Shear-induced platelet adhesion through the interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture is a prerequisite for the physiological hemostatic process or pathological thrombus formation in stenosed arteries. Here we show that shear-induced interaction of platelets with immobilized VWF results in GPIbalpha ectodomain shedding. Washed platelets were exposed to VWF-coated glass capillary or cone-and-plate viscometer at different shear rates, and GPIbalpha ectodomain was shed from platelets, while a small mass of GPIbalpha COOH-terminal peptide, approximately 17 kDa, was increased correspondingly. The extent of GPIbalpha shedding was enhanced with the concentration of immobilized VWF and the time duration of constant shear stress, whereas it was obviously reduced with the decreased number of adherent platelets. Pretreatment of platelets with membrane-permeable calpain inhibitors and metalloproteinase inhibitor abolished shear-induced GPIbalpha shedding. Furthermore, GPIbalpha shedding was obviously diminished by anti-integrin-alphaIIbbeta3 monoclonal antibody SZ21, phosphatidylinositol 3-kinase inhibitor wortmannin, and cell-permeable calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. These results indicate that shear-induced platelet-VWF interaction results in calpain and metalloproteinase-dependent GPIbalpha ectodomain shedding. These findings not only have a physiological implication in understanding the presence of glycocalicin in normal circulation, but also suggest a novel mechanism for the negative regulation of platelet function and the limitation of platelet thrombus infinite formation under pathophysiological flow conditions.