Shear stress-induced binding of von Willebrand factor to platelets

Shear stress-induced platelet aggregation requires von Willebrand factor (VWF), platelet glycoprotein (GP) Ib, GPIIb-IIIa, Ca2+, and adenosine diphosphate (ADP). Recent reports using vWF labeled with either I-125 or fluorescein isothiocyanate (FITC) have demonstrated that in shear-fields, vWF binds to both GPIb, and GPIIb-IIIa. The sequence of the vWF binding to the two platelet receptors has not been precisely determined in these reports, In this study, a flow cytometry technique using a primary anti-vWF antibody and a secondary FITC IgG antibody was used to measure shear stress-induced vWF binding to platelets, Washed normal platelets suspended at 50,000/mu 1 with purified large VWF multimers were exposed to laminar shear stresses of 15 to 120 dynes/cm(2) for 30 sec. At this low platelet count, little or no aggregation occurred in the shear fields. A significant increase in post-shear vWF-positive platelets was consistently observed. Experiments with platelets from normal and severe von Willebrand's disease (vWD) (which lack plasma and platelet a-granule vWF) demonstrated that exogenous vWF predominately contributed to the platelet-vWF binding, Blockade of platelet GPIb with the monoclonal and-CPIb antibody, 6D1, completely inhibited shear stress-induced platelet-vWF attachment, In contrast, blockade of GPIIb-IIIa with monoclonal anti-GPIIb-IIIa antibodies, 10E5 or c7E3, or with the GPIIb-IIIa-blocking tetrapeptide, RGDS, had little or no inhibitory effect on platelet-vWF binding, These data demonstrate that the binding of vWF to GPIb is likely to be the initial shear-induced platelet-ligand binding event. (C) 1997 Elsevier Science Ltd.