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Volume 272, Number 40, Issue of October 3, 1997 pp. 24876-24884
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Distinct Substrate Specificities and Functional Roles for the 78- and 76-kDa Forms of µ-Calpain in Human Platelets

(Received for publication, May 12, 1997, and in revised form, July 7, 1997)

Simone M. Schoenwaelder , Suhasini Kulkarni , Hatem H. Salem , Shinobu Imajoh-Ohmi ^¶ , Wakako Yamao-Harigaya , Takaomi C. Saido and Shaun P. Jackson ^**

From the  Department of Medicine, Monash Medical School, the Australian Centre for Blood Diseases, and the ^** Department of Pathology, Box Hill Hospital, Victoria 3128, Australia, the ^¶ Institute of Medical Science, University of Tokyo, Tokyo 108, and the  Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan

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 pp60^c-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 vitro proteolysis experiments with purified µ-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 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.

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