Comparison of thrombin and ristocetin in the interaction between von Willebrand factor and platelets

It is known that the antibiotic ristocetin exposes the platelet membrane receptor for factor VIII/von Willebrand glycoprotein (FVIII/vWF). Recent reports suggest that low concentrations of thrombin also cause platelet membrane receptors to become available for FVIII/vWF. As a consequence, the suspicion has been raised that thrombin provides similar or equivalent activity in vivo to that observed for ristocetin under in vitro conditions. In this study, we quantitated the extent to which thrombin promotes the binding of FVII/vWF to platelets and determined whether or not this interaction initiates or complements platelet aggregation. With ristocetin present, the amount of 125I-FVIII/vWF that became platelet-bound correlated closely with the onset, rate, and extent of platelet aggregation. In contrast, at every thrombin concentration tested, the amount of 125I- FVIII/vWF that specifically bound to platelets was about 6% of that observed with ristocetin. Significantly, FVIII/vWF did not augment the rate of aggregation of platelets in response to thrombin or initiate platelet aggregation when subaggregating doses of thrombin were used. These observations indicate that the minimal association that occurs between FVIII/vWF and the platelet membrane in the presence of thrombin does not correlate with platelet aggregation and therefore is not analogous to the effects of ristocetin. Whether the low level of binding relates to another process, such as platelet-endothelial interactions, remains unknown.     

Differential effect of the ABO blood group on von Willebrand factor collagen binding activity and ristocetin cofactor assay.

Diagnosis of von Willebrand disease requires a combination of quantitative and qualitative tests including the von Willebrand factor ristocetin cofactor assay (vWF:RCo), the von Willebrand factor collagen binding assay (vWF:CB) and von Willebrand factor antigen quantification (vWF:Ag). Genetic factors, especially the ABO blood group, contribute significantly to the variation in vWF levels and function. Recent studies suggest that ethnicity may be another important modulator. We investigated the effect of the ABO blood group on these tests in 52 blood group O and 54 non-group O Chinese blood donors who were well-matched for sex and age distribution. Group O donors had significantly lower vWF:RCo, vWF:Ag and vWF:CB (P < 0.0001). Reduction in vWF:CB was greater than vWF:RCo in group O donors (53 versus 27%). This led to a lower vWF:CB/vWF:Ag ratio (P < 0.0001) in group O donors whereas the vWF:RCo/vWF:Ag ratio was unaffected (P = 0.97). These variations should be taken into consideration when interpreting these results in the Chinese.     

von Willebrand's disease characterized by increased ristocetin sensitivity and the presence of all von Willebrand factor multimers in plasma

In eight members of one family, platelets in platelet-rich plasma aggregated at much lower ristocetin concentrations than normal. Ivy bleeding time was variously prolonged, and von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity, and factor VIII coagulant activity were decreased. Most of the affected members had had slight to rather severe bleeding symptoms. Platelet-type von Willebrand's disease (vWD) could be ruled out. All multimers of vWF:Ag were found in plasma as well as platelets. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) to the propositus did not cause thrombocytopenia, and platelet-poor plasma obtained immediately after did not aggregate normal platelets. The molecular defect in this family, inherited as an autosomal dominant, resembles the one in type IIB because of the response to ristocetin but differs from IIB because all vWF:Ag multimers are present in plasma and the response to DDAVP is atypical. We conclude that this family has a new subtype of vWD and propose that structural as well as functional criteria should be used for a proper classification of vWD.     

Factor VIII/von Willebrand factor binding to von Willebrand''s disease platelets

A form of von Willebrand's disease has been described with enhanced ristocetin-induced platelet aggregation and anodal migration of the factor VIII/von Willebrand factor protein (type IIb). We studied two families with this form of von Willebrand's disease and macrothrombocytopenia. We have found that these platelets bind more of the normal and intermediate-sized multimers of the factor VIII/von Willebrand factor than normal platelets. Analysis of the binding data show an increased affinity of these vWd platelets for the factor VIII/von Willebrand factor. These findings are consistent with an increased number of platelet receptors, which, either by their native topography or migration on the platelet surface, bind factor VIII/von Willebrand factor protein with greater affinity than normal platelets, platelets of other vWd patients, and large platelets of other etiologies.     

Antibody inhibitors to von Willebrand factor metalloproteinase and increased binding of von Willebrand factor to platelets in ticlopidine-associated thrombotic thrombocytopenic purpura

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) affects 1 in 1600 to 1 in 5000 patients who receive ticlopidine, but little is known about the pathogenesis of this complication. OBJECTIVE: To investigate whether von Willebrand factor (vWF), which has been associated with idiopathic TTP, is involved in the pathogenesis of ticlopidine-associated TTP. DESIGN: Case series. SETTING: Three tertiary care, university-affiliated medical centers. PATIENTS: Seven patients who developed TTP 2 to 7 weeks after initiation of ticlopidine therapy. Controls were 7 consecutive patients without thrombocytopenia who had been receiving ticlopidine for 3 to 5 weeks and 10 randomly selected hospitalized patients. MEASUREMENTS: Platelet-bound vWF in patients' EDTA-anticoagulated whole blood samples; vWF proteinase activity in patients' plasma samples; inhibitory activity of IgG isolated from patients' plasma samples against the proteinase from the controls' plasma samples; and vWF multimeric patterns in patients' EDTA-anticoagulated plasma samples. RESULTS: Binding of vWF to single platelets was increased in the three patients tested during the most thrombocytopenic phase of TTP episodes. Initial plasma samples from all seven patients lacked the largest vWF multimers and were severely deficient in vWF metalloproteinase. IgG molecules, isolated from plasma samples of five patients, inhibited metalloproteinase in plasma samples from the controls. In patients examined, these abnormalities resolved upon the remission that accompanied plasma exchange and discontinuation of ticlopidine therapy. CONCLUSION: In the patients who developed ticlopidine-associated TTP, autoantibodies to the vWF metalloproteinase were formed; this led to the same type of vWF abnormalities observed in patients with idiopathic acute TTP. The findings suggest that failure to process large and unusually large vWF multimers in vivo caused binding of vWF to platelets, systemic platelet thrombosis, and TTP.     

The determination of von Willebrand factor activity by collagen binding assay

A new collagen binding assay has been developed for the determination of the functional activity of human von Willebrand factor based on the following principle: pepsin-digested type III collagen from human placenta was covalently immobilized on a microtitre plate. Binding of collagen to the microtitre plate was carried out in neutral phosphate buffer within 1 h. A collagen concentration of 3 μg mL^–1 was sufficient to achieve optimal coating. After drying, the coated microtitre plates remained stable for months without losing their vWF-binding properties and could be incorporated into a ready-to-use kit. The suitability of various types of collagen for use in this assay was evaluated by simulating the binding of vWF to collagen immobilized on the microtitre plate by using surface plasmon resonance technology with collagen immobilized on a sensor chip. vWF was most effectively bound to pepsin-digested type III collagen from human placenta. The assay thus comprises the following steps: (1) serial dilutions of a vWF reference preparation and vWF-containing samples are prepared and bound to a microtitre plate which is precoated with collagen; (2) vWF is detected with a polyclonal antibody; (3) the substrate reaction is photometrically measured with an ELISA reader. This test is highly specific and sensitive for vWF (detection limit: 10 ng mL^–1). The collagen binding activity measured corresponds to the degree of multimerization of vWF. The high sensitivity and the short time needed to carry it out may make it useful for clinical diagnosis and for the measurement of the functional activity of vWF in factor concentrates. In certain applications it may represent a suitable replacement for the ristocetin cofactor assay.     

Utility of the von Willebrand factor collagen binding assay in the diagnosis of von Willebrand disease

von Willebrand Disease (VWD) is the most common inherited bleeding disorder and also arises as an acquired defect (AVWS). VWD and AVWS are due to quantitative deficiencies and/or qualitative defects in von Willebrand factor (VWF), an adhesive plasma protein with multiple activities. Diagnosis of VWD is problematic, being subject to overdiagnosis, underdiagnosis, and misdiagnosis. This is largely due to limitations in current test procedures and an over‐reliance on these imperfect test systems for clinical diagnosis. VWF essentially acts to assist in the formation of a platelet thrombus to stop blood loss from sites of injury, achieving this by binding to platelets (primarily through the glycoprotein Ib receptor), binding to subendothelial matrix components (primarily collagen), and binding to factor VIII (FVIII), thus protecting FVIII from degradation and enabling its delivery to sites of vascular injury. VWD is classified into six separate types, which may each be differentially managed therapeutically, and this underscores the importance of a correct diagnosis. The current report concisely reviews the utility of a relatively underutilised assay, the VWF collagen binding assay (VWF:CB), in facilitating the correct diagnosis and typing of VWD. In particular, if laboratories do not utilise the VWF:CB, then (i) type 2M VWD will continue to be missed, and/or misdiagnosed as types 2A or 1 VWD, and (ii) types 2A, 2B and PT‐VWD will continue to be missed, or else be misdiagnosed as type 1 VWD or ITP. Am. J. Hematol. 92:114–118, 2017. © 2016 Wiley Periodicals, Inc.     

Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.     

Correlation between von Willebrand factor antigen,von Willebrand factor ristocetin cofactor activity and factor VIII activity in plasma

Background The laboratory diagnosis of von Willebrand Factor (VWF) deficiencies includes qualitative and quantitative measurements of VWF and clotting factor VIII (FVIII). Since the FVIII activity is frequently normal in patients with mild type 1 or 2 von Willebrand disease (VWD), there is controversy whether FVIII testing should accompany VWF Antigen (VWF:Ag) assay. Methods The aim of this study was to explore the correlation between VWF:Ag, VWF ristocetin cofactor activity (VWF:RCo) and FVIII in 213 consecutive patients undergoing screening for VWD. Results Forty-six patients were identified with VWF:Ag levels lower than the diagnostic threshold (54 IU/dl). A significant correlation was observed between VWF:Ag and VWF:RCo (r = 0.892; p < 0.001), VWF:Ag and FVIII (r = 0.834; p < 0.001), VWF:RCo and FVIII (r = 0.758; p < 0.001). Receiver operating characteristic curve analysis of the VWF:Ag assay revealed an area under the curve of 0.978 and 0.957 for detecting life-threatening values of FVIII (<30 IU/dl) and VWF:RCo (<40 IU/dl), respectively. The negative and positive predictive values at the VWF:Ag threshold value of 54 IU/dl were 100% and 33% for detecting life-threatening FVIII deficiencies, 94% and 80% for identifying abnormal values of VWF:RCo. Conclusions Due to the excellent correlation between VWF:Ag and FVIII and to the diagnostic efficiency of VWF:Ag for identifying abnormal FVIII levels in patients with VWF deficiency, routine measurement of FVIII may not be necessary in the initial screening of patients with suspected VWD. However, the limited negative predictive value of VWF:Ag for identifying type 2 VWD does not allow to eliminate VWF:RCo or VWF:FVIIIB assays from the diagnostic workout.     

The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets.

The Arg-Gly-Asp sequence resides in the cell attachment region of fibronectin. Arg-Gly-Asp-containing peptides support fibroblast attachment, inhibit fibroblast adhesion to fibronectin, and inhibit fibronectin binding to thrombin-stimulated platelets. In view of the similarities between the binding of fibronectin, fibrinogen, and von Willebrand factor to stimulated platelets, we have examined the effects of Arg-Gly-Asp-containing peptides on the interaction of these latter two adhesive proteins with platelets. Gly-Arg-Gly-Asp-Ser-Pro was used as a prototype peptide, and this hexapeptide inhibited fibrinogen binding to ADP and thrombin-stimulated platelets in the 10-200 microM range. The inhibition exceeded 90% at high concentrations of peptide and was observed in the presence of either calcium or magnesium. Platelet aggregation was also inhibited by the peptide in this dose range. The hexapeptide inhibited fibrinogen binding to platelets with receptors fixed in an exposed state, indicating direct interference with the ligand-platelet interaction. The peptide was 1/2 to 1/3rd as potent in inhibiting fibrinogen as fibronectin binding to platelets, but fibrinogen and von Willebrand factor binding were inhibited to an identical extent. Conservative amino acid substitutions for the arginine, glycine, or aspartic acid markedly reduced inhibitory activity and the Asp-Gly-Arg sequence was inactive. These results indicate that Arg-Gly-Asp-containing peptides can inhibit the binding of the three adhesive proteins to stimulated platelets, establishing a basic common feature between the interaction of these molecules with platelets.     

Glanzmann thrombasthenia: deficient binding of von Willebrand factor to thrombin-stimulated platelets.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by the failure of platelets to aggregate in response to almost all stimuli. However, thrombasthenic platelets will aggregate with bovine and porcine von Willebrand factor (vWF) and will show normal ristocetin-induced binding and aggregation in the presence of human vWF. In contrast, we now report that the specific binding of vWF to the thrombin-stimulated platelets was less than 20% of normal in three patients with Glanzmann thrombasthenia. Analysis of binding isotherms was based on the assumption of one class of binding sites for vWF on the platelet membrane. Double-reciprocal plots were used to calculate maximal binding at saturation and apparent dissociation constant (Kd). In nine normals, 2.82 +/- 0.64 micrograms (+/- SD) of vWF bound to 10(8) platelets at saturation, with Kd (+/- SD) = 3.65 +/- 1.23 micrograms/ml. In two patients with thrombasthenia binding was markedly decreased and did not approach saturation. In the third patient, binding at saturation corresponded to 0.21 micrograms per 10(8) platelets, with Kd = 3.93 micrograms/ml. These findings suggest that mechanisms underlying the vWF-platelet interaction are incompletely reflected in ristocetin-dependent assay systems. Moreover, these results, in addition to those previously reported for fibronectin, suggest that the platelet defect in Glanzmann thrombasthenia is not limited to decreased binding of fibrinogen but involves several glycoproteins that are known to interact with platelets.     

Altered thrombus formation in von Willebrand factor-deficient mice expressing von Willebrand factor variants with defective binding to collagen or GPIIbIIIa

The role of von Willebrand factor (VWF) in thrombosis involves its binding to a number of ligands. To investigate the relative importance of these particular interactions in the thrombosis process, we have introduced mutations into murine VWF (mVWF) cDNA inhibiting VWF binding to glycoprotein (Gp) Ib, GPIIbIIIa, or to fibrillar collagen. These VWF mutants were expressed in VWF-deficient mice (VWF^–/–) by using an hydrodynamic injection approach, and the mice were studied in the ferric chloride–induced injury model. Expression of the collagen and the GPIIbIIIa VWF-binding mutants in VWF^–/– mice resulted in delayed thrombus growth and significantly increased vessel occlusion times compared with mice expressing wild-type (WT) mVWF (30 ± 3 minutes and 38 ± 4 minutes for the collagen and GPIIbIIIa mutants, respectively, vs 19 ± 3 minutes for WT mVWF). Interestingly, these mutants were able to correct bleeding time as efficiently as WT mVWF. In contrast, VWF^–/– mice expressing the GPIb binding mutant failed to restore thrombus formation and were bleeding for as long as they were observed, confirming the critical importance of the VWF-GPIb interaction. Our observations suggest that targeting the VWF-collagen or VWF-GPIIbIIIa interactions could be an interesting alternative for new antithrombotic strategies.     

Comparative analysis of type 2b von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets

von Willebrand factor (vWF) is a multimeric glycoprotein that forms an adhesive link following vascular injury between the vessel wall and its primary ligand on the platelet surface, glycoprotein Ib (GpIb). Type 2b von Willebrand disease (vWD) is a qualitative form of vWD resulting from enhanced binding of vWF to platelets. Molecular characterization of the vWF gene in patients with type 2b vWD has resulted in identification of a panel of mutations associated with this disorder, all clustered within the GpIb binding domain in exon 28 of the vWF gene. We have expressed six of the most common type 2b vWD mutations in recombinant vWF and show that each mutation produces a similar increase in vWF binding to platelets in the absence or presence of ristocetin. Furthermore, expression of more than one type 2b vWD mutation in the same molecule (cis) or in different molecules within the same multimer (trans) failed to produce an increase in vWF platelet binding compared with any of the individually expressed mutations. Taken together, these data support the hypothesis that the vWF GpIb binding domain can adopt either a discrete "on" or "off" conformation, with most type 2b vWD mutations resulting in vWF locked in the on conformation. This model may have relevance to other adhesive proteins containing type A domains.