von Willebrand factor interaction with subendothelial collagens and platelet surface receptor GPIBalpha under shear conditions

Abstract

The molecular mechanisms of binding interaction among von Willebrand factor, subendothelium and platelets under physiological shear conditions are important medically in hemostasis and thrombosis. By binding both subendothelial collagens and platelet surface receptors GPIbalpha, multimeric vWf acts as a bridge between subendothelium and circulating platelets. In this study, the molecular binding between vWf multimers and human pepsin-digested collagen I, III and VI was characterized using Surface Plasmon Resonance (BIAcore). Compared to binding to pepsin-digested collagen III surfaces, normal vWf multimers (nvWf) bound to human pepsin-digested collagen I with a 5-fold lower binding capacity and a 10-fold higher equilibrium dissociation constant. There was no detectable binding between the vWf multimers and human pepsin-digested collagen VI. vWf preparations enriched in either relatively small vWf (svWf) multimers or in unusually large vWf u1vWf multimers were also studied on collagen III surfaces. Our results demonstrate vWf-collagen binding, which involves multi-step processes, is dependent on vWf molecular mass and the duration of interactions. To study the shear-induced vWf interaction with GPIbalpha, the isolated extracellular domain of GPIbalpha, glycocalicin, was covalently immobilized to carboxylate-modified polystyrene beads. These glycocalicin-coated beads had the ability to bind vWf in the presence of ristocetin, botrocetin or high shear conditions. To evaluate shear stress effects on the interaction between vWf and GPIbalpha, we compared shear-induced vWf binding to glycocalicin-coated beads, Chinese hamster ovary cells expressing the wild type and mutant GPIb-IX complex, and washed human platelets. Compared to binding in the presence of botrocetin and ristocetin, shear stress induces only low-level nvWf binding, whereas shear stress can induce significant ulvWf binding. Our studies demonstrate that the GPIbalpha extracellular domain is necessary and sufficient for interaction with soluble vWf under shear stress. The vWf A1 domain binding to glycocalicin, as well as the botrocetin and ristocetin-induced nvWf binding, were also characterized by using Surface Plasmon Resonance. The results indicate that although the intrinsic binding affinity of vWf and glycocalicin is very weak, botrocetin can increase this affinity dramatically. Compared to botrocetin, ristocetin-induced binding is a highly reversible process with low binding affinity.