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.     

Heparin-associated thrombocytopenia: observations on the mechanism of platelet aggregation

We investigated the mechanism of heparin-mediated platelet aggregation in 11 patients with heparin-associated thrombocytopenia. Severe thrombocytopenia (16,000 to 66,000 platelets/microliters) developed in each patient during heparin therapy, and platelet aggregation occurred in vitro when heparin was added to mixtures of patient plasma and normal platelet-rich plasma. In 10 patients, heparin-initiated platelet aggregation was inhibited by preincubation of mixtures of normal platelet-rich plasma and heparin-associated thrombocytopenia plasma with monoclonal antiglycoprotein Ib antibodies 6D1 or LJ-Ib1. Both antibodies are directed against the von Willebrand factor binding site on glycoprotein Ib and inhibit only ristocetin-induced platelet agglutination. Purified immunoglobulin G (IgG) from patients with heparin-associated thrombocytopenia also supported heparin-induced aggregation, but equivalent amounts of antigen-binding fragments [F(ab')2] did not. We also found that F(ab')2 of LJ-Lb1 did not inhibit heparin-induced platelet aggregation but retained inhibitory activity against ristocetin-induced platelet agglutination. The monoclonal antibody 3G6, directed against the alpha-chain of glycoprotein Ib but not inhibitory of ristocetin-induced platelet agglutination, had no effect on heparin-induced platelet aggregation. Antibodies to von Willebrand factor that inhibit ristocetin-induced platelet agglutination did not inhibit heparin-mediated platelet aggregation, but antibodies to glycoprotein IIb-IIIa blocked aggregation. These data suggest that platelet aggregation in heparin-associated thrombocytopenia may be initiated by an interaction between patient IgG, heparin, and the platelet surface. Platelet activation appears to be mediated by a platelet surface crystallizable fragment (Fc) receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of von Willebrand factor-induced platelet agglutination by ADP does not result from reduced binding of total von Willebrand factor or its larger multimers

Earlier experiments showed that platelet agglutination induced by von Willebrand factor (vWf) plus ristocetin was greatly diminished if adenosine diphosphate (ADP) was added first in the presence of ethylenediaminetetraacetic acid (to prevent aggregation). Platelets treated with ADP and then fixed also agglutinated less than control fixed platelets. The studies reported here demonstrate that ADP did not decrease ristocetin-induced binding of vWf whether binding was measured on suspended platelets with iodine 125-labeled vWf or on suspended or agglutinated platelets with the use of any of three 125I-labeled monoclonal antibodies that bind to vWf but that do not interfere with ristocetin-induced agglutination. Equal amounts of vWf were eluted from ristocetin/vWf-treated platelets when they were resuspended without ristocetin, whether or not the platelets had been exposed to ADP, and the vWf recovered in either case was composed only of large multimers. No evidence for an agglutination site other than glycoprotein Ib could be demonstrated by measuring agglutination of a mixture of platelets fixed after inhibition with antibody against glycoprotein Ib and platelets fixed after inhibition with ADP. We conclude that inhibition of agglutination by ADP must involve the way in which vWf is bound, because it does not result from a decreased amount or from a difference in multimer size of bound vWf.     

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.     

A myeloma paraprotein with specificity for platelet glycoprotein IIIa in a patient with a fatal bleeding disorder.

Impaired platelet aggregation, normal shape change, and agglutination and normal ATP secretion and thromboxane synthesis in response to high concentrations of thrombin or arachidonic acid were found in a patient with multiple myeloma and hemorrhagic tendency. The purified IgG1 kappa or its F(ab1)2 fragments induced similar changes when added in vitro to platelet-rich plasma from normal subjects. In addition, the paraprotein inhibited adhesion to glass microbeads, fibrin clot retraction, and binding of radiolabeled fibrinogen or von Willebrand factor to platelets exposed to thrombin or arachidonic acid without affecting intraplatelet levels of cAMP. The radiolabeled para-protein bound to an average of 35,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets from a patient with Glanzmann's thrombasthenia. Immunoprecipitation studies showed that the platelet antigen identified by the paraprotein was the glycoprotein IIIa. Furthermore, binding of radiolabeled prostaglandin E1 (PGE1) to resting platelets as well as binding of von Willebrand factor to platelets stimulated with ristocetin were entirely normal in the presence of patient's inhibitor. These studies indicate that bleeding occurring in dysproteinemia may be the result of a specific interaction of monoclonal paraproteins with platelets. In addition, our data support the concept that the interaction of fibrinogen and/or von Willebrand factor with the platelet glycoprotein IIb-IIIa complex is essential for effective hemostasis.     

Subcellular platelet factor VIII antigen and von Willebrand factor

Subcellular membrane and granule fractions derived from human platelets contain factor VIIII antigen and von Willebrand factor activity but not factor VII procoagulant activity. Circulating platelets constitute a significant reservoir of plasma factor VIII antigen, containing approximately 15% of the amount of factor VIII antigen present in platelet-poor plasma. The antibiotic ristocetin, which aggregates human platelets in the presence of von Willebrand factor, nonspecifically precipitates platelet membrane factor VIII antigen. Thus normal platelets contain surface-bound as well as internally stored von Willebrand factor, a protein synthesized by endothelial cells which is necessary for normal platelet function in vivo.     

O-linked carbohydrate of recombinant von Willebrand factor influences ristocetin-induced binding to platelet glycoprotein 1b.

By transfecting the full-length cDNA for human von Willebrand factor (vWf) into a line of Chinese hamster ovary cells with a defect in carbohydrate metabolism, we have prepared recombinant vWf specifically lacking O-linked carbohydrates. We have compared this under-glycosylated protein to fully glycosylated recombinant vWf with respect to several structural and binding properties. vWf deficient in O-linked glycans was synthesized, assembled into multimers, and secreted in an apparently normal manner and was not prone to degradation in the extracellular milieu. It did not differ from fully glycosylated vWf in ability to bind to heparin or to collagen type I but did interact less well with glycoprotein 1b on formalin-fixed platelets. This decreased interaction was evidenced in both a lessened overall binding to platelets and in diminished capacity to promote platelet agglutination, in the presence of ristocetin. In contrast, no difference was seen in platelet binding in the presence of botrocetin. These data indicate a possible role for O-linked carbohydrates in the vWf-glycoprotein 1b interaction promoted by ristocetin and suggest that abnormalities in carbohydrate modification might contribute to the altered ristocetin-dependent reactivity between vWf and platelets described for some variant forms of von Willebrand disease.     

Platelet membrane glycoproteins implicated in ristocetin-induced aggregation. Studies of the proteins on platelets from patients with Bernard-Soulier syndrome and von Willebrand''s disease.

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.     

Acquired Bernard-Soulier syndrome. Evidence for the role of a 210,000-molecular weight protein in the interaction of platelets with von Willebrand factor.

A patient with a lymphoproliferative disorder developed bleeding associated with a prolonged bleeding time and a selective defect of platelet aggregation in response to ristocetin. The patient's purified IgG was shown to inhibit aggregation of washed normal platelets by ristocetin and von Willebrand factor (F VIII:vWF). By Western blotting, it was shown that antibody bound specifically to an antigen of Mr 210,000 present on normal platelets but missing on platelets from patients with congenital Bernard-Soulier syndrome (BSS). Binding was effected by the F(ab)2 portion of the IgG, indicating the presence of an autoantibody rather than an immune complex. These results suggest that the 210,000-Mr protein is involved in the interaction of F VIII:vWF with platelets. Furthermore, we have demonstrated the apparent absence of an additional protein on congenital BSS platelets. Heat-aggregated IgG was also shown to bind to the 210,000-Mr protein, suggesting that this protein may function as an Fc receptor on platelets. The relationship of the 210,000-Mr protein to glycoprotein Ib and the precise role of this protein in the interaction of platelets with F VIII:vWF need to be characterized.     

Type IIB von Willebrand factor with normal sialic acid content induces platelet aggregation in the absence of ristocetin. Role of platelet activation, fibrinogen, and two distinct membrane receptors.

Three preparations of purified von Willebrand factor (vWF), obtained from unrelated patients affected by type IIB von Willebrand disease, were found to have normal sialic acid content (between 129 and 170 nmol/mg of vWF, as compared with 158 +/- 17 nmol/mg in four normal preparations) and to induce platelet aggregation in the presence of physiologic levels of divalent cations and without addition of ristocetin. A monoclonal antibody that blocks the vWF binding domain of the platelet glycoprotein (GP)Ib caused complete inhibition of IIB vWF-induced aggregation. In contrast, a monoclonal antibody that blocks the receptor for adhesive proteins on the platelet GPIIb/IIIa complex failed to inhibit the initial response of platelets to high concentrations of IIB vWF. Moreover, IIB vWF caused agglutination of formalin-fixed platelets that was blocked only by the anti-GPIb antibody, suggesting that the binding of vWF to GPIb, even in the absence of ristocetin, results in platelet-platelet interaction that is followed by exposure of the GPIIb/IIIa receptors for adhesive proteins. Endogenous ADP, normally active platelet metabolism and fibrinogen binding to GPIIb/IIIa were necessary for maximal and irreversible platelet aggregation. In the absence of fibrinogen, however, aggregation was mediated by vWF binding to GPIIb/IIIa. A 52/48-kD tryptic fragment containing the GPIb binding domain of normal vWF completely blocked the aggregation induced by all three IIB vWF preparations. The present study defines in detail the mechanisms involved in IIB vWF-induced platelet aggregation. Moreover, it establishes that the GPIb binding domain of normal and IIB vWF are closely related and that desialylation is not required for the direct interaction of IIB vWF with GPIb.     

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.     

Human platelet Fc receptor for immunoglobulin G. Identification as a 40,000-molecular-weight membrane protein shared by monocytes

We have recently shown that human monocytes and U937 cells possess two molecular classes of Fc gamma receptor. One, a 72,000-mol-wt sialoglycoprotein, has high affinity for certain subclasses of human and murine monomeric IgG. The other is a 40,000-mol-wt protein (p40) with low affinity for monomeric IgG but with the capacity to bind IgG aggregates or IgG-coated particles. In the present study, a 40,000-mol-wt single chain protein, apparently identical to p40 from U937 cells, was isolated from surface-radioiodinated human platelets by affinity purification using a murine IgG2b monoclonal antibody to p40. This 40,000-mol-wt protein was not seen when control IgG2b or unrelated murine monoclonal antibodies were employed in place of anti-p40. The same 40,000-mol-wt protein was also recovered from an IgG-Sepharose affinity adsorbent, but not from ovalbumin-or myoglobin-Sepharose. The 72,000-mol-wt Fc gamma receptor of monocytes was not identified on platelets. Monoclonal anti-p40 and Fab fragments derived from this antibody blocked platelet aggregation by heat-aggregated human IgG, whereas a control murine IgG2b protein had little or no inhibitory effect at 500-1,000-fold higher concentrations. A murine IgG1 monoclonal antibody, reactive with an unrelated platelet-specific membrane antigen, did not inhibit platelet responses to aggregated IgG. Anti-p40 did not affect platelet aggregation by thrombin, collagen, or fibrinogen plus ADP. Although anti-p40 did not directly aggregate platelets in the concentrations employed, cross-linking with F(ab')2 goat anti-murine Ig induced apyrase-sensitive aggregation of anti-p40-treated platelets. This indicates that p40 possesses transmembrane linkage for platelet activation and secretion. These observations strongly suggest that this newly recognized 40,000-mol-wt platelet membrane protein serves as an Fc gamma receptor.     

Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor.

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

Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.

The intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P less than 0.01, t test), and this impairment of von Willebrand factor-dependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von Willebrand factor (vWF) levels. We hypothesized that heparin may inhibit vWF-dependent platelet hemostatic functions by directly binding vWF in solution and interfering with vWF-GpIb binding. Using the in vitro techniques of ristocetin-induced platelet agglutination, fluorescent flow cytometric measurement of vWF-platelet binding, and conventional radioligand binding assays we observed that heparin inhibited both vWF-dependent platelet function and vWF-platelet binding in a parallel and dose-dependent manner. Heparin also inhibited platelet agglutination induced by bovine vWF and inhibited the binding of human asialo-vWF to platelets in ristocetin-free systems. The inhibitory potency of heparin was not dependent upon its affinity for antithrombin III, but was molecular weight dependent: homogeneous preparations of lower molecular weight were less inhibitory. Heparin impairment of vWF function may explain why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.     

Platelet Fc Receptor: INCREASED EXPRESSION IN MYELOPROLIFERATIVE DISEASE

The platelet Fc receptor, a membrane receptor for immune complexes or aggregated immunoglobulin (Ig)G, was compared in normal and myeloproliferative platelets. Washed platelets from 11 normal donors and 27 patients were incubated with fluorescein-conjugated ovalbumin-anti-ovalbumin complexes and examined by phase and fluorescence microscopy. Only 3.2±1% of the normal platelets stained, whereas 76±16% of the myeloproliferative platelets stained with the immune complex. The fluorescent staining was mediated by a platelet Fc receptor, as shown by the absence of platelet staining with immune complex containing antibody preincubated with Staphylococcal protein A to block the Fc region. In addition, no staining occurred with antigen or antibody alone or after preincubation of platelets with aggregated IgG. Platelets from normal or myeloproliferative donors did not stain with the immune complexes when the incubation was performed in plasma. The increased expression of Fc receptors on myeloproliferative platelets was corroborated by studies of [¹⁴C]serotonin release by immune complexes or aggregated IgG in 8 patients and 17 normal donors. Serotonin uptake was similar in both groups. Myeloproliferative platelets released significantly more serotonin than normal platelets at each concentration of immune complex or aggregated IgG; in addition, myeloproliferative platelets released serotonin in response to much smaller concentrations of immune complex or aggregated IgG. [¹⁴C]Serotonin release by myeloproliferative platelets was not increased above that of normal platelets when thrombin was used as the stimulus. The results were independent of patient age, sex, therapy, hematocrit, or platelet size. Interaction of circulating immune complexes with platelets bearing increased Fc receptors may contribute to the abnormal hemostasis associated with the myeloproliferative syndromes.     

Evidence that a 210,000-molecular-weight glycoprotein (GP 210) serves as a platelet Fc receptor.

We previously identified a 210,000-mol-wt platelet glycoprotein (GP 210) that is missing from Bernard-Soulier platelets, and found that an antibody against GP 210 inhibits ristocetin-induced platelet agglutination. We now show by immunoblotting that GP 210 binds heat-aggregated rabbit and human IgG, as well as keyhole limpet hemocyanin (KLH)-anti-KLH and ovalbumin (OA)-anti-OA immune complexes. Immune complex binding to GP 210 was preserved on chymotrypsin-treated platelets that lacked glycoprotein Ib (GP Ib). In contrast, ristocetin-induced platelet agglutination resulted in disappearance of immunologically detectable GP 210 and loss of immune complex binding, even though GP Ib remained intact. Purified Fc fragments inhibited binding of anti-GP 210 antibody to intact platelets and to GP 210 on immunoblots. The Fc fragments also blocked immune complex binding to GP 210. Conversely, anti-GP 210 antiserum and F(ab)2 fragments inhibited binding of fluorescein-labeled Fc fragments to intact platelets. We conclude that GP 210 functions as a platelet Fc receptor.     

Platelets and the innate immune system: mechanisms of bacterial‐induced platelet activation

Summary. It has become clear that platelets are not simply cell fragments that plug the leak in a damaged blood vessel; they are, in fact, also key components in the innate immune system, which is supported by the presence of Toll‐like receptors (TLRs) on platelets. As the cells that respond first to a site of injury, they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets, which usually triggers platelet activation and the secretion of antimicrobial peptides. The main platelet receptors that mediate these interactions are glycoprotein (GP)IIb–IIIa, GPIbα, FcγRIIa, complement receptors, and TLRs. This process may involve direct interactions between bacterial proteins and the receptors, or can be mediated by plasma proteins such as fibrinogen, von Willebrand factor, complement, and IgG. Here, we review the variety of interactions between platelets and bacteria, and look at the potential for inhibiting these interactions in diseases such as infective endocarditis and sepsis.     

Thrombotic thrombocytopenic purpura: successful treatment unlocks etiologic secrets

The cause of platelet agglutination in thrombotic thrombocytopenic purpura has been an enigma. Current evidence indicates that the interaction of platelets with a platelet-aggregating factor or unusually large multimers of factor VIII: von Willebrand factor, or both, may cause the abnormal platelet agglutination. Recent success in the treatment of thrombotic thrombocytopenic purpura with intravenous infusion of immunoglobulin suggests that the abnormal platelet agglutination in thrombotic thrombocytopenic purpura may reflect a deficiency of immunoglobulins that normally inhibit platelet-aggregating factors or large multimers of factor VIII: von Willebrand factor.     

Platelets have more than one binding site for von Willebrand factor.

The binding of 125I-von Willebrand factor (125I-vWF) to platelets stimulated by thrombin, ADP, and a combination of ADP + epinephrine (EPI) is specific, saturable, and reversible. Active platelet metabolism and divalent cations are required for binding induced by these stimuli, but not by ristocetin, suggesting the existence of different mechanisms involved in the vWF-platelet interaction. A monoclonal antibody directed against an epitope of membrane glycoprotein (GP) Ib had no effect on the binding of 125I-vWF to normal platelets stimulated by thrombin or a combination of ADP + EPI, but completely blocked ristocetin-induced binding. Binding induced by thrombin to GPIb-blocked platelets was specific. Moreover, thrombin-induced binding of 125I-vWF was increased, rather than decreased, in two patients with the Bernard-Soulier syndrome whose platelets lacked GPIb. Conversely, monoclonal antibodies directed against the GPIIb/IIIa complex had no effect on ristocetin-induced binding of 125I-v-WF to normal platelets, but blocked thrombin- and ADP + EPI-induced binding. To exclude effects mediated by the platelet Fc receptor, a monoclonal IgG directed against an epitope present on human B cells and monocytes, but not expressed on resting or stimulated platelets, was used. It did not affect 125I-vWF binding induced by any of the stimuli. These studies show that platelets have more than one binding site for vWF, and that they may be exposed by different stimuli.     

Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation.

A fluid shear stress of 180 dyn/cm2 was applied for 0.5 and 5 min to platelets in citrated plasma or blood in a cone and plate viscometer with minimal platelet-surface interactions. Platelets aggregated in the shear field if large von Willebrand Factor (vWF) multimers were present. Aggregation did not require ristocetin, other exogenous agents, or desialation of vWF. Unusually large vWF multimers produced by human endothelial cells were functionally more effective than the largest plasma vWF forms in supporting shear-induced aggregation. Shear-induced aggregation was inhibited by monoclonal antibodies to platelet glycoprotein Ib or the IIb/IIIa complex, but was little affected by the absence of fibrinogen. vWF-dependent platelet aggregation under elevated shear stress in partially occluded vessels of the arterial microcirculation may contribute to thrombosis, especially if unusually large vWF multimers are released locally from stimulated or disrupted endothelial cells.