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

The intracellular thiol protease mu-calpain exists as a heterodimeric proenzyme, consisting of a large 80-kDa catalytic subunit and a smaller 30 kDa regulatory subunit, Activation of mu-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 mu-calpain within the cell, Using antibodies that can distinguish among the 80-, 78, and 76-kDa forms of mu-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 pp60(c-src) proteolysis lagged well behind that of PTP-1B and correlated closely with the generation of the fully proteolyzed form of mu-calpain (76 kDa), In citro proteolysis experiments with purified mu-calpain and immunoprecipitated PTP-1B or pp60(c-src) confirmed selective proteolysis of pp60(c-src) by the 76-kDa enzyme, whereas PTP-1B cleavage was mediated by both the 76- and 78-kDa forms of mu-calpain. Studies using selective pharmacological inhibitors against the different autolytic forms of mu-calpain have demonstrated that the initial conversion of the mu-calpain large subunit to the 78-kDa form is responsible for the reduction in platelet-mediated clot retraction, whereas complete proteolytic activation of mu-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 mu-calpain within the cell, that have unique substrate specificities and distinct functional roles.