CHARACTERIZATION OF THE UNIQUE MECHANISM MEDIATING THE SHEAR-DEPENDENT BINDING OF SOLUBLE VON-WILLEBRAND-FACTOR TO PLATELETS

We have studied the mechanism of interaction between soluble von Wille brand factor (vWF), labeled with fluorescein isothiocyanate (FITC), an d platelets exposed 60 shear in a cone-and-plate viscometer. A flow cy tometer calibrated with fluorescent bead standards was used 60 calcula te the number of molecules associated with each platelet in suspension . To validate the methods and reagents used, binding of the same label ed vWF was assessed in the presence of ristocetin or alpha-thrombin an d found to be saturable, with a narrow and symmetric distribution on > 90% of the platelets. As expected, essentially all bound ligand intera cted exclusively with platelet membrane glycoprotein (GP) Ib alpha in the presence of ristocetin and with GP IIb-IIIa after stimulation with alpha-thrombin. In contrast, only a minor proportion (<20%) of the pl atelets exposed to shear were found to bind vWF, with no evidence for saturation and markedly decreased interaction when the platelet count was below 100,000 mu l. Moreover, shear-induced vWF binding was blocke d equally effectively by selected monoclonal antibodies against either GP Ib alpha or GP IIb-IIIa or against the respective binding sites in VWF. Thus, both receptors are involved in the process, possibly throu gh initial transient interactions mediated by GP Ib alpha that lead to platelet activation and subsequent irreversible binding supported by GP IIb-IIIa. While the levels of shear stress theoretically applied to platelets in these experiments are above those thought to occur in th e normal circulation, our findings demonstrate a unique vWF binding me chanism that is not mimicked by other known modulators and correlates with platelet aggregation, Similar processes may occur in response to lower shear stress when platelets are exposed to thrombogenic surfaces and agonists generated at sites of vascular injury during thrombus fo rmation.