Type 2B von Willebrand Disease is a bleeding disorder characterized by thrombocytopenia and abnormal von Willebrand factor (vWF) multimers. In fluid phase, 2BvWF is a hyperactive ligand for platelet GPIb that binds to platelets spontaneously and at low doses of nonphysiologic stimuli. We hypothesized that, despite the increased binding of 2BvWF to platelet GPIb in suspension,r-2BvWF would not mediate increased binding of FFWP to collagen in a model mimicking the in vivo situation at high shear stress. We, therefore, expressed wild type (WT) vWF and 3 recombinant 2BvWF mutations: V553M, R578Q and R578P, respectively. The 2B mutants were shown to have increased binding to FFWP at low concentrations of ristocetin and/or botrocetin. The multimeric structure of each of the expressed mutants was similar to WT vWF. FFWP, 200,000/μl, were suspended in 120 ng/ml of WT or mutant r-vWF. Prior to perfusion experiments, glass coverslips were coated with type III collagen at 30 ng/cm2. The platelet-vWF suspension was perfused through a parallel plate chamber at shear rate of 1200/sec to approximate the shear that exists in small arterioles. Coverslips were stained with a fluorescein-tagged monoclonal anti-platelet GPIIb-IIIa antibody and adherent platelets counted using a 0.3 mm2 grid. The results represent the mean±S.D. of 10 fields. We found the binding to type III collagen of FFWP suspended in WT and each of the three r-2BvWF mutants was similar: WT 50±19/mm2, V553M 58±27/mm2, R578Q 27±10/mm2, and R578P 33±10/mm2. In order to investigate the mechanism leading to this result, we performed a static assay comparing the binding of WT with V553M r-vWF to type III collagen. At 25, 50 and 100 ng/ml of either WT or the V553M mutant, the binding of the V553M mutant to collagen was 45% of the WT. We conclude from these studies that, although 2BvWF binds with increased avidity to platelets in fluid phase, the net effect of this interaction at high shear does not result in increased surface binding of FFWP to collagen.
