Inhibition of calpain blocks platelet secretion, aggregation, and spreading

Previous studies have indicated that the Ca2+-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but speci fic roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpasta tin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrat ing signal sequence from Kaposi's fibroblast growth factor connected to a c alpain-inhibiting consensus sequence derived from calpastatin.Calpastat spe cifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC50 = 20 mu M) during the first 30 s of activation, thrombin-induced pla telet aggregation (IC50 = 50 mu M), and platelet spreading on glass surface s (IC50 = 34 mu M). Calpastat-Ala, a mutant peptide in which alanine is sub stituted at conserved calpastatin residues, lacks calpain inhibitory activi ty and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secret ion, aggregation and spreading, but require 3-10fold higher concentrations than calpastat for biological activity. Together, these findings demonstrat e that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation followi ng thrombin receptor stimulation than had been previously detected.