Pathological Shear Regulates ADAM10 Activity on Circulating Platelets

Abstract 2194

Metalloproteinase-mediated ectodomain shedding of platelet-specific receptors for collagen (GPVI) and von Willebrand factor (GPIbα of the GPIb-IX-V complex) is triggered by either ligand-induced platelet activation-dependent pathways, or by activation-independent pathways mediated by Factor Xa or induced by the thiol-modifying agent, N-ethylmaleimide. We recently reported that shed soluble GPVI (sGPVI) was elevated in plasma of 159 ischaemic stroke patients compared with 159 community-based controls (P=0.0168), and in 29 patients with disseminated intravascular coagulation compared with healthy donors (n=25, P=0.002), consistent with a pathophysiological role for GPVI shedding from human platelets. Our new studies now show that transient exposure of human platelets to arterial or pathological shear rates of 3000–10,000 s⁻¹ for 1–5 min ex vivo in a cone-plate viscometer, in the absence of GPVI ligand or platelet activation, activated sheddases producing a 2- to 3-fold increase in plasma sGPVI and a corresponding loss of surface GPVI from sheared platelets. Shear-induced GPVI shedding was blocked by GM6001 or GI254023, a selective inhibitor of ADAM10. In contrast to shear-induced platelet aggregation, shedding was unaffected by inhibitors of aggregation (VWF-blocking anti-GPIbα mAb, AK2, or the αIIbβ3 antagonist, RGD peptide) or by the absence of VWF in a patient with von Willebrand's disease Type III (VWF antigen levels <1%). Further, shedding of GPVI increased for up to 10 min after cessation of a short exposure of platelets to shear even when signalling, secretion and aggregation was blocked by inhibiting intracellular kinases (PP2, piceatannol), thromboxane generation (aspirin), ADP (apyrase) and calcium flux (BAPTA). Together, the combined results provide the first evidence that receptor sheddase activity can be regulated by hydrodynamic shear stress independent of cellular activation. This may represent a novel protective mechanism for down-regulating platelet reactivity as a response to pathological shear.