Inhibition of Calpain Blocks Platelet Secretion, Aggregation, and Spreading

Abstract

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 ␣-granule secretion (IC 50 ‫؍‬ 20 M) during the first 30 s of activation, thrombin-induced platelet aggregation (IC 50 ‫؍‬ 50 M), and platelet spreading on glass surfaces (IC 50 ‫؍‬ 34 M). 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.

Genetic variation of the platelet- surface integrin GPIIb-IIIa ( PIA1/A2- SNP) shows a high association with Type 2 diabetes mellitus

“…Agonistdependent calpain activation is recognized as part of the outside-in stimulus response pathways being implicated in the procoagulant activity of activated platelets [17]. Recently, this enzyme has been shown to proteolyse structural and signalling proteins involved in cytoskeleton remodelling and platelet activation events downstream to α 2 β 3 -integrin (GPIIb-IIIa) activation [18,19]. Unfortunately, we do not have parallel genomic calpain data available in this limited cohort study, but our results of a tight genetic association of the α 2 β 3 -integrin (GPIIb-IIIa) physiology (not exclusively restricted to platelets) with Type 2 diabetes could provide an additional pathway as to how genetic variations in the calpain system might functionally operate.…”

Calpain Cleavage Promotes Talin Binding to the β3Integrin Cytoplasmic Domain

“…Several reports implicate calpain in the activation of integrins (33,39). Indeed, calpain-deficient platelets exhibit a defect in platelet aggregation, an integrin ␣ IIb ␤ 3 activationdependent process (33).…”

Haplotype Combinations of Calpain 10 Gene Polymorphisms Associate With Increased Risk of Impaired Glucose Tolerance and Type 2 Diabetes in South Indians

“…Studies using cysteine protease inhibitors have implicated members of the calpain family as being involved in either the promotion (28) or inhibition of protein secretion (29), depending on the cell type. A role of calpains in insulin secretion from mouse pancreatic islets has been demonstrated using protease inhibitors affecting exocytosis of insulin (30,31).…”