SHEAR STRESS-INDUCED BINDING OF VON-WILLEBRAND-FACTOR TO PLATELETS

Shear stress-induced platelet aggregation requires von Willebrand fact or (VWF), platelet glycoprotein (GP) Ib, GPIIb-IIIa, Ca2+, and adenosi ne diphosphate (ADP). Recent reports using vWF labeled with either I-1 25 or fluorescein isothiocyanate (FITC) have demonstrated that in shea r-fields, vWF binds to both GPIb, and GPIIb-IIIa. The sequence of the vWF binding to the two platelet receptors has not been precisely deter mined in these reports, In this study, a flow cytometry technique usin g a primary anti-vWF antibody and a secondary FITC IgG antibody was us ed to measure shear stress-induced vWF binding to platelets, Washed no rmal platelets suspended at 50,000/mu 1 with purified large VWF multim ers were exposed to laminar shear stresses of 15 to 120 dynes/cm(2) fo r 30 sec. At this low platelet count, little or no aggregation occurre d in the shear fields. A significant increase in post-shear vWF-positi ve platelets was consistently observed. Experiments with platelets fro m normal and severe von Willebrand's disease (vWD) (which lack plasma and platelet a-granule vWF) demonstrated that exogenous vWF predominat ely contributed to the platelet-vWF binding, Blockade of platelet GPIb with the monoclonal and-CPIb antibody, 6D1, completely inhibited shea r stress-induced platelet-vWF attachment, In contrast, blockade of GPI Ib-IIIa with monoclonal anti-GPIIb-IIIa antibodies, 10E5 or c7E3, or w ith the GPIIb-IIIa-blocking tetrapeptide, RGDS, had little or no inhib itory effect on platelet-vWF binding, These data demonstrate that the binding of vWF to GPIb is likely to be the initial shear-induced plate let-ligand binding event. (C) 1997 Elsevier Science Ltd.