Distinct substrate specificities and functional roles for the 78- and 76-kDa forms of mu-calpain in human platelets.

Abstract The intracellular thiol protease μ-calpain exists as a heterodimeric proenzyme, consisting of a large 80-kDa catalytic subunit and a smaller 30-kDa regulatory subunit. Activation of μ-calpain requires calcium influx across the plasma membrane and the subsequent autoproteolytic conversion of the 80-kDa large subunit to a 78-kDa “intermediate” and a 76-kDa fully autolyzed form. Currently, there is limited information on the substrate specificities and functional roles of these distinct active forms of μ-calpain within the cell. Using antibodies that can distinguish among the 80-, 78-, and 76-kDa forms of μ-calpain, we have demonstrated a close correlation between the autolytic generation of the 78-kDa enzyme and the proteolysis of the non-receptor tyrosine phosphatase, PTP-1B, in ionophore A23187-stimulated platelets. Time course studies revealed that pp60c- src proteolysis lagged well behind that of PTP-1B and correlated closely with the generation of the fully proteolyzed form of μ-calpain (76 kDa). In vitroproteolysis experiments with purified μ-calpain and immunoprecipitated PTP-1B or pp60c- src confirmed selective proteolysis of pp60c- src by the 76-kDa enzyme, whereas PTP-1B cleavage was mediated by both the 76- and 78-kDa forms of μ-calpain. Studies using selective pharmacological inhibitors against the different autolytic forms of μ-calpain have demonstrated that the initial conversion of the μ-calpain large subunit to the 78-kDa form is responsible for the reduction in platelet-mediated clot retraction, whereas complete proteolytic activation of μ-calpain (76 kDa) is responsible for the shedding of procoagulant-rich membrane vesicles from the cell surface. These studies demonstrate the existence of multiple active forms of μ-calpain within the cell, that have unique substrate specificities and distinct functional roles.