Inhibition of calpain blocks platelet secretion, aggregation, and spreading

Kevin Crocet, Robert Flaumenhaft, Marc Rivers, Bruce Furie, Barbara C. Furie, Ira M. Herman, David A. Potter

Research output: Contribution to journalArticlepeer-review

114 Scopus citations


Previous studies have indicated that the Ca2+-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific 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, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced α-granule secretion (IC50 = 20 μM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC50 = 50 μM), and platelet spreading on glass surfaces (IC50 = 34 μM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.

Original languageEnglish (US)
Pages (from-to)36321-36327
Number of pages7
JournalJournal of Biological Chemistry
Issue number51
StatePublished - Dec 17 1999


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