Effect of monoclonal antibodies against von Willebrand factor and platelet glycoproteins IIb/IIIa on the platelet retention test

The platelet retention test provides a measure of the number of platelets retained in a column of glass beads and is one of the few in vitro platelet function tests that is abnormal in von Willebrand's disease (vWd). In a two-stage test, 1 mL of blood (designated A) was passed through the column, followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which platelet retention was measured. With normal blood as A and B, retention is very high in all 5 mL of blood B. In the first stage, platelets adhere to the glass beads; this requires fibrinogen but not von Willebrand factor (vWf). The platelet-platelet adhesion in the second stage requires vWf, is dependent on release of ADP, and fails to occur if thrombasthenic platelets are tested. Retention was normal when blood from a patient with afibrinogenemia was used as blood B. We have now used monoclonal antibodies to elucidate further the mechanism of platelet retention. Five antibodies to different epitopes on vWf essentially abolished retention in the one- stage test and in the second stage of the two-stage test, but had no effect on the first stage. Thus, the entire vWf molecule must be free of antibody to function in the platelet-platelet adhesion of the second stage of this test. Binding of the antigen-antibody complex to the platelet Fc receptor was not responsible, as Fab and F(ab')2 fragments of one of the antibodies were as effective as intact antibody, and as neither heat-aggregated IgG nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2 fragments of 6D1, an antibody to platelet GP Ib that prevents binding of vWf to platelets, also inhibited the second phase of retention. An antibody that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8) inhibited both the first and second stages of retention, whereas LJ-P5, an antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused slight inhibition of retention when normal or afibrinogenemic blood was used as blood B and was reported to cause only partial inhibition of ADP- induced platelet aggregation in this afibrinogenemic patient. The results suggest that vWf is altered during rapid passage of blood through the glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which then binds the altered vWf or fibrinogen, either of which can induce platelet aggregation (platelet-platelet adhesion) and thus retention in the column.     

Platelet aggregation induced by type IIb platelet von Willebrand factor

Platelet lysates from five patients with a form of type IIb von Willebrand's disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWd's platelet-rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard-Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmann's thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate-induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.     

Effect of calcium ion concentration on the ability of fibrinogen and von Willebrand factor to support the ADP-induced aggregation of human platelets

To investigate the suggestion that von Willebrand factor (vWf) can substitute for fibrinogen in supporting ADP-induced aggregation of human platelets, we studied platelet reactions in two media: (1) a high calcium medium, Tyrode-albumin solution containing calcium ions in the physiological range of 2 mmol/L, and (2) a low calcium medium, modified Tyrode-albumin solution from which calcium salt was omitted (calcium ion concentration approximately 20 mumol/L). In the high calcium medium vWf even at concentrations up to six times as high as physiological, showed little or no potentiation of ADP-induced platelet aggregation, whereas fibrinogen strongly potentiated reversible aggregation without thromboxane formation or release of granule contents. In the low calcium medium, either vWf or fibrinogen supported biphasic aggregation in response to ADP, with thromboxane formation and release of granule contents. Aspirin and the thromboxane receptor blocker BM 13.177 inhibited these secondary responses to von Willebrand factor, indicating that they require thromboxane A2 formation and feedback amplification by thromboxane A2. A monoclonal antibody, 10E5, to the platelet glycoprotein IIb/IIIa complex inhibited both primary and secondary aggregation. Although vWf supports ADP-induced aggregation when the concentration of ionized calcium is in the micromolar range, it does not support ADP-induced aggregation in the presence of a concentration of ionized calcium in the physiological range, indicating that vWf probably cannot substitute for fibrinogen in supporting ADP- induced aggregation in vivo.     

Role of ADP in platelet aggregation at high shear: studies in a patient with congenital defect of platelet responses to ADP

Summary. The in vitro measurement of platelet aggregation (PA) at the high shear levels that can be found in the microcirculation may provide useful informations on primary haemostasis, which is usually explored in vivo with the skin bleeding time (BT). PA at high shear requires von Willebrand factor (vWf) and the platelet glycoprotein (GP) complexes Ib/IX/V and IIb/IIIa; controversial results have been reported on its requirement of released adenosine diphosphate (ADP). Due to its dependence on vWf, PA at high shear may be affected by the vasopressin analogue DDAVP, which increases the plasma vWf levels and shortens the prolonged BT of patients with congenital or acquired defects of platelet function. We studied PA at high shear, BT and plasma vWf levels in a patient with congenital impairment of platelet responses to ADP before and after the i.v. infusion of 0.3 μg/kg DDAVP. Two methods to study PA at high shear were used: shear-induced PA (SIPA) and the filter aggregation test. With both methods, PA at high shear of the patient was impaired. The infusion of DDAVP increased plasma vWf levels, shortened the prolonged BT and potentiated PA at high shear of the patient. In conclusion, PA at high shear is impaired in a patient with congenital defect of platelet responses to ADP and prolonged BT and is potentiated by DDAVP. Our results suggest that released ADP plays an important role in PA at high shear and that potentiation of PA at high shear by DDAVP may be one mechanism by which the drug shortens the prolonged BT of patients with congenital or acquired defects of platelet function.     

Monoclonal antibodies to von Willebrand''s factor: reactivity with porcine and human antigens

Monoclonal antibodies to porcine von Willebrand's factor (vWf) were obtained from hybridomas derived from the fusion of NS-1 plasmacytoma cells and spleen cells from immunized BALB/c mice. Twenty hybrids were positive in a radioimmunoassay based on the ability of immobilized hybridoma antibodies to bind porcine vWf as detected by the subsequent binding of 125I-polyclonal rabbit anti-vWf. Growth of the hybridomas as ascitic tumors yielded fluids having titers in this assay ranging from 8 x 10(7) to 8 x 10(11). All the antibodies were positive when normal, but not von Willebrand's, plasma was used as the source fo antigen in this RIA, and when normal, but not von Willebrand's, acetone-fixed platelets were used in an indirect immunofluorescence assay. Two of these antibodies inhibited both ristocetin-induced platelet aggregation and platelet aggregating factor. In a second radioimmunoassay, only 13 were of the 20 antibodies were reactive with human vWf, and two of these would not react with human antigen in the presence of 2M Nacl. Bases on these assays, six reactivity groups of antibodies could be identified.     

Analysis of platelet von Willebrand factor antigen

Platelet lysates were obtained from suspensions of normal washed platelets by freeze-thawing or Triton X-100 lysis. The resultant platelet lysates contained 0.34 +/- 0.15 U/10(9) platelets (n = 8) of von Willebrand factor antigen (vWf:Ag) as determined by radioelectroimmunoassay using a monospecific antibody to vWf:Ag. The vWf:Ag level was higher in platelet lysates prepared from freshly drawn blood than from outdated platelet packs. Platelet lysates from patients with severe von Willebrand's disease type I (n = 2) did not contain detectable vWf:Ag. When normal platelet lysates were analyzed by radiocrossed immunoelectrophoresis in agarose using a monospecific polyclonal antibody to plasma vWf:Ag, two immunochemically identical precipitin peaks were seen. One of the platelet vWf:Ag peaks corresponded in its electrophoretic mobility to plasma vWf:Ag, while the other peak, i.e. platelet vWf:Ag-peak II, migrated to a more anodal position. The presence of the platelet vWf:Ag-peak II suggests structural differences between plasma and platelet vWf:Ag and illustrates previously unrecognized heterogeneity of platelet vWf:Ag.     

Heat-treated factor VIII/von Willebrand factor concentrate in platelet-type von Willebrand's disease

Cryoprecipitate has proved to correct the hemostatic defects in von Willebrand's disease (vWD) and platelet-type vWD. However, recent studies have revealed that transmission of the AIDS retrovirus (HIV) occurs through exposure to blood products including cryoprecipitate. Treatment with heat-treated factor VIII/von Willebrand factor (vWf) concentrates may have certain advantages over treatment with nonheated products, if these preparations are efficacious in these disorders. We found that a commercially available factor VIII/vWf concentrate, Haemate P, contained the high-molecular-weight multimers of vWf and had a ratio of ristocetin cofactor (RCof) to vWf antigen (vWf:Ag) close to unity. In addition, its capacity to directly induce aggregation of platelet-type vWD platelets in vitro was similar to that for cryoprecipitate. When infused into a patient with platelet-type vWD, Haemate P shortened the prolonged bleeding time and caused spontaneous platelet aggregation in vitro with a mild diminution of platelet count. These results indicate that some of the heat-treated factor VIII/vWf concentrates may provide a safer, yet still effective, treatment for platelet-type vWD.     

Characterization of the defect of the factor VIII/von Willebrand factor protein in von Willebrand's disease

The factor VIII/von Willebrand factor (f.VIII/vWf) protein was purified from the plasma of a patient with von Willebrand's disease (vWd). The patient had all of the classic laboratory findings of vWd except for the ristocetin-induced platelet aggregation of his own platelet-rich plasma. The disease has been documented in three generations. Comparison of the purified normal and vWd f.VIIi/vWf protein revealed several abnormalities, including decreased concentration of f.VIII/vWf antigen; decreased specific vWf activity; absence of the larger molecular forms of the f.VIII/vWf protein; carbohydrate deficiencies affecting the sialic acid, penultimate galactose and N-acetylglucosamine moieties; and decreased binding of the f.VIII/vWf protein to its platelet receptor. These studies indicate the multiplicity of biochemical and functional abnormalities associated with the f.VIII/vWf protein in vWd. f.VIII/vWf protein to normal f.VIII/vWf protein that had been treated with 2-mercaptoethanol (2-ME) to reduce the multimer size and then treated with specific exoglycosidases to remove the sialic acid and penultimate galactose residues revealed similar biologic properties.     

von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.     

Platelet release abnormality associated with a variant of von Willebrand's disease.

A family with a platelet release abnormality (PRA) is described. The only son also showed a reduced rate of platelet aggregation in response to ristocetin, markedly reduced levels of von Willebrand's factor (vWf, ristocetin cofactor), and increased mobility of factor VIII-like antigen, features which were suggestive of von Willebrand's disease (vWd). No inhibition of vWf was found in his plasma. Family studies showed no evidence of vWd in the mother. The father's investigations showed a low rate of ristocetin aggregation on one of the two occasions when it was tested and low vWf on two of four occasions. Despite repeated testing, the findings in the father did not conclusively rule out the possibility of mild vWd, and it was impossible to determine whether the vWd in the son was inherited or arose as a mutation. The findings in this family suggest a possible relationship between abnormalities of the factor VIII complex and defective platelet function.     

Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood

We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti-GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.     

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

Pregnancy-induced worsening of thrombocytopenia in a patient with type IIB von Willebrand's disease.

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)     

Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.

Von Willebrand factor (vWF) was purified from the plasma of a patient with type IIB von Willebrand disease (vWF from such a patient, IIB vWF) who had a normal platelet count and showed no evidence of spontaneous platelet aggregation. Large multimers of IIB vWF were absent from purified preparations and from plasma. Ristocetin-induced platelet aggregation was enhanced by purified IIB vWF. The aggregation of washed normal platelets mixed with IIB vWF (0.4 microgram/ml) required lower amounts of ristocetin than the aggregation of normal platelets mixed with the same concentrations of normal vWF. Moreover, purified IIB vWF alone induced aggregation of platelet-rich plasma at concentrations as low as 10 micrograms of IIB vWF/ml in the absence of any other agonist. Aggregation was blocked by a monoclonal antibody against the platelet membrane glycoprotein, GPIb, as well as by an anti-GPIIb/IIIa antibody. Washed platelet suspensions were promptly aggregated by IIB vWF only when fibrinogen and CaCl2 were added to the mixture. Purified IIB vWF induces the binding of fibrinogen to platelets. Such binding was blocked by the anti-GPIb monoclonal antibody as well as by the anti-GPIIb/IIIa monoclonal antibody that inhibited aggregation. A second anti-GPIIb/IIIa antibody, which has the property of blocking vWF but not fibrinogen binding to platelets, blocked neither aggregation nor fibrinogen binding induced by IIB vWF. These studies demonstrate that platelet aggregation is triggered by the initial interaction of IIB vWF with GPIb which is followed by exposure of fibrinogen binding sites on GPIIb/IIIa. Fibrinogen binds to these sites and acts as a necessary cofactor for the aggregation response.     

Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa

Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.     

In vitro correction of the abnormal multimeric structure of von Willebrand factor in type IIa von Willebrand''s disease.

Type IIa von Willebrand's disease (vWd) has been characterized by the absence of the largest and a reduction in the intermediate-sized multimers of the plasma and platelet von Willebrand factor (vWf) and by the diminished response of the platelet-rich plasma of these patients to ristocetin. Other recently demonstrated abnormalities include the presence of an abnormal triplet structure of vWf. We have studied the plasma and platelets from three patients with this form of vWd and have found that both their plasma and platelets manifest the previously described abnormalities. Because of the heterogeneity of the multimeric structure of the vWf in these patients, we considered the possibility that postsynthetic events may have modified the vWf. When blood was collected in 5 mM EDTA or 5 mM EDTA/leupeptin/N-ethylmaleimide, the abnormal multimeric structure of the plasma and platelet vWf was partially normalized in that the intermediate and the largest vWf multimers were increased, the abnormal multimer structure was no longer as apparent, and the fastest migrating band (an abnormality seen only in the type IIa vWd plasma and platelets) disappeared. The enzymatic activity responsible for this degradation can be classified as a calcium-dependent protease. Studies of normal radiolabeled vWf incubated with platelet lysates from normal subjects and these patients revealed that the patients' platelets did not contain increased amounts of calcium-dependent protease activity as assessed by degradation of normal vWf. These data suggest that patients with type IIa vWd synthesize an abnormal vWf protein that is susceptible to in vitro proteolytic degradation and that proteolytic degradation can play a significant role in the phenotypic expression of vWd by modifying the plasma and platelet vWf multimeric structure.     

Discrepant increase in factor VIII: C and von Willebrand factor after DDAVP infusion in a patient with variant von Willebrand's disease.

We have studied a patient with von Willebrand's disease (vWd) whose von Willebrand factor (vWf) multimer patterns showed significant decreases of all but the major fast moving vWf multimer (promoter). Bleeding time (BT) was very prolonged, there was almost no ristocetin-induced platelet aggregation (RIPA) and vWf levels were very low. The factor VIII: C/vWf: Ag ratio appeared to be higher than normal because of the relatively increased concentration of factor VIII: C. The infusion of DDAVP normalized BT, improved RIPA and restored normal factor VIII: C levels, these effects lasted for 5 h even though only a slight increase of vWf: Ag and vWf: RCoF was observed. RIPA was completely inhibited by an anti-glycoprotein (GP) Ib monoclonal antibody that recognizes the ristocetin-induced vWf binding site. Plasma vWf multimer analysis revealed only slight increases of all components and an additional, more pronounced representation of vWf protomer. These data suggest that the patient has an abnormal vWf molecule characterized by a greater ability to carry factor VIII than would be expected from the vWf levels. Furthermore, since the vWf protomer was the only significant vWf component present both before and after DDAVP infusion we hypothesize that some of the haemostatic functions of the patient's vWf may depend on it.     

Platelet aggregation and pseudothrombocytopenia induced by 1-desamino-8-D-arginine vasopressin (DDAVP) in type IIB von Willebrand's disease patient

Our study shows that the thrombocytopenia described in type IIB von Willebrand's disease (vWd) after 1-desamino-8-D-arginine vasopressin (DDAVP) infusion is, at least partially, a pseudothrombocytopenia. There was a discrepancy in platelet counts in blood anticoagulated with EDTA (less than 10 x 10(3)/microliters) or citrate (55 x 10(3)/microliters) in one patient with type IIB vWd and chronic thrombocytopenia (80 x 10(3)/microliters) after DDAVP infusion. Furthermore, DDAVP induced a normalization of patient's prolonged bleeding time. Spontaneous platelet aggregation (SPA) observed in platelet-rich plasma before DDAVP infusion was inhibited completely by monoclonal antibodies which block binding of fibrinogen, vWf and fibronectin to GPIIb-IIIa. SPA was partially inhibited by a monoclonal antibody which blocks the binding of vWf to GPIb. After DDAVP, in contrast, SPA partially persisted in the presence of anti-GPIIb-IIIa monoclonal antibodies but was completely inhibited by anti-GPIb monoclonal antibody. Therefore GPIb and GPIIb-IIIa complex seem to play a different role in SPA before and after DDAVP infusion into type IIB vWd.     

Platelet Activation at High and Low Shear is Followed by Inactivation: The Clinical Relevance

Platelets contribute vitally to the complex processes involved in haemostasis and thrombosis. Even more complex is their contribution to atherogenesis. Many studies have been devoted to analysing the processes involved in platelet activation since, clearly, prevention of activation may have clinical value. There are now at least two systems of platelet activation under intensive study: (a) agonist (e.g. ADP and thrombin) induced platelet activation when fibrinogen is the ligand; this process occurs at low shear forces and is aspirin sensitive; (b) secondly, in marked contrast, at high shear forces, shear itself activates the platelets and von Willebrand's factor (vWf) is the ligand, and this process is aspirin insensitive.(1).     

Spontaneous platelet aggregation in type IIB Tampa von Willebrand disease is inhibited by the 52/48-kDa fragment of normal von Willebrand factor, which contains the GPIb binding domain

The association of Type IIB von Willebrand disease (vWD) with chronic persistent thrombocytopenia and spontaneous platelet aggregation has recently been recognized. It has been shown that IIB von Willebrand factor (vWF) can initiate platelet aggregation by binding to the platelet glycoprotein (GP) lb receptor and inducing exposure of the GpIIb/IIIa fibrinogen receptor. In this study we demonstrate the increased binding of Type IIB Tampa vWF with normal platelets when compared with nonthrombocytopenic Type IIB vWF. Studies further demonstrate that spontaneous platelet aggregation initiated by IIB Tampa vWF can be blocked by a 52/48-kDa fragment of normal vWF, which contains the binding domain.