A new von Willebrand variant (type I, New York): increased ristocetin-induced platelet aggregation and plasma von Willebrand factor containing the full range of multimers

We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.     

Von Willebrand disease associated with familial thrombocytopenia and increased ristocetin-induced platelet aggregation

Two cases of von Willebrand disease (vWD) associated with familial thrombocytopenia were reported. The proband (daughter) and her father showed thrombocytopenia with large platelets and decreased von Willebrand factor activity (VIIIR:WF). Factor VIII procoagulant activity (VIII:C) and factor VIII-related antigen (VIIIR:AG) were normal, but both patients revealed an increased ristocetin-induced platelet aggregation and a qualitative abnormality of the factor VIII protein, which was characterized by fast electrophoretic mobility of VIIIR:AG and an abnormal elution of factor VIII-related activities on Sepharose 2B. DDAVP was hemostatically effective even in this thrombocytopenic patient undergoing a dental extraction.     

Platelet receptors for human Factor VIII/von Willebrand protein: functional correlation of receptor occupancy and ristocetin-induced platelet aggregation.

Previous studies of von Willebrand disease indicate that a deficiency of blood clotting Factor VIII/von Willebrand factor (FVIII/vWF) activity is responsible for the failure of platelets to participate fully in the initial stages of hemostasis. We have recently identified specific FVIII/vWF binding sites on platelets, suggesting that the interaction of these sites with FVIII/vWF may be functionally important in the development of platelet clumps. We have now studied how different ristocetin concentrations, various known platelet aggregation inhibitors, and the exposure of platelets to proteases affect the ability of platelets to bind FVIII/vWF and to form aggregates. Our results demonstrate a highly significant linear correlation between the degree of FVIII/vWF receptor binding and the extent of ristocetin-induced platelet aggregation. Because neither FVIII/vWF binding nor platelet aggregation occurs after platelets are exposed to low concentrations of proteases, the FVIII/vWF receptors must be in the platelet membrane. We conclude that the interaction between FVIII/vWF protein and its receptors on the platelet membrane is an important mechanism by which platelet aggregation occurs during primary phase hemostasis.     

A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.     

von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib.

von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.     

Epinephrine augments von Willebrand factor-dependent shear-induced platelet aggregation.

BACKGROUND. Shear-induced platelet aggregation (SIPA) is an important mechanism in thrombogenesis. von Willebrand factor (vWF) binding to platelet glycoprotein Ib (GP Ib) has been found to be crucial for platelet aggregation under the high shear force probably generated in stenosed coronary artery. The physiological significance of vWF-dependent SIPA has not been clarified. METHODS AND RESULTS. Blood samples were collected from 23 normal volunteers. SIPA was continuously monitored using a modified cone-plate viscometer adapted for measuring the transmitted light intensity of the material. The effects of low concentrations of epinephrine, ADP, and collagen on SIPA under both low shear (12 dyne/cm2) and high shear (108 dyne/cm2) force were investigated. All agonists tested enhanced SIPA under low shear force, whereas only epinephrine augmented SIPA under high shear force. The maximum extents of SIPA under high shear force in the absence and presence of epinephrine (10 ng/ml) were 37.9 +/- 11.5% and 59.7 +/- 13.9%, respectively. The antagonist of the alpha 2-adrenergic receptor yohimbine (1 microgram/ml) antagonized the effects of epinephrine. The monoclonal antibody NMC-4 against vWF, which was shown to inhibit its binding to GP Ib, completely abolished SIPA under high shear force, even in the presence of epinephrine. However, this antibody only partially inhibited SIPA under low shear force. CONCLUSIONS. Our findings suggest that epinephrine is the agonist that enhances SIPA mediated by vWF through its specific receptor. This may be clinically important because occlusion of the coronary artery often occurs in stenosed atherosclerotic vessels under sympathetic stimulation.     

Characterization of 25 monoclonal antibodies to factor VIII-von Willebrand factor: relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium

We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.     

Enhanced ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib in patients with steroid-responsive nephrotic syndrome

To elucidate the mechanism of enhanced ristocetin-induced platelet aggregation (RIPA) in steroid-responsive nephrotic syndrome (SRNS), plasma levels of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RCof) were examined in 6 patients and the amount of ristocetin-induced vWF binding to platelets was determined. At the initial or relapse stage, the plasma vWF:Ag level was 415 +/- 137% and the RCof level was 364 +/- 117%. The ratio of RCof/vWF:Ag was 0.90 +/- 0.15 and no abnormalities of vWF:Ag multimers were observed, indicating that neither functional nor structural abnormalities were present in patient's plasma. The amount of ristocetin-induced normal vWF binding to nephrotic washed platelets, when ristocetin was used at concentrations of 0.5, 0.75, and 1.0 mg/ml, was 152-163% above the binding to normal platelets. In addition, nephrotic washed platelets resuspended in either normal or nephrotic plasma aggregated at a low concentration of ristocetin (0.75 mg/ml) which did not induce aggregation of normal platelets. In accordance with these observations, the decrease of Alcian blue 8GX binding to platelets, reflecting diminished surface negative charge, was also observed. These results appear to indicate that the plasma vWF level and the altered surface-negative charge in platelets both contribute to heightened vWF binding to GPIb, thus lowering the ristocetin concentration required for RIPA in SRNS.     

Botrocetin- and polybrene-induced platelet aggregation in platelet-type von Willebrand disease

It has been reported that botrocetin, a Bothrops venom factor, induces platelet aggregation dependent on von Willebrand factor (vWF), and that platelet aggregation induced by Polybrene, a synthetic polycation, is enhanced by vWF. This report describes the platelet aggregability on stimulation with botrocetin and Polybrene in four patients with platelet-type von Willebrand disease (vWD) who showed increased platelet aggregation with low concentrations of ristocetin as the result of a platelet abnormality. Enhanced platelet aggregability with botrocetin was observed in platelet-rich plasma (PRP) from the patients. Platelet aggregation induced by botrocetin in a mixture of normal washed platelets and patient plasma was either decreased or normal, being dependent on the amount of plasma vWF. In contrast with ristocetin and botrocetin, Polybrene did not cause increased aggregation of patient PRP. Polybrene aggregated normal washed platelets less extensively in the presence of patient plasma than normal plasma. These studies demonstrated that botrocetin induced heightened interaction between platelets and vWF, but Polybrene did not, in platelet-type vWD, and that the enhanced responsiveness of patient platelets to botrocetin is related to an intrinsic platelet abnormality.     

Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor.

A platelet-aggregating activity was found in many snake venoms, predominantly those of the genus Bothrops, that is apparent only in the presence of the platelet-aggregating von Willebrand factor of plasma. It is designated "venom coagglutinin." The coagglutinin can be largely separated from the thrombin-like enzyme of the venoms by ion-exchange chromatography. The venom factor acts on formaldehyde-fixed platelets and is effective with decalcified, heparinized, and afibrinogenemic plasmas but not with severe von Willebrand disease plasmas or with normal plasmas in which the von Willebrand factor has been neutralized by specific antibodies. Use of this coagglutinin permits the assay of von Willebrand factor without the many disadvantages of the ristocetin test. The coagglutinin is active with human, dog, pig, and bovine plasmas and with platelets of any one of these species. This broad-spectrum activity without regard to species contrasts with the ristocetin-resistance of many combinations of plasma and platelets from various species. The assay provides a procedure for studying human, porcine, and canine von Willebrand disease. The lack of species specificity of the coagglutinin suggests that it may be a universal activator of the von Willebrand factor-platelet reaction.     

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.     

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.     

Helicobacter pylori binds von Willebrand factor and interacts with GPIb to induce platelet aggregation

BACKGROUND & AIMS: Clinical studies have suggested an association between cardiovascular disease and infection with Helicobacter pylori. We examined the effect of H. pylori on platelets and the mechanism of the interaction. METHODS: Three of 5 strains of H. pylori induced platelet aggregation with a lag time of 5 +/- 2 minutes that was independent of the toxigenic genes cagA and vacA. Aggregation was inhibited completely by aspirin and a glycoprotein (GP) IIb/IIIa antagonist. Aggregation also was inhibited by monoclonal antibodies that prevented the von Willebrand factor (vWF) interaction with GPIb. vWF-coated H. pylori bound to cells transfected with GPIbalpha but not to mock transfected cells and this was inhibited by an antibody to GPIb. RESULTS: The interaction with platelets appeared to be mediated by vWF because platelet aggregation was blocked by an antibody to vWF. Moreover, a strain of H. pylori that induced platelet aggregation bound vWF to a greater extent than a nonaggregating strain. Aggregation also required IgG and could be inhibited by an antibody to the platelet IgG receptor (FcgammaRIIA). CONCLUSIONS: Some strains of H. pylori induce platelet activation mediated by H. pylori-bound vWF interacting with GPIb, and supported by IgG. These platelet-H. pylori interactions may contribute to the pathogenesis of H. pylori-associated peptic ulcer disease and to the association between H. pylori infection and cardiovascular disease, whereas local platelet effects may contribute to the pathogenesis of H. pylori-associated peptic ulcer disease.     

A platelet defect in a patient with eosinophilic leukaemia: absent ristocetin-induced platelet aggregation associated with a reduced platelet sialic acid content.

Platelets from a patient with eosinophilic leukaemia were not aggregated by ristocetin. The defect was not corrected by normal human plasma and was due to a platelet abnormality. The patient's platelets also showed a diminished sensitivity to aggregation by bovine factor VIIIVWF. The defect was not associated with a prolonged bleeding time. No abnormalities were detected in ADP, collagen or thrombin-induced platelet aggregation. Biochemical studies showed that the platelets were deficient in sialic acid. This deficiency was associated with a reduced staining for glycoprotein I following SDS-polyacrylamide gel electrophoresis. The results suggest an acquired platelet surface abnormality.     

A plasma inhibitor of ristocetin-induced platelet aggregation in patients with sickle hemoglobinopathies

Because of the high prevalence of thrombotic complications in patients with sickle cell anemia (SCA), we investigated platelet function in patients with sickle hemoglobinopathies. Platelet aggregation induced by epinephrine, ADP, and collagen, except for absent secondary wave in 3 of 10 patients with SCA, was qualitatively normal. However, ristocetin-induced platelet aggregation (RIPA) with a final concentration 1.12 mg/ml was markedly abnormal-absent or virtually absent in 9 of 10 patients with SCA, 3 of 3 patients with hemoglobin S-C disease, and 2 of 3 patients with sickle trait. All 8 controls used in these experiments repeatedly demonstrated normal RIPA. Addition of normal plasma failed to correct abnormal RIPA in sickle hemoglobinopathy patients. All patients demonstrated normal RIPA with a ristocetin dose of 2.24 mg/ml and aggregated with bovine fibrinogen. Recombinant mixing experiments demonstrated that washed SCA platelets support RIPA (1.12 mg/ml) when resuspended in normal plasma or high dilutions of SCA plasma, but not in undiluted SCA plasma. Washed normal platelets do not support RIPA (1.12 mg/ml) when resuspended in SCA plasma. These findings suggest the presence of a plasma inhibitor of RIPA in patients with sickle hemoglobinopathies.     

von Willebrand disease "Vicenza" with larger-than-normal (supranormal) von Willebrand factor multimers

When normal volunteers or patients with type I von Willebrand disease (VWD) are given desmopressin (DDAVP), a set of larger-than-normal (supranormal) von Willebrand factor (VWF) multimers, similar to those present in VWF-containing cells such as platelets megakaryocytes and endothelial cells, appear transiently in postinfusion plasma. In two kindreds with mild lifelong bleeding symptoms transmitted as an autosomal dominant trait, all ten symptomatic members (but none of the five asymptomatic members) had a supranormal multimeric structure for plasma VWF, apparently identical to that seen for postdesmopressin normal plasma. Plasma factor VIII coagulant activity (VIII:C), VWF antigen (VWF:Ag), ristocetin-induced platelet agglutination, and ristocetin cofactor (RiCof) activity were low. Platelet VWF:Ag and RiCof levels (tested for three patients only) were normal. Bleeding times were normal or slightly prolonged. The patients' platelet multimeric structure was the same as that for normal platelets. After desmopressin infusion the plasma VWF multimeric structure remained supranormal as for preinfusion plasma, with VIII:C VWF:Ag and RiCof increasing markedly over baseline values and disappearing at a normal rate. Examination of the VWF subunit composition from three of these patients indicated that proteolytic processing of their VWF did not differ from normal. This study describes the first variant of VWD with a supranormal multimeric structure.     

Generation and breakdown of soluble ultralarge von Willebrand factor multimers

Ultralarge von Willebrand factor (ULVWF) multimeric strings are rapidly secreted by, and anchored to, stimulated endothelial cells (EC), and are hyperadhesive to platelets until cleavage by ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In ADAMTS-13-deficient familial and autoantibody-mediated thrombotic thrombocytopenic purpura (TTP), there is severely restricted cleavage of EC-anchored ULVWF-platelet strings. The small amount of active enzyme released from their EC cleaves ULVWF strings minimally just above EC surfaces, thus generating soluble ULVWF multimers that are 2.5 to 50 times longer than plasma von Willebrand factor (VWF) forms. Soluble ULVWF multimers (detected in TTP and several other disorders) are also hyperadhesive to platelets and can cause excessive platelet adhesion/aggregation. Without exogenous chemicals or extreme shear stress, soluble ULVWF multimers cannot be cleaved by ADAMTS-13 but can be de-assembled (reduced) in vitro, by a free thiol-containing molecule (>30 kD) present in the cryosupernatant fraction of plasma that is not ADAMTS-13, thrombospondin-1, albumin, cysteine, or glutathione. This reduction may prevent occlusion of the microvasculature by embolic soluble ULVWF multimers (± adherent/aggregated platelets). New inhibitors of platelet adhesion to EC-anchored ULVWF multimeric strings and soluble ULVWF include an aptamer, a nanobody, and N-acetylcysteine.     

beta2-Glycoprotein I inhibits von Willebrand factor dependent platelet adhesion and aggregation

Patients with antiphospholipid syndrome are characterized by the association of thrombosis or pregnancy morbidity and the presence of antiphospholipid autoantibodies. Particularly, anti-beta(2)-glycoprotein (beta(2) GPI) autoantibodies correlate with thrombosis, suggesting an antibody-induced gain of prothrombotic function and/or an antibody-induced loss of antithrombotic function of beta(2) GPI. In the search for potential antithrombotic properties of beta(2) GPI, we found that beta(2) GPI inhibits von Willebrand factor (VWF)-induced platelet aggregation. In addition, platelet adhesion to a VWF-coated surface was decreased by 50% in the presence of beta(2) GPI (P < .03). beta(2) GPI binds to the A1 domain of VWF but preferably when the A1 domain is in its active glycoprotein Ibalpha-binding conformation. Anti-beta(2) GPI antibodies isolated from a subset of antiphospholipid syndrome patients neutralized the beta(2) GPI-VWF interactions and thus the inhibitory activity of beta(2) GPI. In comparison to healthy individuals, the amounts of active VWF in circulation were increased 1.5-fold (P < .001) in patients positive for lupus anticoagulant (LAC) due to anti-beta(2) GPI antibodies. Thus, beta(2) GPI is a biologically relevant inhibitor of VWF function by interfering with VWF-dependent platelet adhesion. Anti-beta(2) GPI autoantibodies neutralize this inhibitory function and are associated with increased levels of active VWF. This mode of action could contribute to the thrombosis and consumptive thrombocytopenia observed in patients with anti-beta(2) GPI antibodies.