVIIIC), factor VIII-related antigen (FVIIIRAg) and von Willebrand factor. The antibodies directed against FVIIIRAg was demonstrated by the inhibitory effect of a platelet eluate on Ristocetin-induced aggregation of normal platelets. This effect was not shown by the patient's platelet-poor plasma alone, nor could it be demonstrated in platelet eluates from 13 other patients who had antibodies to FVIIIC but in whom there was no evidence of an acquired vWs.

Topics: Aged; Antibodies; Antigens; Blood Coagulation Tests; Chemical Precipitation; Cryoglobulins; Factor VIII; Female; Hemophilia A; Humans; Male; Middle Aged; Plasmapheresis; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Defective ADP-induced aggregation was observed in in vitro streptokinases(SK)-treated normal platelet-rich plasma. Classic haemophilia and normal platelet poor plasma (PPP) treated with SK inhibit the aggregation of washed platelets; plasmin-treated normal human serum also shows an inhibitory effect on platelet aggregation. However, SK-treated von Willebrand plasmas do not inhibit the aggregation of washed platelets. This confirms the fact that the anti-aggregating effect is mainly linked to the digested factor VIII) but not to the digested fibrinogen. Defective ristocetin-induced platelet aggregation has also been observed in SK-treated plasmas. The presence of normal PPP does not modify the inhibition of the ADP-induced aggregation of washed platelets in SK-treated PPP. However, it does correct the ristocetin-induced aggregation. These results suggest that the inhibition of the ADP-induced aggregation is caused by the factor VIII degradation products, while the inhibition of the ristocetin-induced aggregation appears because of a defective von Willebrand activity of the factor VIII molecule.

Topics: Adenosine Diphosphate; Antigens; Factor VIII; Humans; Peptide Hydrolases; Platelet Aggregation; Ristocetin; Streptokinase; von Willebrand Diseases

Topics: Blood; Humans; Hydrogen-Ion Concentration; Platelet Aggregation; Ristocetin; Time Factors; von Willebrand Diseases

Present methods for assay of platelet aggregating agents use freshly prepared platelets. Much time is spent in daily preparation of platelets and standardization presents problems. The preparation of fixed washed platelets (FWP) and their use in two bioassays are described in this report. Washed human platelets were fixed for 48 hours with 4 per cent paraformaldehyde, washed twice in phosphate buffer, pH 6.4, and stored at 4 degrees C. Aggregation of FWP was studied with a macroscopic test and a light absorbance measurement. FWP did not aggregate with adenosine diphosphate, collagen, adrenalin, and thrombin. FWP aggregated with bovine or porcine plasma, poly-L-lysine, and ristocetin with normal human plasma but not with von Willebrand's disease plasma. These observations confirm the direct aggregating effect of these agents. Macroscopic aggregation times were dependent on the amount of aggregating agent (bovine plasma, normal human plasma). A quantitative assay for bovine platelet aggregating factor (PAF) and von Willebrand factor (vWF) with FWP was developed. The ability of FWP to aggregate remained unchanged after 1 month of storage at 4 degrees C. Ristocetin alone caused a decrease in light transmission of FWP suspensions, depending upon the concentration of ristocetin, but did not cause aggregation. FWP constitute a stable reagent suitable for quantitative measurement of PAF and vWF.

Topics: Animals; Biological Assay; Blood Coagulation Factors; Blood Platelets; Blood Preservation; Cattle; Formaldehyde; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Light; Lysine; Peptides; Platelet Adhesiveness; Platelet Aggregation; Polymers; Ristocetin; Swine; von Willebrand Diseases

Previously we showed that von Willebrand patients could be separated into 2 groups on the basis of ristocetin-induced platelet aggregation and factor VII (antigen and activity). Ristocetin-induced platelet aggregation and factor VIII exhibited a positive correlation. This paper deals with 5 out of 31 examined von Willebrand patients in which ristocetin-induced platelet aggregation was not in correlation either with factor VIII activity or antigen. The importance of these findings is discussed.

Topics: Antigens; Factor VIII; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Stimulation, Chemical; von Willebrand Diseases

Topics: Animals; Anticoagulants; Binding Sites; Blood Coagulation; Blood Platelets; Carbohydrates; Chromatography; Disulfides; Electrophoresis, Polyacrylamide Gel; Factor VIII; Glycoproteins; Goats; Hemophilia A; Humans; Immune Sera; Macromolecular Substances; Male; Molecular Weight; Platelet Aggregation; Precipitin Tests; Rabbits; Ristocetin; Sodium Dodecyl Sulfate; von Willebrand Diseases

Topics: Adult; Blood Platelets; Cell Aggregation; Factor VIII; Female; Hemophilia A; Humans; Male; Middle Aged; Platelet Adhesiveness; Ristocetin; Time Factors; von Willebrand Diseases

Normal subjects, patients with various bleeding disorders, and patients with von Willebrand's disease were studied. All patients with von Willebrand's disease had decreased levels of ristocetin-Willebrand factor (range, 0 to 41%) as compared with all other subjects (range, 79 to 202%). Ristocetin-induced platelet aggregation of platelet-rich plasma was abnormal in all patients with von Willebrand's disease tested, and it was possible to correct this abnormal response by addition of normal platelet-poor plasma. Abnormal ristocetin-induced platelet aggregation was seen in patients with intrinsic platelet disorders or, on some occasions, in normal patients who had ingested aspirin. Ristocetin-induced platelet aggregation is not diagnostic, but it may be useful as a simple screening test for patients with possible von Willebrand's disease. In conjunction with other tests, the assay for ristocetin-Willebrand factor will be useful in diagnosis and evaluation of these patients.

Topics: Anticoagulants; Antigens; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Platelets; Factor VIII; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Ristocetin was used to study platelet aggregation in platelet-rich plasma and to assay the von Willebrand factor activity of factor VIII (VIII-VWF). Ristocetin-induced platelet aggregation (RIPA) was decreased in 13 of 18 patients with von Willebrand's disease (VWD) who had decreased plasma levels of VIII-VWF. The five patients with normal RIPA appeared to have mild VWD but did not constitute a separate subclass. RIPA was also abnormal in some patients with intrinsic platelet defects, but in no case was the defect corrected by normal plasma. The latter type of correction appears to be specific for VWD. Aspirin ingestion inhibited the second phase of RIPA (at low concentrations of ristocetin only) but did not affect the initial phase of aggregation or the level of VIII-VWF. We also studied a group of patients who had both abnormalities of the factor VIII complex and intrinsic platelet defects, such as impaired collagen-induced aggregation, as well. The findings in these patients and in those with typical von Willebrand's disease appear to comprise a spectrum of disorders (the von Willebrand syndrome) in which some abnormality of the factor VIII complex is associated with impaired platelet function. At present, ristocetin would appear to be a useful reagent for evaluating patients with bleeding disorders and for studying patients with the von Willebrand syndrome.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aspirin; Blood Coagulation Tests; Blood Platelets; Collagen; Epinephrine; Factor VIII; Glass; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Drug Interactions; Humans; Platelet Adhesiveness; Platelet Aggregation; Polygeline; Polymers; Ristocetin; von Willebrand Diseases

Topics: Blood Platelets; Collagen; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

The purified factor VIII-related protein we have previously characterized from normal cryoprecipitate possesses both procoagulant activity and vWf activity. We have attempted to isolate and characterize this protein from three patients with severe vWd. This protein is absent or markedly diminished in amount in these vWd patients, as judged by gel filtration, polyacrylamide-gel electrophoresis, and immunoprecipitation assays. Likewise, the procoagulant and vWf activities are deficient. As vWf activity is one of the major biologic functions of either the normal or hemophilic factor VIII-related protein, the purified protein should be designated the f VIII/vWf protein.

Topics: Animals; Antigens; Blood Coagulation Tests; Blood Proteins; Chromatography, Gel; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Factor VIII; Female; Humans; Immune Sera; Immunoelectrophoresis; Male; Platelet Aggregation; Rabbits; Ristocetin; Sodium Dodecyl Sulfate; von Willebrand Diseases

Normal human plasma contains a component or components which interfere with ristocetin-induced platelet aggregation. Preliminary examination suggests a protein (or proteins) which binds ristocetin and competes more effectively for ristocetin than do the proteins involved in ristocetin-induced platelet aggregation. The presence of this protein in normal human plasma also prevents ristocetin-induced precipitation of plasma proteins at levels of ristocetin necessary to produce platelet aggregation (0.5-2.0 mg/ml). Serum contains an apparent two0fold increase of this component when compared with plasma. Heating serum at 56 degrees for one hour results in ad additional 2 to 4 forl increase. The presence of a ristocetin-binding protein in normal human plasma requires that this protein be saturated with ristocetin before ristocetin-induced platelet aggregation will occur. Variations in the ristocetin-binding protein(s) will cause apparent discrepancies in ristocetin-induced platelet aggregation in normal human plasmas.

Topics: Adsorption; Aluminum Hydroxide; Binding, Competitive; Blood Proteins; Chemical Precipitation; Hot Temperature; Humans; Platelet Adhesiveness; Platelet Aggregation; Protein Binding; Ristocetin; von Willebrand Diseases

The Factor VIII/von Willebrand factor protein was characterized in two unrelated patients with von Willebrand's disease in whom procoagulant and Factor VIII/von Willebrand factor antigen levels were normal. In both patients evidence of an abnormal protein was observed on crossed antigen-antibody electrophoresis. In one patient the Factor VIII/von Willebrand factor protein eluted from Sepharose 4B in a position and distribution identical to normal with normal levels of procoagulant activity and antigen. However, the partially purified Factor VIII/von Willebrand factor protein had markedly reduced von Willebrand factor activity in a ristocetin assay. In the second patient the peak of Factor VIII/von Willebrand factor protein, antigen, and procoagulant activity eluted from a Sepharose 4B column with an estimated molecular weight of approximately half that of normal. This protein had no von Willebrand factor activity. In both patients the reduced Factor VIII/von Willebrand factor protein subunit was indistinguishable from normal on polyacrylamide gel electrophoresis. These studies indicate that in some patients with von Willebrand's disease there is a qualitative defect of the Factor VII/von Willebrand factor protein; the total amount of protein, antigen, and procoagulant activity are normal while the von Willebrand factor activity is deficient.

Topics: Antibodies; Antigens; Blood Coagulation Tests; Chemical Precipitation; Chromatography, Gel; Chymotrypsin; Coagulation Protein Disorders; Counterimmunoelectrophoresis; Electrophoresis, Polyacrylamide Gel; Female; Fibrinogen; Hemophilia A; Humans; Male; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Factor VIII; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Platelet aggregation by various inductors was studied in citrated and heparinized plasma of the following groups of subjects: Normal, hemophilia A, combined factor V and factor VIII deficiency, v. Willeprand's disease and congenital afibrinognemia. The results may be summarized as follows: A-platelet aggregation in citrated plasm 1) platelet aggregation by common inductors ADP, adrenalin and collagen was normal in all groups of subjects but for the patients with congential afibrinogenemia in whom adrenalin induced aggregation was absent or markedly refuced whereas ADP and collagen gave slightly reduced or near normal aggregation curves. 2) platelet aggregation by ristocetin was normal in all groups of subjects but for v. Willebrand's disease in which it was absent. B-platelet aggregation in heparized plasma. 1) platelet aggregation by common inductors resulted to be normal in all groups of subjects except in congenital afibrinogenemia. In this latter case the pattern was still mildly defective but here was an increased aggregation as compared to citrated plasma. These findings have been interpretemmon inductors. 2) platelet aggregation by ristocetin resulted to be absent in all groups of subjects investigated. The possible mechanism of action of the inhibitory effect exercised py heparin with regard to restocetin is discussed.

Topics: Adenosine Diphosphate; Afibrinogenemia; Collagen; Epinephrine; Factor V Deficiency; Hemophilia A; Hemorrhagic Disorders; Heparin; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Adolescent; Adult; Antigens; Blood Coagulation Disorders; Child; Child, Preschool; Factor VIII; Female; Humans; Male; Middle Aged; Platelet Aggregation; Ristocetin; Thrombocytopenia; von Willebrand Diseases

Topics: Adolescent; Adult; Antigens; Blood Platelets; Child; Child, Preschool; Factor VIII; Female; Humans; Male; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Five patients with an original diagnosis of von Willebrand's disease are described because of their levels of factor VIII related protein, Ristocetin-induced platelet aggregation and/or family studies differed from the main group of patients with classical von Willebrand's disease. Two had normal levels of factor VIII related protein with reduced Ristocetin aggregation when this was tested in platelet rich plasma. In one, however, this was due to a plasma defect and in the other to a platelet abnormality. After cryoprecipitate infusion all abnormal tests were corrected in both these patients. The first patient, however, failed to show a secondary rise of factor VIII whereas the second showed a secondary rise of both factor VIII and of factor VIII related protein. The other three cases, who were all very severely affected, have been separated from the main group as none of their families was segregating for classical von Willebrand's disease. It is suggested that the term von Willebrand's disease should be confined to those patients who have reduced factor VIII related protein and Ristocetin aggregation, and that von Willebrand's syndrome should be used for the various sub-groups that are emerging.

Topics: Adult; Factor VIII; Female; Humans; Middle Aged; Pedigree; Platelet Aggregation; Ristocetin; Syndrome; von Willebrand Diseases

Percent aggregation and the aggregation rate of platelet rich plasma (PRP) in response to ristocetin (1.75 mg/ml) were measured in 20 normals and 16 patients with von Willebrand's disease (v Wd), with and without the addition of acetylsalicylic acid (ASA). Percent aggregation did not clearly distinguish between normals and patients with vWd. Aggregation rate was normal in only 2 of 16 patients, and after incubation of PRP with ASA 1 of these 2 remained normal. The corrective effect of dilutions of platelet poor plasma (PPP) on the ristocetin response of washed platelets (von Willebrand's factor, vWf) was measured in 21 normals and 12 patients with vWd. All patients with vWd had abnormal levels. There was a significant correlation between aggregation rate and vWf in patients with vWd but not in normals. Both tests appear to measure closely related defects, and the aggregation rate is as specific as the vWf level for the diagnosis of clinically affected patients.

Topics: Antigens; Aspirin; Blood Coagulation Factors; Dose-Response Relationship, Drug; Factor VIII; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Blood Proteins; Female; Humans; Immunoglobulin G; Male; Platelet Aggregation; Ristocetin; von Willebrand Diseases

In 18 insulin-dependent diabetics (6 without retinopathy, 6 with proliferative retinopathy and 6 with proliferative retinopathy treated by hypophysectomy) matched for age and duration of diabetics, in vitro haemostasis was studied using ADP induced platelet aggregation, ristocetin induced platelet aggregation which allows von Willebrand factor (VIII VWF) assay, and determination of antihemophilic factor procoagulant activity (VII AHF). Using gel filtration-isolated platelets, the ADP induced hyperaggregation previously reported in diabetics with severe retinopathy untreated by hypophysectomy appeared to be related to a platelet and not a plasma factor; the normal results of thrombin induced aggregation suggests that the presumed abnormal platelet factor is related to the platelet plasma membrane. High level of plasma VII VWF was observed in diabetics with proliferative retinopathy while the VII AHF level was within normal limits.

Topics: Adenosine Diphosphate; Adolescent; Adult; Blood Coagulation Factors; Diabetic Retinopathy; Factor VIII; Female; Humans; Hypophysectomy; Male; Middle Aged; Platelet Aggregation; Ristocetin; Thrombin; von Willebrand Diseases

Platelet adhesion to a thrombogenic surface and adhesion-induced aggregation were investigated using a perfusion system at a blood flow rate similar to that observed in arteries. Morphometric measurements revealed diminished adhesion of platelets but normal surface-induced aggregation with blood of patients with von Willebrand's disease and with Bernard-Soulier syndrome. In contrast, surface-induced aggregation was defective with blood of patients with storage pool disease, thrombasthenia and with blood of healthy volunteers after Aspirin ingestion. These findings may explain the defective hemostasis in these patients. They suggest that platelet adhesion and aggregation are governed by different mechanisms.

Topics: Aorta; Aspirin; Endothelium; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Absorption; Animals; Antibodies; Blood Platelets; Factor VIII; Female; Fibrinogen; Fluorescent Antibody Technique; Goats; Hemophilia A; Humans; Immunoelectrophoresis; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Serotonin; Serum Albumin; von Willebrand Diseases

Topics: Antigens; Blood Coagulation Tests; Factor VIII; Female; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Antigens; Blood Coagulation Tests; Factor VIII; Humans; Immunologic Deficiency Syndromes; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Antigens; Factor VIII; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Antigens; Factor VIII; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Blood Coagulation Tests; Blood Proteins; Chemical Precipitation; Ethanol; Factor VIII; Humans; Immunoelectrophoresis; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Blood Coagulation; Factor VIII; Female; Humans; Male; Platelet Adhesiveness; Ristocetin; Vasopressins; von Willebrand Diseases

Topics: Adenosine Diphosphate; Albumins; Aspirin; Carbon Radioisotopes; Chromatography, Gel; Dose-Response Relationship, Drug; Factor VIII; Fibrinogen; gamma-Globulins; Hemophilia A; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Serotonin; von Willebrand Diseases

Topics: Adult; Azathioprine; Blood Coagulation Tests; Collagen Diseases; Factor VIII; Female; Hemorrhagic Disorders; Humans; Jaundice; Methylprednisolone; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Splenomegaly; von Willebrand Diseases

Topics: Blood Coagulation Tests; Blood Platelet Disorders; Consanguinity; Factor VII; Female; Genes, Recessive; Heterozygote; Homozygote; Humans; Immunoassay; Male; Pedigree; Ristocetin; von Willebrand Diseases

Topics: Antigens; Blood Coagulation Tests; Factor VIII; Female; Humans; Male; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Blood Coagulation Tests; Factor VIII; Gastrointestinal Hemorrhage; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Arginine; Blood Coagulation Tests; Blood Platelets; Factor VIII; Hemophilia A; Humans; Ristocetin; Vasopressins; von Willebrand Diseases

Topics: Animals; Antigens; Blood Coagulation Disorders; Blood Platelets; Factor VIII; Humans; Immunoelectrophoresis; Purpura, Thrombocytopenic; Rabbits; Ristocetin; Syndrome; von Willebrand Diseases

Topics: Adenosine Diphosphate; Blood Platelets; Factor VIII; Female; Fibrinogen; Humans; Male; Platelet Adhesiveness; Ristocetin; Stimulation, Chemical; von Willebrand Diseases

Topics: Blood Coagulation Tests; Blood Proteins; Chromatography, Gel; Epinephrine; Factor VIII; Female; Humans; Immunoelectrophoresis; Male; Phenotype; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Adenosine Diphosphate; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Humans; Platelet Adhesiveness; Purpura, Thrombocytopenic; Ristocetin; Thrombin; von Willebrand Diseases

Topics: Adult; Blood Coagulation; Blood Coagulation Tests; Factor VIII; Hemostasis; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Platelet-active "von Willebrand factor" is a poorly characterized activity of a plasma-protein macromolecular complex. A new simple assay for von Willebrand factor is based on the dose response relation of the factor and the ristocetin platelet aggregation time. This assay uses the "snowstorm" macroscopic endpoint. A multiply transfused subject with von Willebrand's disease was observed to have a circulating inhibitor that blocks normal ristocetin aggregation of platelets, but not ADP-, epinephrine-, or collagen-induced aggregation. The inhibitor was not adsorbed by normal platelets, and was stable to heating at 56 degrees for 30 min and to repeated freezings and thawings. This inhibitor also prevents action on human platelets by platelet-aggregating factor of bovine plasma, indicating this bovine factor activity is a function of von Willebrand factor. Three inhibitors were compared: (1) the von Willebrand factor inhibitor specifically blocked von Willebrand factor activity, (2) a human antibody from a hemophiliac inhibited only antihemophilic factor activity, and (3) a rabbit antiserum to a preparation of human antihemophilic factor inhibited both activities. The active site for von Willebrand factor on the macromolecular complex appears to be spaced some distance from the antihemophilic factor site.

Topics: Adenosine Diphosphate; Adult; Animals; Antibodies; Antigen-Antibody Reactions; Biological Assay; Blood Coagulation Factors; Blood Platelets; Collagen; Dose-Response Relationship, Drug; Epinephrine; Factor VIII; Female; Freezing; Hot Temperature; Humans; Immune Sera; Macromolecular Substances; Male; Neutralization Tests; Platelet Aggregation; Rabbits; Ristocetin; von Willebrand Diseases

Topics: Animals; Antigens; Blood Transfusion; Cattle; Chromatography, Gel; Cryoglobulins; Electrophoresis; Factor VIII; Freezing; Homozygote; Hydrogen-Ion Concentration; Male; Molecular Weight; Platelet Aggregation; Rabbits; Ristocetin; Thrombin; Time Factors; von Willebrand Diseases

Topics: Antigens; Densitometry; Factor VIII; Hemophilia A; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Animals; Antibodies; Chemical Precipitation; Dialysis; Factor VIII; Hemophilia A; Humans; Immunoglobulin G; Injections, Subcutaneous; Methods; Platelet Adhesiveness; Platelet Aggregation; Rabbits; Ristocetin; von Willebrand Diseases

Topics: Antigen-Antibody Complex; Antigens; Blood Coagulation Factors; Blood Coagulation Tests; Chromatography, Gel; Factor VIII; Humans; Hydrogen-Ion Concentration; Immunoassay; Neutralization Tests; Pedigree; Platelet Adhesiveness; Ristocetin; Temperature; von Willebrand Diseases

Topics: Antigens; Blood Coagulation Tests; Child; Factor VII; Factor VIII; Female; Homozygote; Humans; Molecular Weight; Pedigree; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Adenosine Diphosphate; Adult; Animals; Antigen-Antibody Reactions; Binding Sites; Blood Coagulation Tests; Collagen; Epitopes; Factor VIII; Female; Hemophilia A; Humans; Platelet Adhesiveness; Rabbits; Ristocetin; von Willebrand Diseases

Factor VIII corrects both the clotting defect in hemophilia A and an abnormality of platelet aggregation in von Willebrand's disease. These two activities of factor VIII (antihemophilic factor and von Willebrand factor) are both detected in the void volume when human plasma or cryoprecipitate is chromatographed on Bio-Gel 5M under conditions of isotonic salt concentration. In contrast, antihemophilic factor procoagulant activity is detected with proteins of lower molecular weight when the chromatography is performed with a buffer containing 0.8M NaCl. In this way, the two activities of factor VIII can be dissociated. It remains to be determined whether these components are separate molecules associated as a complex of high molecular weight in plasma or whether they are subunits of a complex macromolecule.

Topics: Animals; Blood Coagulation; Chromatography; Epitopes; Factor VIII; Hemophilia A; Humans; Isotonic Solutions; Molecular Weight; Osmolar Concentration; Platelet Adhesiveness; Rabbits; Radioimmunoassay; Ristocetin; Sodium Chloride; Thrombin; von Willebrand Diseases

The antibiotic ristocetin, in concentrations of 1.0-1.5 mg/ml, aggregated normal platelets in citrated platelet-rich plasma by a mechanism in which the release reaction played only a minor role. Platelet aggregation by ristocetin in a concentration of 1.2 mg/ml was absent or markedly decreased in 10 patients with von Willebrand's disease. Lesser degrees of abnormality were obtained with a concentration of 1.5 mg/ml. The magnitude of the defect in ristocetin-induced platelet aggregation correlated well with the degree of abnormality of the bleeding time and the levels of antihemophilic factor (AHF, VIII(AHF)) procoagulant activity. In all patients, the defect in ristocetin-induced platelet aggregation was corrected in vitro by normal plasma. Correction was also obtained with a fraction of normal cryoprecipitate that eluted in the void volume with VIII(AHF) after chromatography on a gel that excludes molecules larger than 5 x 10(6). A similar fraction, devoid of VIII(AHF) activity, obtained from patients with von Willebrand's disease had no corrective effect, but fractions obtained from patients with hemophilia were just as effective as those obtained from normal subjects. The correction activity of plasma and partially purified factor VIII was inhibited by a rabbit antibody to human factor VIII but not by a human antibody against VIII(AHF) procoagulant activity. The studies provide further evidence that patients with von Willebrand's disease are deficient in a plasma factor that is necessary for normal platelet function. The activity of this factor appears to be associated with factor VIII but is unrelated to VIII(AHF) procoagulant activity.

Topics: Adenosine; Adenosine Diphosphate; Antibodies; Blood Coagulation Tests; Blood Platelet Disorders; Blood Platelets; Carbon Radioisotopes; Chromatography; Collagen; Cyanides; Edetic Acid; Factor VIII; Female; Glucose; Hemophilia A; Heparin; Humans; In Vitro Techniques; Male; Platelet Adhesiveness; Purpura, Thrombocytopenic; Ristocetin; Serotonin; von Willebrand Diseases

Topics: Animals; Antigens; Blood Coagulation Factors; Factor VIII; Humans; Immunodiffusion; Immunoelectrophoresis; Platelet Adhesiveness; Rabbits; Ristocetin; von Willebrand Diseases

Topics: Adenosine Diphosphate; Blood Platelets; Factor VIII; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

The platelets of three patients with the hereditary giant platelet syndrome of Bernard and Soulier failed to aggregate in response to either ristocetin or bovine fibrinogen. The results of aggregation experiments using mixtures of platelets and plasma suggest that a reaction between a plasma factor deficient in von Willebrand's disease and a platelet component lacking in our patients, and leading to platelet aggregation independently of adenosine diphosphate (ADP), is essential for normal haemostasis.

Topics: Adenosine Diphosphate; Adolescent; Blood Platelet Disorders; Child; Collagen; Female; Fibrinogen; Hemorrhage; Humans; Plasma; Platelet Adhesiveness; Prothrombin; Purpura, Thrombocytopenic; Ristocetin; Syndrome; Thrombin; von Willebrand Diseases

Topics: Blood Coagulation; Blood Platelet Disorders; Blood Platelets; Factor VIII; Hemorrhage; Humans; Platelet Adhesiveness; Purpura, Thrombocytopenic; Ristocetin; von Willebrand Diseases

Topics: Factor VIII; Genes; Humans; Platelet Adhesiveness; Ristocetin; Sex Chromosomes; von Willebrand Diseases

Topics: Animals; Factor VIII; Humans; Immune Sera; In Vitro Techniques; Platelet Adhesiveness; Rabbits; Ristocetin; von Willebrand Diseases

In a previous paper, we showed that the abnormality of ristocetin-induced platelet aggregation in platelet-rich plasma in 10 patients with von Willebrand's disease could be corrected by a factor in normal plasma that was present in the same fractions as factor VIII procoagulant activity (antihemophilic factor, AHF, VIII(AHF)) when prepared by chromatography on Bio-Gel 5 M (Bio-Rad Laboratories, Richmond, Calif.). This observation suggests that patients with this disorder are deficient in a plasma factor, associated with the factor VIII molecule, that is necessary for normal platelet function. In the present paper, we describe, an assay for this factor, the von Willebrand factor (VIII(VWF)), based on the observation that a log-log relationship exists between the amount of ristocetin-induced aggregation of washed, normal platelets and the concentration of normal plasma present in the test system. We assayed the activity of VIII(VWF) as well as antihemophilic factor procoagulant activity (VIII(AHF)) and factor VIII antigen (VIII(AGN)) in 15 patients with von Willebrand's disease and 20 normal subjects. A highly significant correlation (r approximately 0.80) between VIII(VWF) and both VIII(AHF) was found in normal subjects and in patients with von Willebrand's disease. This finding, in addition to the observation that agarose gel chromatography fractions that have VIII(AHF) procoagulant activity also have VIII(VWF) activity, strongly suggests that the von Willebrand factor is associated with the factor VIII molecule. VIII(VWF) in normal plasma was not inhibited by human anti-VIII, and VIII(VWF) levels were normal in hemophilic plasma. Thus, the VIII(VWF) site on the factor VIII molecule appears to be different from that determining VIII(AHF). Finally, the activity of VIII(VWF) appeared to correlate better with the bleeding time than either VIII(AHF) or VIII(AGN). This suggests that VIII(VWF) assayed in this study may be the "anti-bleeding factor" that is deficient in von Willebrand's disease. These findings are consistent with a decreased synthesis of the factor VIII molecule in von Willebrand's disease and suggest the possibility of additional abnormalities of the site on the molecule that determines the activity of VIII(VWF).

Topics: Adult; Animals; Antibodies; Antigens; Blood Platelets; Chromatography; Factor VIII; Female; Hemophilia A; Humans; In Vitro Techniques; Male; Middle Aged; Platelet Adhesiveness; Rabbits; Ristocetin; von Willebrand Diseases

Topics: Blood Proteins; Factor VIII; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Topics: Afibrinogenemia; Blood Platelet Disorders; Female; Fibrinogen; Humans; Male; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases