ristocetin and Hemorrhage

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

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

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: 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

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

Bleeding phenotype is reported in β-thalassemia patients. However, the underlying etiology remains elusive. We aimed to assess coagulation profile and the platelet aggregation in β-thalassemia children with bleeding diathesis. Fifty β-thalassemia children with a positive bleeding history were recruited. Bleeding phenotype was explored through full history taking and thorough clinical examination. Complete blood count, prothrombin time, international normalized ratio, and platelets aggregometry were performed for children with negative workup. Mucosal bleeding was manifest among most of our patients (96%). Two-third of patients had decreased aggregation with ristocetin (68%), adenine di-phosphate (64%), and arachidonic acid (64%). While half of the patients (48%) had deficient response to epinephrine. Collagen, ristocetin, and arachidonic acid induced aggregation were negatively correlated to frequency of blood transfusion (P=0.021, r=-0.325; P<0.001, r=-0.465; P=0.018, r=-0.333, respectively). Aggregation to collagen and epinephrine demonstrated a negative correlation with age (P=0.04, r=-0.287; P=0.03, r=-0.315). Deferiprone was associated with a deficient response to ristocetin and collagen when compared with deferasirox or no chelation (P=0.021 and 0.006, respectively). Impaired ristocetin response was linked to hydroxyurea (P=0.035). Platelets function defect should be considered in β-thalassemia patients with bleeding symptoms.

Topics: Arachidonic Acid; beta-Thalassemia; Blood Platelets; Collagen; Epinephrine; Hemorrhage; Hemostasis; Humans; Platelet Aggregation; Ristocetin

Topics: Autoantibodies; Blood Loss, Surgical; Blood Platelets; Female; Hemorrhage; Humans; Middle Aged; Platelet Aggregation; Platelet Function Tests; Platelet Glycoprotein GPIIb-IIIa Complex; Ristocetin; Thrombasthenia

Bleeding because of impaired platelet function is a major side effect of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. We quantitatively assessed ristocetin-induced platelet aggregation (RIPA) in 64 patients with chronic lymphocytic leukemia (CLL) under ibrutinib at 287 time points. Eighty-seven bleeding episodes in 39 patients were registered (85 Common Toxicity Criteria (CTC) grade 1 or 2, 2 CTC grade 3) during a median observation period of 10.9 months. At times of bleeding, RIPA values were significantly lower (14 vs 28 U; P<0.0001). RIPA was impaired in patients receiving concomitant antiplatelet therapy or anticoagulation (14 vs 25 U, P=0.005). A gradual decline of median RIPA values was observed with increasing bleeding severity. Importantly, no CTC grade 2 or 3 bleeding were observed with RIPA values of >36 U. Sequential monitoring indicated a decrease of RIPA values from a median of 17 to 9 U within 2 weeks after initiation of treatment as well as an increase above the critical threshold of 36 U within 7 days when ibrutinib was paused. Low RIPA values were similar during treatment with another BTK inhibitor, CC292. Quantitative assessment of platelet function is a practical tool to monitor bleeding tendency under BTK-inhibitor therapy.

Topics: Adenine; Adult; Aged; Aged, 80 and over; Drug Monitoring; Female; Hemorrhage; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Piperidines; Platelet Aggregation; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Ristocetin

High-molecular-weight (HMW) von Willebrand factor (vWF) multimers are crucial for primary hemostasis. Increased shear stress from ventricular assist devices can provoke premature degradation of HMW vWF multimers. Whether similar loss of vWF multimers occurs during extracorporeal membrane oxygenation (ECMO) is not clear.. We conducted a prospective observational study in a clinical cohort of patients who required ECMO for intractable cardiac and/or respiratory failure. The primary end point was the quantity and quality of HMW vWF multimer bands before, during, and after ECMO support. To investigate further changes in primary hemostasis, we also measured vWF antigen activity (vWF:Ag), vWF ristocetin cofactor activity (vWF:RCo), and factor VIII in 38 patients who required ECMO support before initiation of ECMO (baseline), after 24 and 48 hours on ECMO, and 24 hours after termination of ECMO therapy.. Compared with baseline, vWF:Ag and vWF:RCo decreased after 24 hours of ECMO (mean ± SD, vWF:Ag, 307% ± 152% to 261% ± 138%, P = 0.002; vWF:RCo 282% ± 145% to 157% ± 103%, P < 0.0001) and remained lower during ongoing support (vWF:Ag 265% ± 128%, P = 0.025; vWF:RCo 163% ± 94%, P < 0.0001). After termination of ECMO, vWF:Ag was greater than baseline (359% ± 131%, P = 0.004) and vWF:RCo was similar to baseline levels (338% ± 142%, P = 0.046). Compared with baseline, the calculated vWF:RCo/vWF:Ag ratio decreased after 24 hours on support (0.96 ± 0.23 to 0.61 ± 0.17, P ≤ 0.0001) and remained lower during 48 hours on ECMO (0.63 ± 0.18, P ≤ 0.0001). After termination of ECMO support (0.94 ± 0.19, P = 0.437), values rapidly returned to baseline. The number of HMW vWF multimers (n) decreased from baseline after 24 hours on ECMO (21 ± 1.4 to 14 ± 1.8, P ≤ 0.0001) and after 48 hours on ECMO (15 ± 2.1, P ≤ 0.0001). Twenty-four hours after termination of ECMO support, HMW vWF multimeric pattern had returned to baseline values (21 ± 1.8, P = 0.551).. Loss of HMW vWF multimer bands occurred in patients undergoing ECMO support and resolved after the termination of ECMO. Although not detectable with coagulation screening tests, a vWF:RCo/vWF:Ag ratio <0.7 during ECMO was highly indicative for loss of HMW vWF multimers. Our findings may at least in part explain increased bleeding tendency during ECMO therapy. Administration of vWF concentrates may support restoration of primary hemostasis in patients with relevant bleeding during ECMO support.

Topics: Adult; Aged; Blood Coagulation; Extracorporeal Membrane Oxygenation; Female; Hemorrhage; Hemostasis; Humans; Male; Middle Aged; Molecular Weight; Oxygen; Prospective Studies; Ristocetin; Shear Strength; Treatment Outcome; von Willebrand Factor

This study evaluated platelet function for an extended period of time in patients with a HeartMate II continuous-flow left ventricular assist device (Thoratec Corporation, Pleasanton, Calif) with light transmission aggregometry and investigated the potential role of this test in clinical management.. Twenty-four patients were studied prospectively after implantation. Mean duration of support was 8.5 months. Platelet functions were assessed with light transmission aggregometry induced by thrombin receptor agonist peptide, ristocetin, or arachidonic acid. All patients received an aspirin regimen that was progressively increased until arachidonic acid-triggered platelet aggregation dropped lower than 20%. Plasma levels of von Willebrand factor were also determined when ristocetin-induced platelet agglutination was impaired.. Intensity of platelet aggregation with thrombin receptor agonist peptide was little changed in patients with a HeartMate II relative to control subjects. Aspirin dose greater than 160 mg/d was progressively required in 46% of patients. Ristocetin-induced platelet agglutination was impaired in 4 patients in association with a lack of high molecular weight von Willebrand factor multimers. Three patients had thromboembolic events (12.5%) and 8 (33%) suffered from major bleeding complications.. High platelet reactivity during treatment with aspirin is common in patients with a HeartMate II. Moreover, light transmission aggregometry may detect impaired ristocetin-induced platelet agglutination, enabling dosage of aspirin to be adjusted. Our strategy showed no major improvements in terms of thrombosis rate when compared with published data, although bleeding frequency was somewhat reduced. Benefits of light transmission aggregometry testing need to be assessed in a larger randomized study with a longer follow-up.

Topics: Aged; Arachidonic Acid; Aspirin; Drug Administration Schedule; Drug Monitoring; Female; France; Heart Failure; Heart-Assist Devices; Hemorrhage; Humans; Male; Middle Aged; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Function Tests; Predictive Value of Tests; Prospective Studies; Prosthesis Design; Risk Factors; Ristocetin; Thromboembolism; Time Factors; Treatment Outcome; Ventricular Function, Left

Bernard-Soulier syndrome is a severe bleeding disease due to a defect of GPIb/IX/V, a platelet complex that binds the von Willebrand factor. Due to the rarity of the disease, there are reports only on a few cases compromising any attempt to establish correlations between genotype and phenotype. In order to identify any associations, we describe the largest case series ever reported, which was evaluated systematically at the same center.. Thirteen patients with the disease and seven obligate carriers were enrolled. We collected clinical aspects and determined platelet features, including number and size, expression of membrane glycoproteins, and ristocetin induced platelet aggregation. Mutations were identified by direct sequencing of the GP1BA, GP1BB, and GP9 genes and their effect was shown by molecular modeling analyses.. Patients all had a moderate thrombocytopenia with giant platelets and a bleeding tendency whose severity varied among individuals. Consistent with expression levels of GPIbα always lower than 10% of control values, platelet aggregation was absent or severely reduced. Homozygous mutations were identified in the GP1BA, GP1BB and GP9 genes; six were novel alterations expected to destabilize the conformation of the respective protein. Except for obligate carriers of a GP9 mutation with a reduced GPIb/IX/V expression and defective aggregation, all the other carriers had no obvious anomalies.. Regardless of mutations identified, the patients' bleeding diathesis did not correlate with thrombocytopenia, which was always moderate, and platelet GPIbα expression, which was always severely impaired. Obligate carriers had features similar to controls though their GPIb/IX/V expression showed discrepancies. Aware of the limitations of our cohort, we cannot define any correlations. However, further investigations should be encouraged to better understand the causes of this rare and underestimated disease.

Topics: Adolescent; Adult; Amino Acid Sequence; Bernard-Soulier Syndrome; Blood Platelets; Cell Shape; Child; Child, Preschool; Female; Genetic Association Studies; Genetic Markers; Hemorrhage; Homozygote; Humans; Italy; Male; Membrane Glycoproteins; Middle Aged; Molecular Sequence Data; Platelet Aggregation; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Point Mutation; Polymerase Chain Reaction; Ristocetin; Thrombocytopenia; von Willebrand Factor; Young Adult

Many serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to microgravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of -8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibalpha surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions.

Topics: Adenosine Diphosphate; Animals; Blood Platelets; Collagen; Cytoskeleton; Hemorrhage; Hemostasis; Hindlimb Suspension; Humans; Hypergravity; Membrane Glycoproteins; Membrane Proteins; Mice; Models, Animal; P-Selectin; Platelet Adhesiveness; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Ristocetin; Space Flight; Thrombosis; Time Factors; von Willebrand Factor; Weightlessness Simulation

This study investigated the influence of the mechanical blood pump HeartMate II (HMII) (Thoratec Corporation, Pleasanton, California) on blood coagulation and platelet function.. HMII is an implantable left ventricular assist device used for the treatment of heart failure. Patients treated with HMII have increased bleeding tendencies.. We measured agonist-induced platelet aggregation in 16 patients on HMII support.. The von Willebrand factor (vWF)-dependent ristocetin-induced platelet aggregation was impaired in 11 of the 16 patients, of which 12 had experienced at least 1 minor or major bleeding episode. The impaired ristocetin-induced platelet aggregation was associated both with decreased specific activity of plasma vWF, presumably due to lack of high molecular weight vWF multimers, as well as with attenuated function of the platelets themselves.. The results imply that HMII treatment is associated with impaired platelet aggregation, which may contribute to an increased tendency to bleed.

Topics: Adult; Anti-Bacterial Agents; Blood Platelets; Female; Heart Failure; Heart-Assist Devices; Hemorrhage; Humans; International Normalized Ratio; Male; Middle Aged; Platelet Aggregation; Platelet Aggregation Inhibitors; Ristocetin; Stroke Volume; Ventricular Dysfunction, Left; Ventricular Function, Left; von Willebrand Factor; Young Adult

The Fab-fragment of 6B4, a murine monoclonal antibody targeting the human platelet glycoprotein (GP) Ibalpha and blocking the binding of von Willebrand factor (VWF), is a powerful antithrombotic. In baboons, this was without side effects such as bleeding or thrombocytopenia. Recently, we developed a fully recombinant and humanized version of 6B4-Fab-fragment, h6B4-Fab, which maintains its inhibitory capacities in vitro and ex vivo after injection in baboons. We here investigated the antithrombotic properties, the effect on bleeding time and blood loss and initial pharmacokinetics of h6B4-Fab in baboons. The antithrombotic effect of h6B4-Fab on acute platelet-mediated thrombosis was studied in baboons where thrombus formation is induced at an injured and stenosed site of the femoral artery, allowing for cyclic flow reductions (CFRs) which are measured on an extracorporeal femoral arteriovenous shunt. Injection of 0.5 mg/kg h6B4-Fab significantly reduced the CFRs by 80%, whereas two extra injections, resulting in cumulative doses of 1.5 and 2.5 mg/kg, completely inhibited the CFRs. Platelet receptor occupancy, plasma concentrations and effects ex vivo were consistent with what was previously observed. Finally, minimal effects on bleeding time and blood loss, no spontaneous bleeding and no thrombocytopenia were observed. We therefore conclude that h6B4-Fab maintains the antithrombotic capacities of the murine 6B4-Fab, without causing side effects and therefore can be used for further development.

Topics: Acute Disease; Animals; Anti-Bacterial Agents; Bleeding Time; Blood Platelets; Constriction, Pathologic; Disease Models, Animal; Femoral Artery; Hemorrhage; Humans; Immunoglobulin Fab Fragments; Mice; Papio; Platelet Adhesiveness; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Regional Blood Flow; Ristocetin; Stress, Mechanical; Thrombocytopenia; Thrombosis

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

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

Velocardiofacial syndrome (VCFS) is a common, phenotypically heterogeneous developmental disorder caused by an interstitial microdeletion within human chromosome 22q11. The deleted chromosomal region in >90% of VCFS patients includes the GPIb beta gene, encoding for one subunit of the platelet GPIb-V-IX receptor, which is critical for platelet adhesion under shear, and important in aggregation and thrombin-mediated activation. Complete loss of GPIb-V-IX due to autosomal recessive inheritance of two GPIb alpha, Ib beta or GP9 gene mutations, results in a severe bleeding disorder, Bernard-Soulier syndrome (BSS). In this study, twenty-one confirmedVCFS patients were analyzed for platelet morphological and functional alterations, resulting from the heterozygous loss of one GPIb beta gene allele. Compared to unaffected family members, VCFS patients showed a significant decrease in platelet count; VCFS platelet size and mean platelet volume were increased, but not as markedly as in BSS. As expected from obligatory heterozygotes for GPIb beta deficiency, VCFS patients showed reduced platelet GPIb-V-IX surface expression and total GPIb content, but with considerable variation between cases. Platelet function tested using the PFA-100 trade mark analyzer was impaired in 70% of patients. Platelet aggregation was reduced in response to a GPIb-dependent agonist, ristocetin, in 50% of VCFS patients, with 35% showing a reduced response to thrombin receptor activating peptide. Genomic screening was performed to exclude mutations of the subunit genes, indicating that these platelet abnormalities were due to GPIb beta heterozygosity and not spontaneous BSS. In conclusion, many VCFS patients have in-vitro defects in platelet function that may increase their risk of bleeding during surgery.

Topics: Blotting, Western; DiGeorge Syndrome; DNA Mutational Analysis; Flow Cytometry; Genotype; Hemorrhage; Humans; Loss of Heterozygosity; Peptide Fragments; Phenotype; Platelet Aggregation; Platelet Count; Platelet Function Tests; Platelet Glycoprotein GPIb-IX Complex; Ristocetin

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

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

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

Widespread hemorrhagic manifestations commonly occur in patients with severe heat stroke. The pathogenesis of hemostatic disorders in these patients is not fully understood, although it is believed to be multifactorial in origin. The present investigation was designed to study the changes in blood platelets caused by heat stress in an experimental model of five merino sheep. The experiments were performed in two groups of five merino sheep each. In one group the sheep were subjected to a combination of heat (elevated environmental temperature) and exertional stress, and allowed to proceed throughout the experiment until a state of near collapse was reached (Task A). In the other group (Task B) the animals were heated in the same manner as those in Task A and also subjected to exertional heat; however, when the temperature reached 43.6 +/- 0.2 degrees C, the critical core temperature (CCT), they were subjected to evaporative cooling in a climatic chamber. Serial changes in the platelet counts and platelet functions were measured throughout the duration of the experiments. At the core temperature (CT) of 42.1 degrees C and above there was a significant impairment of adhesion of platelets to glass beads. During the early phases of elevation of CT, platelets showed hyperaggregation in the presence of different agonists (such as, collagen, ADP, ristocetin); this was followed by hypoaggregation when the CCT was raised above 43.6 +/- 0.2 degrees C. However, these impairments of platelet functions occurring at elevated CT and CCT were found to reverse to normal within 24 hours after the animals were cooled to 39 degrees C. It was also found that the hyperaggregation of platelets to different agonists induced by raised CT could be partially prevented by prior in vitro treatment of platelets with apyrase, a known enzyme destroying of ADP. The results of these experiments indicate that heat stress induced by exposing merino sheep to elevated controlled temperature directly activates the platelets. This may be an important contributing factor in causing altered hemostasis in heat stroke activated directly by heat. This mechanism may be operating in altered hemostasis in heat stroke.

Topics: Animals; Apyrase; Arachidonic Acid; Blood Platelets; Collagen; Heat Stress Disorders; Hemorrhage; Platelet Aggregation; Platelet Count; Platelet Function Tests; Ristocetin; Sheep

1. A murine anti-human vWF monoclonal antibody, AJvW-2, was developed that inhibited the interaction between platelet glycoprotein Ib (GPIb) and von Willebrand factor (vWF) during the ristocetin- (IC50 = 0.7 +/- 0.1 microgram ml-1) and botrocetin- (IC50 = 1.8 +/- 0.3 microgram ml-1) induced aggregation of human platelets. 2. AJvW-2 inhibited the high shear stress (10.8 N m-2) induced aggregation of human platelets dose-dependently with an IC50 = 2.4 +/- 0.3 micrograms ml-1, but had no effect on low shear stress induced platelet aggregation (1.2 N m-2) up to 100 micrograms ml-1. 3. AJvW-2 also inhibited the high shear stress (5.0 N m-2) induced adhesion of human platelets to collagen I with the same efficacy (IC50 = 2.4 +/- 0.3 micrograms ml-1), but no effect at low shear conditions (1.5 N m-2). 4. AJvW-2 inhibited the botrocetin-induced aggregation of platelets from guinea-pig, rat, rabbit, dog and pig at the same concentration range as human platelets; it likewise also inhibited the high shear stress induced aggregation and adhesion to collagen I of guinea-pig platelets. 5. AJvW-2 prevented arterial thrombus formation in guinea-pigs at a dose of 100 micrograms kg-1 without prolonging the template bleeding time, whereas the GPIIb/IIIa antagonists lamifiban mediated inhibition of thrombosis at 1000 micrograms kg-1 was accompanied by a significant prolongation of the bleeding time. 6. These results suggest that AJvW-2 is a potent inhibitor of the GPIb-vWF interaction and a potential novel antithrombotic agent with lower bleeding risk than GPIIb/IIIa antagonists.

Topics: Animals; Anti-Bacterial Agents; Antibodies, Monoclonal; Bleeding Time; Blood Coagulation; Collagen; Crotalid Venoms; Dogs; Fibrinolytic Agents; Guinea Pigs; Hemorrhage; Humans; Mice; Mice, Inbred BALB C; Platelet Adhesiveness; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Rabbits; Rats; Ristocetin; Thrombosis; von Willebrand Factor

The whole venom of Pseudechis papuanus, in addition to its anticoagulant activity, powerfully inhibited platelet aggregation induced by ADP, adrenaline, collagen, ristocetin and thrombin. High levels of phospholipase A2 (PLA2) activity were detected. A mild procoagulant activity was also observed. Following exposure of platelets to P. papuanus venom, platelet factor 3 (procoagulant platelet phospholipid) showed decreased cofactor activity in factor X activation by Russell's viper, venom suggesting that the venom PLA2 plays a major role in the inhibition of the coagulation mechanism. In vivo rodent assays confirmed the inhibitory effect on platelets and the haemorrhagic and neurotoxic activities. It is possible that PLA2 is responsible for anticoagulation and that this, combined with the effect on platelet aggregation, a mild procoagulant and a moderately potent haemorrhagin, is responsible for the haemorrhagic diathesis observed in systemically envenomed patients. Polyvalent (Australia-Papua New Guinea) Commonwealth Serum Laboratories antivenom, currently used for clinical treatment of snakebite in Papua New Guinea, proved highly effective against P. papuanus venom in rodent and in vitro assays, despite the absence of this particular venom from the immunising mixture.

Topics: Adenosine Diphosphate; Animals; Anticoagulants; Blood Platelets; Collagen; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Epinephrine; Factor X; Hemorrhage; Homeostasis; Humans; Injections, Intravenous; Injections, Subcutaneous; Male; Mice; Phospholipases A; Phospholipases A2; Platelet Aggregation Inhibitors; Platelet Factor 3; Rats; Rats, Sprague-Dawley; Ristocetin; Snake Venoms; Thrombin

In 20 patients with acute myocardial infarction (AMI) treated with streptokinase (SK, n = 7), recombinant single-chain tissue plasminogen activator (rt-PA, n = 7) or urokinase (UK, n = 6), the behavior of plasma von Willebrand factor (vWF) was studied before and 1.5, 3, 24, 48, and 72 hours after beginning thrombolytic therapy. vWF antigen (vWF:Ag) was high in plasma, especially after SK. The ristocetin cofactor (RiCof) activity of vWF, high before therapy, tended to decrease soon after therapy. This pattern of vWF changes was paralleled by the early loss of higher molecular weight multimers. By immunoblotting of immunopurified and reduced vWF and monoclonal antibody epitope mapping, we found that vWF was degraded after thrombolysis, especially after SK, as indicated by the higher values of two plasmin-generated fragments of 176 and 145 Kd. There were more plasmin-generated fragments in the five patients who had bleeding complications than in the remaining 15 who did not. In conclusion, quantitative and qualitative changes of vWF compatible with proteolytic degradation of the protein occur during thrombolytic therapy. Such degradation, roughly proportional to the degree of the general lytic state induced by each agent, might be a cofactor of the bleeding complications occurring in treated patients.

Topics: Fibrinolysin; Hemorrhage; Humans; Immunoblotting; Myocardial Infarction; Peptide Fragments; Recombinant Proteins; Ristocetin; Streptokinase; Thrombolytic Therapy; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator; 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

Topics: Adult; Albinism; Factor VIII; Female; Hemorrhage; Hemorrhagic Disorders; Humans; Platelet Aggregation; Postoperative Complications; Ristocetin; Syndrome

A mild hemorrhagic tendency was observed in a group of beta-thalassemia major patients. This included easy bruising and frequent epistaxis. A consistent platelet anomaly manifested by diminished platelet aggregation to ADP, collagen, ristocetin and epinephrine was found in these patients, and could be responsible in part for the hemorrhagic phenomena.

Topics: Adenosine Diphosphate; Adolescent; Adult; Blood Coagulation Tests; Child; Child, Preschool; Collagen; Epinephrine; Female; Hemorrhage; Humans; Male; Platelet Aggregation; Ristocetin; Thalassemia

Topics: Anemia, Hemolytic; Hemorrhage; Humans; Infant; Male; Ristocetin

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