CALPAIN CLEAVAGE OF FOCAL ADHESION PROTEINS REGULATES THE CYTOSKELETAL ATTACHMENT OF INTEGRIN ALPHA(IIB)BETA(3) (PLATELET GLYCOPROTEIN IIB IIIA) AND THE CELLULAR RETRACTION OF FIBRIN CLOTS/

The intracellular thiol protease calpain catalyzes the limited proteol ysis of various focal adhesion structural proteins and signaling enzym es in adherent cells. In human platelets, calpain activation is depend ent on fibrinogen binding to integrin alpha(IIb)beta(3) and subsequent platelet aggregation, suggesting a potential role for this protease i n the regulation of postaggregation responses. In this study, we have examined the effects of calpain activation on several postaggregation events in human platelets, including the cytoskeletal attachment of in tegrin alpha(IIb)beta(3), the tyrosine phosphorylation of cytoskeletal proteins, and the cellular retraction of fibrin clots. We demonstrate that calpain activation in either washed platelets or platelet-rich p lasma is associated with a marked reduction in platelet-mediated fibri n clot retraction. This relaxation of clot retraction was observed in both thrombin and ionophore A23187-stimulated platelets. Calcium dose response studies (extracellular calcium concentrations between 0.1 mu M and 1 M) revealed a strong correlation between calpain activation an d relaxed clot retraction. Furthermore, pretreating platelets with the calpain inhibitors calpeptin and calpain inhibitor I prevented the ca lpain-mediated reduction in clot retraction. Relaxed fibrin clot retra ction was associated with the cleavage of several platelet focal adhes ion structural proteins and signaling enzymes, resulting in the dissoc iation of talin, pp60(c-scr), and integrin alpha(IIb)beta(3) from the contractile cytoskeleton and the tyrosine dephosphorylation of multipl e cytoskeletal proteins. These studies suggest an important role for c alpain in the regulation of multiple postaggregation events in human p latelets. The ability of calpain to inhibit clot retraction is likely to be due to the cleavage of both structural and signaling proteins in volved in modulating integrin-cytoskeletal interactions.