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At least 19 recordsLinked to original sources
PubMed1979

Studies on the mechanism of ristocetin-induced platelet agglutination: binding of ristocetin to platelets.

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

Alkylation↗
PubMed1977

Studies on the mechanism of ristocetin-induced platelet agglutination. Effects of structural modification of ristocetin and vancomycin.

The mechanism by which ristocetin induces platelet agglutination in the presence of the von Willebrand factor was studied by chemically altering ristocetin and a similar antibiotic, vancomycin, by reaction with a water-soluble carbodiimide in the presence of glycine methyl ester at pH 4.75. Altering ristocetin's phenolic groups (which are thought to be important in its peptide-binding properties) resulted in a loss of both platelet-agglutinating and antibiotic activities. Restoring the phenolic groups with hydroxylamine restored both activities. Vancomycin has antibiotic and peptide-binding properties similar to ristocetin's, but differs structurally in having a free carboxyl group and thus a less positive charge at neutral pH. It does not induce platelet agglutination and actually inhibits ristocetin-induced agglutination. Reacting vancomycin with the water-soluble carbodiimide resulted in alteration of phenolic groups and permanent conversion of the carboxyl to a neutral derivative. Restoring the phenolic groups with hydroxylamine (but leaving the carboxyl neutralized) produced a compound with charge properties similar to ristocetin's which induced platelet agglutination as ristocetin does. These data suggest both a binding requirement (mediated through phenolic groups) and a strong positive charge requirement for ristocetin-induced agglutination. The data are consistent with a model wherein positively charged ristocetin binds, via its phenolic groups, to sites on the platelet surface and reduces the platelet's negative charge. This could reduce the electrostatic repulsion between platelets and/or between platelets and the negatively charged von Willebrand factor, and permit the macromolecular von Willebrand factor to cause agglutination by bridging between platelets.

Carbodiimides↗
PubMed1978

The effects of ristocetin and von Willebrand factor on platelet electrophoretic mobility.

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.

Antibodies↗
PubMed1977

Inhibition of ristocetin-induced platelet agglutination by vancomycin.

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

Binding, Competitive↗
PubMed1978

Interaction of platelet membrane receptors with von Willebrand factor, ristocetin, and the Fc region of immunoglobulin G.

The agglutination of human platelets by ristocetin and von Willebrand factor was inhibited by aggregated immunoglobulin (Ig)G and by Fc fragments of IgG, but not by Fab, F(ab')(2) or pFc fragments of IgG. Because this inhibition occurred with formalin-fixed platelets as well as with normal platelets, a generalized aggregation of fluid membrane components by Fc fragments was not responsible for this inhibition of ristocetin and von Willebrand factor-induced agglutination. Reciprocal inhibition of platelet Fc receptors was produced by prior incubation of platelets with von Willebrand factor and ristocetin. Sucrose density gradient ultracentrifugation studies demonstrated that aggregated IgG did not form fluid-phase complexes with von Willebrand factor and ristocetin. Furthermore, passage of von Willebrand factor and ristocetin through a column of immobilized heat-aggregated IgG did not alter platelet agglutinating activity which indicates that aggregated IgG did not inactivate von Willebrand factor or ristocetin. Thus, it was likely that the IgG-mediated interference with platelet agglutination by ristocetin and von Willebrand factor did not occur in the fluid phase but at the platelet surface. These studies suggest that the platelet membrane Fc receptor may be either a part of, or sterically related to, the membrane glycoprotein I complex that interacts with von Willebrand factor, and that occupation of one of these surface components blocks the availability of the other.

Blood Coagulation Factors↗
PubMed1976

The pH dependence of quantitative ristocetin-induced platelet aggregation: theoretical and practical implications-a new device for maintenance of platelet-rich plasma pH.

Quantitative ristocetin-induced platelet aggregation of normal platelet-rich plasma (PRP) decreased with time after PRP preparation. An increase in p H of the PRP with time proved to be responsible for this finding. Diffusion of CO2from the plasma is the prime determinant of the change in pH. Since a complex combination of factors influences CO2 diffusion (surface area-to-volume relationship, capping, mixing, etc.) The change in pH is variable with time. Thus, quantitative ristocetin aggregation should be pH controlled. A simple device for maintaining PRP pH constant by control of the ambient pCO2 was designed and found effective in keeping both pH and quantitative ristocetin aggregation constant over a prolonged period of time. It can be adapted for use in platelet aggregation studies employing other reagents. The pH dependence of ristocetin-induced platelet aggregation is consistent with other data supporting an elctrostatic interaction between the platelet, von Willebrand factor, and ristocetin. We favor a model wherein ristocetin neutralizes some of the platelet's negative change and permits the von Willebrand factor to bridge sites on separate platelets to induce agglutination.

Blood Coagulation Tests↗
PubMed1978

Ristocetin precipitation test: a new simple test for detection of fibrin monomer and fibrin degradation products.

The ristocetin precipitation test was designed as a simplified test to detect fibrin monomers and fibrinogen/fibrin degradation products (FPD/fdp). The ristocetin precipitation test is positive in plasma samples containing either fibrin monomer (greater than 5--10 microgram/ml) or early fdp (greater than 50--100 microgram/ml). The ristocetin precipitation test is negative in plasma with fibrinogen concentrations to 1,000 mg/dl or fibrinogen degradation products FDP) and late fdp to 400 microgram/ml. The ristocetin precipitation test is positive in plasmas collected from rabbits after the infusion of thrombin (2.7 u/kg) or thrombin and streptokinase (10,000 u/kg); the test is negative in plasmas from animals treated with streptokinase or saline solution alone. The ristocetin precipitation test is negative in normal human plasmas and plasmas from patients who have primary firbinogenolysis, but positive in plasmas from patients with disseminated intravascular coagulation. These results suggest that the restocetin precipitation test can be a useful test for the detection of plasma fibrin monomers and early fdp.

Animals↗
PubMedDate not supplied

Precipitation of fibrin monomers and fibrin degradation products by ristocetin.

Ristocetin, at relatively low concentrations (1.0 mg/ml-1.5 mg/ml), can selectively precipitate fibrin monomers and fibrin degradation products (fdp) from plasma without effect on fibrinogen or fibrinogen degradation products (FDP). 125I-labeled fibrin monomers and fibrin degradation products were precipitated by ristocetin when their plasma concentrations were greater than 0.25 microgram/ml and 50 microgram/ml, respectively. In order to obtain a visible precipitated, 2 microgram/ml of fibrin monomers of 50 to 100 microgram/ml of fibrin degradation products were necessary. These effects were optimally observed under the following conditions: (1) temperature, 20 C to 37 C; (2) pH, 7.0 to 7.5; and (3) incubation time, 15 to 60 minutes. Late-fibrin degradation products are approximately eight times less sensitive to ristocetin-induced precipitation than early-fibrin degradation products. Plasma medium is essential for the differentiation of fibrin monomers and fibrin degradation products from fibrinogen and fibrinogen degradation products by ristocetin. These results suggest that the specific detection of fibrin monomers and fibrin degradation products in plasma may be easily performed by ristocetin.

Anticoagulants↗
PubMed1975

Initial membrane reaction in the biosynthesis of peptidoglycan. Spin-labeled intermediates as receptors for vancomycin and ristocetin.

Phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla:undecaprenyl phosphate, phospho-MurNAc-pentapeptide transferase) catalyzes the initial membrane reaction in the biosynthesis of peptidoglycan. The spin-labeled nucleotide, UDP-MurNAc-Ala-DGlu-Lys (Nepsilon-2,2,5,5-tetramethyl-N-oxyl-pyrroline-3-carbonyl)-DAla-DAla, was used as a substrate by this enzyme for the synthesis of membrane-associated undecaprenyl-diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Tempyo)-DAla-DAla. The spin-labeled substrate and product complex with the antibiotics vancomycin and ristocetin. The association constants for the spin-labeled nucleotide are 6.2 times 10(5) and 6.2 times 10(4) M-1 for vancomycin and ristocetin, respectively. The association constants for the spin-labeled lipid intermediate are 3.0 times 10(4) and 2.1 times 10(4) M-1 for vancomycin and ristocetin, respectively. These results indicate that the acyl-DAla termini of membranes-associated spin-labeled undecaprenyl-diphosphate-MurNAc-pentapeptide are accessible to vancomycin and ristocetin and that the association constants are smaller than those determined for the corresponding antibiotic spin-labeled UDP-MurNAc-pentapeptide complexes.

Binding Sites↗
PubMed1979

Studies on the mechanism of ristocetin-induced platelet aggregation: binding of factor VIII to platelets.

The effect of ristocetin on the binding of [125I]factor VIII to platelets was studied. High and low affinity F.VIII binding sites exist on platelets. The high affinity sites bind 13 times more F.VIII than the low affinity sites. Ristocetin increased the binding of F.VIII to both types of binding sites by increasing the affinity of F.VIII for the platelet and increasing the total number of platelet binding sites. Chymotrypsin-treated platelets were not aggregated by ristocetin and F.VIII: these platelets have less of the major platelet membrane glycoproteins and bind much less [125I]F.VIII than do buffer-treated platelets with and without ristocetin.

Antigens↗
PubMed1978

Ristocetin and the thrombin clotting time.

The addition of the antibiotic ristocetin to plasma accelerated the thrombin clotting time (TCT) in 20 out of 22 subjects. Prior incubation of ristocetin with thrombin or plasma did not alter its effect on the TCT. Ristocetin accelerated clotting greatly at low but not at high levels of thrombin. A simple linear correlation between heparin concentrations and the TCT was demonstrated when ristocetin at 2.5 mg per ml was added to plasma containing between 0.05 and 0.5 unit of heparin per ml. There are implications for assay procedures involving heparin and the TCT.

Blood Coagulation Tests↗
PubMed1977

Immunoinhibition of ristocetin-induced platelet aggregation.

Human platelets washed and fixed in paraformaldehyde aggregate in the presence of the antibiotic ristocetin and normal plasma. This aggregation response is abolished after digestion of the fixed platelets with chymotrypsin. Antisera to fixed washed platelets were produced in rabbits and absorbed with chymotrypsin-treated, fixed washed platelets. Monovalent Fab fragments obtained from the isolated gamma-globulin fractions of the antisera blocked ristocetin-induced aggregation of fixed washed platelets in buffer and normal platelets in platelet-rich plasma. By double-antibody immunoprecipitation, it was shown that the antibody which blocked the ristocetin reaction interacted with a platelet membrane surface protein of mol wt 155,000. The results suggest that the glycoprotein I complex on the surface of the human platelet mediates ristocetin-induced von Willebrand factor-dependent platelet aggregation.

Antibodies↗
PubMed1976

Platelet-bound ristocetin aggregation factor in normal subjects and patients with von Willebrand's disease.

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

Blood Platelets↗
PubMed1975

Studies on the mechanism of ristocetin-induced platelet aggregation.

Adenine nucleotide metabolism and the release reaction were studied during ristocetin-induced platelet aggregation. Decreasing platelet ATP by incubation with metabolic poisons did not decrease ristocetin-induced aggregation. ADP and ATP were released from platelets during ristocetin-induced aggregation, and ATP was converted to hypoxanthine. However, these occurred after aggregation was almost complete. Aggregation was inhibited by p-choromercuribenzoic acid. By studying platelet suspensions, we were able to determine that this effect was on platelets and not on the plasma cofactor needed for aggregation. We postulate that ristocetin and its cofactor aggregate platelets by binding platelet membranes and that the platelet plays a passive role in this reaction.

Adenine Nucleotides↗
PubMed1979

Interaction of lectins with human platelets. Effects on platelet stimulation by thrombin and ristocetin.

Wheat germ agglutinin induced aggregation and secretion of fresh platelets. Aggregation, but not secretion of serotonin by platelets in plasma, by the lectin was inhibited by 5 mM EDTA. Further, the lectin-induced stimulation of fresh platelets was blocked by prostaglandin E1. Thus, this lectin stimulates platelets by a mechanism which closely mimics thrombin activation and is independent of intercellular crosslinking. Lentil lectin did not stimulate platelets. Each platelet contained about 6 . 10(-5) binding sites for the lectins with an apparent dissociation constant of 3.0 . 10(-7) M. Wheat germ agglutinin, which binds mainly to glycoprotein I (Mr 150 000), increased the subsequent binding of thrombin to fixed platelets while lentil lectin was without effect. It appears that thrombin and wheat germ agglutinin bind to independent but interacting sites. Wheat germ agglutinin, but neither thrombin nor lentil lectin, inhibited the agglutination of platelets by ristocetin. Further, rat platelets were not aggregated by either ristocetin or wheat germ agglutinin. It appears that the interaction sites of ristocetin and wheat germ agglutinin on platelets are overlapping.

Animals↗
PubMed1979

Re-evaluation of plasmas from patients previously diagnosed as having von Willebrand's disease with the factor VIII-related antigen and ristocetin cofactor assay.

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

Adolescent↗
PubMed1976

The effect of ionophore on platelet aggregation in von Willebrand's disease and in congenital afibrinogenemia. A comparison with ristocetin.

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

Afibrinogenemia↗