Shedding of Procoagulant Microparticles from Platelets through Integrin Outside-In Signaling Involving WASP and the Actin Cytoskeleton.

Platelet activation by potent calcium-mobilizing agents, like collagen and thrombin or ionomycin, results in Ca²⁺- and calpain-dependent shedding of microparticles (MP) that are active in coagulation. Here, we describe and characterize a different mechanism of MP formation by platelets under storage in the absence of agonists. This shedding relies on integrin αIIbβ3 activity, since clinically used integrin blockers, like tirofiban or abciximab, reduce storage-induced MP shedding by 83±5.7% (p=0.006); inhibition of intracellular integrin signaling via Src kinase (PP1) or phosphoinositide 3-kinase (PI-3 kinase, wortmannin) inhibits this process by 52.3±14.3% (p=0.035) and 39±7% (p=0.005), respectively. Calpain inhibition suppresses MP formation by only 35±3.8% (p=0.003), while calpain activity is also not increased during storage. Levels of cytosolic Ca²⁺ concentration remain at 25–30 nM in the stored platelets. Treatment of platelets with recombinant WASP (Wiskott-Aldrich Syndrome Protein) peptide, activating the Arp2/3 complex and promoting actin nucleation, affects storage-induced MP shedding (p=0.001). Rho and Rac1, belonging to the family of Rho GTP-binding proteins, are involved in actin cytoskeleton regulation and platelet spreading. However, these GTP-binding proteins do not seem to be involved in MP shedding, as treatment of platelets with recombinant Rac1 peptide or with Rho kinase inhibitor is ineffective, as in the absence of these GTP-binding proteins. Furthermore, incubation of platelets with cytochalasin D, latrunculin B or jasplakinolide, which compounds affect actin filament polymerization, increases the amount of procoagulant MP (p<0.032). Interestingly, this effect is greatly reverted in the presence of integrin blockers or wortmannin. We determine that coagulation-active MP are abundantly present in stored platelet preparations that are used for transfusion purposes. The contribution of these MP to the coagulation process was studied after transfusion of these preparations to thrombocytopenic patients. Thrombin generation measurements indicate that the transfused MP initiate up to 25 nM concentrations of thrombin, which potentiate the procoagulant effect of platelets in these patients. Taken together, we conclude that the process of integrin-mediated MP shedding from apparently resting platelets is a uniquely regulated cellular response that operates independently of Ca²⁺ elevation and calpain activation, and contributes to coagulant activity in a clinically relevant way. It involves αIIbβ3 outside-in signaling, leading to WASP-mediated destabilization of the actin cytoskeleton, but independently of Rho and Rac1.