Poster Board III-960
Shear-induced platelet adhesion through the interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture is the first step for the physiological hematosis or pathologic thrombus formation in stenosed arteries. However, the role of shear stress in the regulation of platelet function remains poorly defined. Methods: Washed platelets were exposed to VWF-coated surface at different shear conditions, provided by syringe pump connecting with glass capillary or cone-and-plate viscometer. GPIbα shedding was investigated by flow cytometry and western blot.
GPIbα ectodomain was shed from platelets, while a small mass of GPIbα C-terminal peptide around 17 kDa was increased correspondingly. Although GPIbα ectodomain shedding was found under all shear conditions, the extent of GPIbα shedding was maximum at 250/s and dramatically reduced with increased shear rate, which was consistent with the number of stable adhesive platelets. There was a correlation between the levels of platelet adhesion and the extent of GPIbα shedding. Furthermore, GPIbα shedding increased with the concentration of immobilized VWF and time duration (within 1 minute) of constant shear stress. The potential protease(s) and signaling pathway involved in this process were investigated. Pretreatment of platelets with membrane-permeable calpain inhibitors (MDL28170, calpain inhibitor I and II) and metalloproteinase inhibitor (GM6001) abolished shear-induced GPIbα shedding. Though platelets showed partial activation detected by PAC-1 binding, GPIbα shedding was not impacted by apyrase and PGE1. However, integrin αIIbβ3 antibody (SZ-21) and phosphoinositide 3-kinase inhibitors (wortmanine) obviously inhibited GPIbα shedding. Conclusions: These results indicate that shear-induced platelet-VWF interaction results in calpain and metalloproteinase-dependent GPIbα ectodomain shedding. Fluid shear stress and VWF always exist in both the normal circulation of blood and pathological medical conditions, particularly, the interaction of GPIbα with VWF under flow initiates platelet adhesion and thrombus formation. Thus, the current finding that shear-induced the interaction of GPIbα with VWF incurs GPIbα ectodomain shedding not only has physiological implication in understanding the presence of glycocalicin in normal circulation, but also suggests a novel mechanism for the negative regulation of platelet function and the limitation of platelet thrombus growth under pathophysiological flow conditions.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.