Abstract
To clarify the possible role of calpain (calcium activated neutral protease; EC 3.4.22.17) in Ca 2+ homeostasis of human platelets, we investigated the effects of cell permeable calpain inhibitors, calpeptin and E-64d (EST), on the restoration of cytoplasmic Ca 2+ ([Ca 2+] i) in both Fura-2 and aspirin (ASA) loaded platelets. Although neither calpeptin (30 μM) nor EST (250 μM) altered the increase of [Ca 2+] i in thrombin (1 U/ml) stimulated platelets, both calpain inhibitors delayed the decrease of [Ca 2+] i back towards the basal level. These observations suggested that calpain might be involved in Ca 2+ restoration. Then, the activity of Ca 2+-ATPase was examined in thrombin (2 U/ml) stimulated platelets. Thrombin produced a rapid rise in Ca 2+-ATPase activity by 2-fold at 8 s of incubation, which then returned to below the basal activity within 2 min. Calpeptin inhibited transient Ca 2+-ATPase activation induced by thrombin in a dose related manner. Ca 2+-ATPase of isolated platelet membranes was digested by purified human platelet calpain-I and Ca 2+-ATPase activity was investigated. With a short incubation (8–15 s), Ca 2+-ATPase activity was increased about 2-fold and then it decreased below the basal level at longer incubations or at a higher calpain/membrane ratio. The initial rate of Ca 2+ uptake was also increased by about 2-fold with a short incubation (8–15 s). For molecular characterization of the Ca 2+-ATPase, the formation of the enzyme-phosphate complex (EP) was investigated. The membrane bound intact 105 kD Ca 2+-ATPase was converted by calpain to a fragment of approximately 50 kD. These results indicate that calpain might be involved in Ca 2+ restoration through the activation of dense tubular system Ca 2+-ATPase by limited proteolysis.
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