ristocetin and Purpura--Thrombocytopenic

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

We investigated the characteristics of the antiplatelet autoantibodies in 60 patients with ITP. Using flow cytometry, the binding of monoclonal antibodies to the platelet glycoprotein (GP) IIb/IIIa complex and to GPIb was examined in these patients. The extent of binding was decreased in 15 patients (anti-GPIIb/IIIa in 12 patients and both anti-GPIIb/IIIa and anti-GPIb in 3 patients). Western blotting revealed that 10 of these 15 patients had either anti-GPIIb or anti-GPIIIa and 2 had anti-GPIb autoantibodies, ADP-induced aggregation of normal platelets was inhibited by autoantibodies in 12 of 60 patients, and 11 of these had anti-GPIIb/IIIa antibodies. Ristocetin-induced aggregation was inhibited in 4 of these patients, and 2 with prominent inhibition had anti-GPIb antibodies. There was a significant relationship between platelet-associated IgG value and ATP secretion. These results suggest that some antiplatelet autoantibodies can affect platelet function and thus have an influence on the pathophysiology of ITP.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adolescent; Adult; Aged; Aged, 80 and over; Autoantibodies; Blood Platelets; Blotting, Western; Child; Collagen; Female; Flow Cytometry; Humans; Male; Middle Aged; Platelet Aggregation; Platelet Membrane Glycoproteins; Purpura, Thrombocytopenic; Ristocetin

Von Willebrand factor (vWF) has been implicated to function as a cofactor in platelet aggregation induced by thrombotic thrombocytopenic purpura (TTP) plasma. To investigate further this role of vWF, we have used rabbit monospecific anti-FVIII/vWF antibodies and a monoclonal antibody to platelet glycoprotein Ib (GP Ib) that blocks the ristocetin-induced platelet aggregation. The monoclonal anti-platelet GP Ib antibody inhibited the platelet aggregation induced by ristocetin in the presence of normal plasma, but not that by any of the five TTP plasma samples. The TTP plasma samples from five patients were incubated with the monospecific antibodies to FVIII/vWF. In all of the samples, the FVIII/vWF:Ag was drastically reduced; however, there was almost no effect on the platelet-aggregating activity. Therefore, it is concluded that vWF is unlikely to play a major role in platelet aggregation induced by majority of TTP plasmas and that the site of platelet GP Ib, to which vWF binds in the presence of ristocetin, is not involved in TTP plasma-induced aggregation.

Topics: Antibodies, Monoclonal; Antibody Specificity; Humans; Platelet Aggregation; Purpura, Thrombocytopenic; Ristocetin; von Willebrand Factor

The case of a patient with thrombocytopenia and giant platelets is presented. Platelet function tests including bleeding time, aggregation, availability of PF3, and serotonin uptake and release were abnormal. A bone marrow aspirate showed megakaryocytes that while not increased in number, were young in appearance. Electron microscopy of platelets revealed a dilated surface-connecting canalicular system and a prominent membrane complex. No similar findings were encountered among the patient's relatives. The various diagnostic possibilities in this patient are discussed.

Topics: Adenosine Diphosphate; Adolescent; Autoantibodies; Blood Coagulation Tests; Blood Platelets; Collagen; Epinephrine; Female; Humans; Platelet Aggregation; Purpura, Thrombocytopenic; Ristocetin

Human Factor VIII desialylated by treatment with Vibrio cholerae neuraminidase (ASVIII) aggregated human platelets in the absence of ristocetin in platelet-rich plasma and, to a lesser extent, in washed platelet suspensions. Aggregation is accompanied by thromboxane formation and is completely inhibited by EDTA. Aspirin blocks the second phase of aggregation and abolishes thromboxane production. Subaggregating doses of ASVIII and of either ADP, epinephrine, or collagen produce prompt and complete platelet aggregation. Bernard-Soulier syndrome platelets either did not aggregate with ASVIII (Two cases) or showed markedly decreased aggregation (one cases). Factor VIII complex was prepared from the plasma of two patients with variant von Willebrand's disease (sialic acid content 142 and 75 nmol/mg, respectively); neither protein generated platelet-aggregating activity upon desialylation. [3H]ASVIII binds rapidly to platelets and 37 degrees C, while tritiated, fully sialylated factor VIII binds to a negligible extent. As little as 1--2 micrograms ASVIII bound/10(9) platelets is capable of inducing platelet aggregation. ASVIII may be a useful tool for investigating platelet-Factor VIII interactions in the absence of ristocetin. Furthermore, desialylated Factor VIII might play a physiologic role in Factor VIII-mediated platelet reactions in vivo.

Topics: Asialoglycoproteins; Blood Platelets; Factor VIII; Humans; Platelet Aggregation; Protein Binding; Purpura, Thrombocytopenic; Ristocetin; Sialic Acids; Structure-Activity Relationship; Syndrome

An oscillatory pattern of platelet agglutination-disagglutination in response to Ristocetin (R) at narrow concentration ranges was observed in citrated platelet rich plasma (PRP) of 10 patients with Glanzmann's thrombasthenia. The cyclic pattern decreased in intensity over time, was reproducible, and was not pH dependent. Formalin-fixed thrombasthenic platelets agglutinated with R but did not show a cyclic pattern. Incubation with 2.5 microM ADP inhibited R oscillation response, but small increases in R dose overcame this inhibition. The addition of ATP or creatine phosphate/creatine phosphokinase to thrombasthenic platelets inhibited by ADP restored the R oscillation response. In the platelets of a single patient, intracellular levels of ADP and ATP were shown to diminish during an oscillation response to R. There was an increase in AMP levels during the same period of time. The changes in these three intracellular nucleotides were gradual over time and did not vary with phases of the oscillation. Acetyl salicylic acid (ASA), at concentrations shown to block cyclooxygenase activity in control platelets, enabled thrombasthenic platelets to respond to R with full agglutination without oscillations. Lower concentrations of ASA in the PRP gave a return of the oscillation response. Our data suggest that the disagglutination phase of the R response of thrombasthenic platelets is not a function of the known glycoprotein membrane defect, but depends on materials originating in the platelet whose release is blocked by ASA.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adolescent; Adult; Aspirin; Child; Child, Preschool; Creatine Kinase; Dose-Response Relationship, Drug; Female; Humans; Infant; Male; Periodicity; Phosphocreatine; Platelet Aggregation; Purpura, Thrombocytopenic; Ristocetin; Time Factors

Ristocetin will induce the agglutination of platelets in the presence of von Willebrand factor. In previous studies, an electrostatic mechanism was proposed for this phenomenon wherein first the platelet's surface charge is reduced by the binding of ristocetin and then the von Willebrand factor acts as a bridge between platelets. To test this hypothesis, the effects of ristocetin and von Willebrand factor, singly and together, on the electrophoretic mobility of normal, trypsinized, and Bernard-Soulier platelets was measured. Ristocetin alone, at concentrations of 0.5 mg/ml or more, produced a statistically significant reduction in the electrophoretic mobility of fresh or fixed platelets. Control experiments showed that the reduction was not due to changes in the ionic milieu of the solution. Therefore, the decrease in platelet mobility is evidence for binding of ristocetin to the platelet surface. Bernard-Soulier and trypsinized platelets also had reductions in mobility with ristocetin, suggesting that ristocetin binds to the platelet at sites other than the binding site for von Willebrand factor. The presence of plasma from a patient with von Willebrand's disease did not alter the reduction in mobility of normal platelets by ristocetin. However, the reduction was markedly enhanced in the presence of normal plasma. This enhancement did not occur with Bernard-Soulier platelets and was inhibited by anti-Factor VIII/von Willebrand factor antiserum or trypsinization of the platelets. Thus, the enhanced reduction appears to be associated with the binding of von Willebrand factor to the platelet surface. These studies indicate that platelets undergo two changes with ristocetin and von Willebrand factor, both of which facilitate agglutination: reduction in net surface charge and binding of von Willebrand factor, a large molecule which can serve as a bridge between platelets. In parallel studies, bovine von Willebrand factor, without ristocetin, agglutinated and reduced the electrophoretic mobility of normal but not Bernard-Soulier or trypsinized platelets; this indicates a similar mechanism of agglutination.

Topics: Antibodies; Binding Sites; Blood Coagulation Factors; Blood Platelets; Electrophoresis; Humans; Movement; Purpura, Thrombocytopenic; Ristocetin; Syndrome; von Willebrand Factor

Topics: Adenosine Diphosphate; Blood Platelets; Factor VIII; Humans; In Vitro Techniques; Platelet Aggregation; Purpura, Thrombocytopenic; Ristocetin; Trypsin; von Willebrand Factor

Topics: Adenosine Diphosphate; Antigen-Antibody Reactions; Aorta; Blood Platelets; Endothelium; Factor VIII; Glycoproteins; Humans; Isoantibodies; Platelet Adhesiveness; Platelet Aggregation; Purpura, Thrombocytopenic; Ristocetin; Syndrome

Topics: Adenosine Diphosphate; Adolescent; Factor VIII; Female; Humans; Platelet Aggregation; Purpura, Thrombocytopenic; Ristocetin; Syndrome

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

Bernard-Soulier syndrome is a rare constitutional thrombopathy, the main clinical feature is a bleeding tendency which is variable. The long bleeding time, in spite of a normal or little decreased platelet count, has been recently explained: the giant platelets have a defective adhesion to subendothelium. This abnormal adhesion could be related with an abnormal pattern of platelet membrane glycoproteins. Interactions between vessel wall, von Willebrand's factor and platelets are discussed.

Topics: Adenosine Diphosphate; Blood Coagulation Factors; Blood Platelets; Cell Membrane; Cell Survival; Collagen; Factor VIII; Factor XI; Glycoproteins; Humans; Platelet Adhesiveness; Platelet Aggregation; Purpura, Thrombocytopenic; Ristocetin; Serotonin; Sialic Acids; Syndrome; Thromboplastin; von Willebrand Factor

Topics: Adolescent; Animals; Blood Cell Count; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Cattle; Chemical Precipitation; Child; Chromatography, Gel; Endothelium; Factor VIII; Fibrinogen; Hemostasis; Humans; Male; Platelet Adhesiveness; Purpura, Thrombocytopenic; Ristocetin; Syndrome

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

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

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