Summary. Background: Calpains are implicated in a wide range of cellular functions including the maintenance of hemostasis via the regulation of cytoskeletal modifications in platelets.
Objectives: Determine the functional role of calpain isoforms in platelet spreading.
Methods and Results: Platelets from calpain-1−/− mice show enhanced spreading on collagen- and fibrinogen-coated surfaces as revealed by immunofluorescence, differential interference contrast (DIC) and scanning electron microscopy. The treatment of mouse platelets with MDL, a cell permeable inhibitor of calpains 1/2, resulted in increased spreading. The PTP1B-mediated enhanced tyrosine dephosphorylation in calpain-1−/− platelets did not fully account for the enhanced spreading as platelets from the double knockout mice lacking calpain-1 and PTP1B showed only a partial rescue of the spreading phenotype. In non-adherent platelets, proteolysis and GTPase activity of RhoA and Rac1 were indistinguishable between the wild-type (WT) and calpain-1−/− platelets. In contrast, the ECM-adherent calpain-1−/− platelets showed higher Rac1 activity at the beginning of spreading, whereas RhoA was more active at later time points. The ECM-adherent calpain-1−/− platelets showed an elevated level of RhoA protein but not Rac1 and Cdc42. Proteolysis of recombinant RhoA, but not Rac1 and Cdc42, indicates that RhoA is a calpain-1 substrate in vitro.
Conclusions: Potentiation of the platelet spreading phenotype in calpain-1−/− mice suggests a novel role of calpain-1 in hemostasis, and may explain the normal bleeding time observed in the calpain-1−/− mice.