ristocetin and Thrombocytopenia

Topics: Binding Sites, Antibody; Blood Platelets; Cell Differentiation; Cell Migration Inhibition; Clot Retraction; Complement Fixation Tests; Gold; Heparin; Humans; Immunoglobulin G; Intradermal Tests; Platelet Aggregation; Platelet Factor 3; Prednisone; Quinidine; Ristocetin; Serotonin; Thrombocytopenia; Thromboembolism; Urea; Valproic Acid

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

Essentials The role of von Willebrand factor (VWF) domains in regulating platelet adhesion was studied in vivo. Multimeric VWF with spacers at the N- and C-terminus of VWF-A1 were systematically tested. N-terminal modified VWF avidly bound platelet GpIbα, causing VWD Type2B like phenotype in mice. Novel anti-D'D3 mAbs suggest that changes at the D'D3-A1 interface may be biologically relevant.. Background Previous ex vivo studies using truncated VWF (von Willebrand factor) suggest that domain-level molecular architecture may control platelet-GpIbα binding function. Objective We determined if this is the case with multimeric VWF in vivo. Methods Full-length human VWF ('hV') was modified with a 22-amino acid mucinous stretch at either the N-terminus of VWF-A1 to create 'hNV' or C-terminus to yield 'hCV'. This extends the physical distance between VWF-A1 and the adjacent domains by ~6 nm. Similar mucin inserts were also introduced into a human-murine chimera ('h[mA1]V') where murine-A1 replaced human-A1 in hV. This yielded 'h[mA1]NV' and 'h[mA1]CV', with N- and C-terminal inserts. The constructs were tested ex vivo and in vivo. Results Mucin insertion at the N-terminus, but not C-terminus, in both types of constructs resulted in >50-fold increase in binding to immobilized GpIbα. N-terminal insertion also resulted in greater shear-induced platelet activation, more thrombus formation on collagen, enhanced platelet accumulation and slower platelet translocation on immobilized VWF in microfluidics assays. Hydrodynamic injection-based expression of h[mA1]NV, but not h[mA1]V or h[mA1]CV, in VWF

Topics: Animals; Blood Platelets; DNA, Complementary; Female; HEK293 Cells; Hemostasis; Humans; Hydrodynamics; Male; Mice; Microfluidics; Mucins; Phenotype; Platelet Activation; Platelet Adhesiveness; Platelet Function Tests; Protein Binding; Protein Domains; Protein Folding; Ristocetin; Shear Strength; Thrombocytopenia; Thrombosis; von Willebrand Factor

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

We aimed here to elucidate the role of adhesive platelet ligands and endothelial involvement during the acute phase of Puumala hantavirus (PUUV) infection. Nineteen hospital-treated patients with serologically confirmed diagnosis of acute PUUV infection were included. Patient charts were reviewed for clinical and basic laboratory data. Plasma levels of von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), factor VIII (FVIII:C) and a disintegrin and metalloproteinase with a thrombospondin type 1 domain 13 (ADAMTS13) activities as well as fibrinogen and fibronectin were measured three times acutely and once during the recovery phase. VWF:Ag and VWF:RCo were nearly three-fold higher acutely compared with recovery (median 252 vs. 88%, and mean 267 vs. 98%, respectively; P<0.001 for both), whereas FVIII:C was only slightly elevated (median 118 vs. 88%, P=0.002) and remarkably failed to show association with VWF in the acute phase. ADAMTS13 activity and fibronectin concentration were lower in the acute compared with the recovery phase (median 56 vs. 63%, P=0.003, and median 221 vs. 330 μmol/l, P=0.001, respectively). Fibrinogen raised acutely (mean 5.0 vs. 3.3 g/l, P<0.001), negatively correlating with the platelet count (r=-0.468, P=0.043). Markedly upregulated fibrinogen and VWF together with decreased levels of ADAMTS13 activity and fibronectin were observed during acute PUUV infection. VWF and FVIII:C did not associate during the acute phase, whereas thrombocytopenia correlated negatively with fibrinogen. These findings imply several rearranged interactions between platelets and their ligands.

Topics: Acute-Phase Reaction; ADAM Proteins; ADAMTS13 Protein; Adult; Blood Coagulation Tests; Blood Platelets; Convalescence; Factor VIII; Fibrinogen; Fibronectins; Finland; Hemorrhagic Fever with Renal Syndrome; Humans; Immunochemistry; Ligands; Male; Middle Aged; Platelet Activation; Platelet Adhesiveness; Puumala virus; Ristocetin; Thrombocytopenia; von Willebrand Factor

Little is known about the role of platelets in relation to ischemia/reperfusion injury (IRI) of the liver graft especially in children. Thrombocyte function was prospectively analysed in 21 consecutive pediatric liver transplantation (pLT) patients by platelet aggregometry secondary to adenosine diphosphate (ADP), collagen, and the von Willebrand factor activator ristocetin (VWF:rco). Post-OP serum levels of ALT were used to divide patients into groups with high (highHD, n = 8) and low (lowHD, n = 13) hepatocellular damage. Clinically, highHD-patients showed impaired plasmatic coagulation and elevated serum bilirubin levels early after pLT when compared with lowHD-patients. Further, platelet counts markedly decreased between pre-OP and postreperfusion (postrep.) in the highHD group (P = 0.003) and did not recuperate by POD6. In lowHD individuals thrombocytopenia improved from both pre-OP (P < 0.05) and postrep. (P < 0.001) respectively towards POD6. Experimental thrombocyte testing revealed that before graft reperfusion only ADP-dependent platelet aggregation correlated with reperfusion injury, thrombocytopenia and early graft function. During the first 48 h after graft reperfusion, all inducers tested demonstrated elevated platelet aggregation levels in the highHD group. Our data suggest a possible role of platelets and their aggregative status in liver IRI subsequent to clinical pLT. Reperfusion-independent ADP-triggered platelet function may be a determinant for IRI in the pediatric hepatic graft recipient.

Topics: Adenosine Diphosphate; Adolescent; Adult; Child; Child, Preschool; Female; Humans; Liver Transplantation; Male; Middle Aged; Platelet Aggregation; Reperfusion Injury; Ristocetin; Thrombocytopenia; von Willebrand Factor

Von Willebrand disease (VWD)-type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

Topics: ADAMTS13 Protein; Amino Acid Substitution; Animals; Anti-Bacterial Agents; Bleeding Time; Blood Platelets; Disease Models, Animal; Half-Life; Humans; Metalloendopeptidases; Mice; Mice, Mutant Strains; Mutation, Missense; Platelet Aggregation; Protein Multimerization; Ristocetin; Severity of Illness Index; Thrombocytopenia; Thrombosis; von Willebrand Disease, Type 2; von Willebrand Factor

Light transmission platelet aggregation tests are important for diagnosing platelet function defects. However, uncertainties exist about the best procedures to determine aggregation reference intervals. We investigated methods for determining reference intervals for light transmission aggregation tests, using the % maximal aggregation values for prospectively collected data on healthy control samples. Reference intervals for samples tested at 250 x 10(9) platelets/l were determined by mean +/- 2 standard deviations and non-parametric analyses. To establish reference intervals for tests on thrombocytopenic subjects, regression analyses were used to estimate 95% confidence limits for % maximal aggregation, according to sample platelet counts, using data for control samples diluted to match the platelet count of undiluted thrombocytopenic patient platelet-rich plasma samples. For samples tested at 250 x 10(9) platelets/l, non-parametric analyses described 95% of data for healthy control samples better than mean +/- 2 standard deviations. For samples tested at lower counts, to match thrombocytopenic samples, the % maximal aggregation was influenced by platelet count and derived limits were wider at very low platelet counts for almost all agonists. With ristocetin, it proved feasible to test samples with very low platelet counts to exclude Bernard-Soulier syndrome and type 2B von Willebrand disease. Non-parametric analyses should be the preferred method to establish light transmission aggregation reference intervals for samples tested at normal platelet counts. The derived limits for thrombocytopenic samples provide guidance for evaluating thrombocytopenic platelet function disorders, including which agonists to test, based on the sample platelet count.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Arachidonic Acid; Blood Platelets; Cell Size; Collagen; Data Interpretation, Statistical; Epinephrine; Feasibility Studies; Humans; Light; Platelet Aggregation; Platelet Count; Platelet Function Tests; Prospective Studies; Reference Values; Ristocetin; Statistics, Nonparametric; Thrombocytopenia

Sebastian syndrome is characterized by enlarged platelets and Döhle-like body leukocyte inclusions. This syndrome is an MYH-9-related disease, a group that also includes May-Hegglin anomaly and Fechtner syndrome. The differential diagnosis of the MYH-9 diseases requires ultrastructural studies. Certain in vitro aggregation responses may be abnormal in these conditions.. A 6-month-old boy presented with macrothrombocytopenia but no overt bleeding tendency. Giant platelets and Döhle-like body leukocyte inclusions were present in blood smears from both the patient and his mother. Electron microscopy confirmed ultrastructural features consistent with Sebastian syndrome. Platelet aggregation studies were normal except for an impaired response to the agonist ristocetin.. In this patient peripheral blood analyses and platelet aggregation studies revealed disease features shared with the Bernard-Soulier syndrome, but this syndrome was excluded by cellsurface glycoprotein analysis.

Topics: Bernard-Soulier Syndrome; Blood Platelets; Child, Preschool; Diagnosis, Differential; Humans; Male; Microscopy, Electron; Platelet Aggregation; Ristocetin; Syndrome; Thrombocytopenia

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

Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37 degrees C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F(2) strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.

Topics: ADAM Proteins; ADAMTS13 Protein; Animals; Anthrax; Anti-Bacterial Agents; Bacterial Proteins; Blood Platelets; Cell Communication; Collagen; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelial Cells; Hemostasis; Humans; Leukopenia; Metalloendopeptidases; Metalloproteases; Mice; Plasma; Platelet Aggregation; Protein Binding; Protein Structure, Tertiary; Ristocetin; Spores, Bacterial; Substrate Specificity; Thrombocytopenia; Thrombosis; Time Factors; Urea; 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

To diagnose a patient with Bernard-Soulier syndrome (BSS) and investigate her gene abnormality.. Platelet size and structure were studied under light and electron microscopies. Platelet membrane glycoproteins (GP) were measured by flow cytometry. PCR and DNA sequencing were used to identify gene abnormality.. The patient had thrombocytopenia with giant platelets. Ristocetin-induced platelet agglutination was absent. GP I b/IX complex in the platelet membrane was significantly decreased, which was resulted from an Ala139 Thr substitution in the transmembrane domain of GPIX.. Ala139 Thr mutation of the GPIX gene in this patient is a novel missense mutation, which has not been reported in BSS.

Topics: Adult; Alanine; Bernard-Soulier Syndrome; Cell Size; Female; Humans; Membrane Proteins; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Point Mutation; Protein Structure, Tertiary; Ristocetin; Threonine; Thrombocytopenia

Spontaneous EDTA-independent cold platelet agglutination is a rare phenomenon that produces pseudothrombocytopenia when blood samples are analyzed in automated cell counters. We report a case of platelet cold agglutinins and an analysis by flow cytometry. A 49 year old woman presented with abnormal vaginal bleed secondary to uterine fibroids. Platelet clumping was observed in blood samples taken in EDTA-, heparin- and citrate-containing tubes. In flow cytometric tests, patient serum agglutinated 16% of normal platelets at 22 degrees C, and 7% of platelets after incubation at 37 degrees C; in contrast, 3% and < 1% of platelets were agglutinated at 22 and 37 degrees C, respectively, after incubation with normal serum. Minimal agglutination (< 10%) was observed with patient serum at a titre of 1:5 or at temperatures > 30 degrees C. After incubation at 4 degrees C, IgM antibody and C3 were increased on the patient's platelets; no significant amount of IgM or C3 was detected on normal platelets. The specificity of the platelet cold agglutinin was determined by competitive inhibition by monoclonal anti-CD41(GPIIbIIIa). Before the addition of monoclonal antibody, patient's serum agglutinated 16% of normal platelets at 22 degrees C; after addition of anti-CD41 only 2% of the platelets were agglutinated. This blocking effect was not observed with anti-CD42. The patient's platelets functioned normally as determined by CD62 and CD63 expression in response to thrombin, normal platelet aggregation in response to collagen, ADP, and ristocetin, and a normal template bleeding time. In summary, platelet agglutination by a platelet cold agglutinin was quantitated by flow cytometry, the responsible antibody was characterized as a low titre IgM with minimal activity > 30 degrees C, and competitive binding studies supported the GPIIbIIIa complex as the binding site for the antibody. Since the antibody did not affect platelet function, we believe that these patients will not suffer complications from their platelet cold agglutinin, but it could pose a problem under circumstances such as cardiac surgery with hypothermia.

Topics: Adenosine Diphosphate; Agglutinins; Antibody Specificity; Antigens, Human Platelet; Blood Platelets; Cold Temperature; Collagen; Cryoglobulins; Edetic Acid; False Positive Reactions; Female; Flow Cytometry; Humans; Immunoglobulin M; Leiomyomatosis; Middle Aged; Platelet Aggregation; Platelet Function Tests; Platelet Glycoprotein GPIIb-IIIa Complex; Ristocetin; Thrombocytopenia; Uterine Hemorrhage; Uterine Neoplasms

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

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

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

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

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

The Bernard-Soulier syndrome is an inherited bleeding disorder that is due to a deficiency in platelet glycoprotein Ib. Bernard-Soulier platelets fail to agglutinate in response to ristocetin despite normal levels of factor VIII:von Willebrand factor. We report a patient who developed severe refractory thrombocytopenia postsurgically while receiving procainamide therapy. Thrombocytopenia was immune mediated since the patient's platelets bore high levels of antiplatelet antibody. Radioimmunoprecipitation studies demonstrated that the autoantibodies had specificity for platelet glycoproteins Ib and V as well as platelet HLA. The patient's plasma as well as purified immunoglobulin G completely inhibited the ristocetin-induced aggregation of normal platelets but did not inhibit adenosine diphosphate-induced aggregation. The laboratory studies revealed that this patient suffered from antibody-mediated thrombocytopenia with unusual characteristics that we have called pseudo-Bernard-Soulier syndrome.

Topics: Aged; Antilymphocyte Serum; Autoantibodies; Bernard-Soulier Syndrome; Blood Platelet Disorders; Female; Humans; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; Thrombocytopenia

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

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

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

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

10 cases of pernicious anaemia are reported in which 6 had abnormal aggregation with epinephrine, 3 with fibrinogen, 2 with ADP, and 2 with ristocetin. 5 patients had thrombocytopenia and 3 of these had a prolongation of the bleeding time. These abnormalities were normalized after vitamin B12 treatment. After treatment, platelet size changed toward a smaller diameter, but platelet size, however, was not significantly different from the normal platelet size distribution.

Topics: Adenosine Diphosphate; Aged; Anemia, Pernicious; Blood Cell Count; Blood Coagulation Tests; Blood Platelet Disorders; Collagen; Epinephrine; Female; Fibrinogen; Hemoglobins; Humans; Male; Middle Aged; Platelet Aggregation; Ristocetin; Thrombocytopenia; Vitamin B 12

Direct-current transcatheter electrocoagulation in vitro produced substantial clots in thrombocytopenic blood in dogs. The splenic artery was occluded in 3 dogs following ristocetin-induced thrombocytopenia. This procedure may produce effective vessel occlusion in patients with platelet-poor blood.

Topics: Animals; Dogs; Electrocoagulation; Embolization, Therapeutic; In Vitro Techniques; Ristocetin; Splenic Artery; Thrombocytopenia

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: Adenosine Diphosphate; Aged; Blood Cell Count; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Platelets; Blood Proteins; Collagen; Electrophoresis; Female; Fibrinogen; Gastrointestinal Hemorrhage; Humans; Male; Microscopy, Electron; Middle Aged; Platelet Aggregation; Ristocetin; Sialic Acids; Stimulation, Chemical; Thrombocytopenia

Topics: Acute Disease; Adenosine Diphosphate; Blood Cell Count; Blood Coagulation Factors; Blood Coagulation Tests; Blood Platelet Disorders; Blood Platelets; Cell Survival; Epinephrine; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Phospholipids; Platelet Adhesiveness; Remission, Spontaneous; Ristocetin; Thrombocytopenia

Topics: Acetazolamide; Anticonvulsants; Arsphenamine; Chloramphenicol; Chlorothiazide; Colchicine; Gold; Hydantoins; Hypoglycemic Agents; Meprobamate; Perchlorates; Phenothiazines; Phenylbutazone; Pyrimethamine; Quinacrine; Quinidine; Quinine; Ristocetin; Streptomycin; Sulfonamides; Thrombocytopenia; Toxicology

Topics: Anti-Bacterial Agents; Blood Platelets; Humans; Ristocetin; Thrombocytopenia