Regulation of plasma membrane Ca2+-ATPase in human platelets by calpain

Abstract

The plasma membrane Ca2+-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca2+ in resting human platelets by extruding Ca2+ from the cytoplasm across the plasma membrane. Since PMCA is the main agent of Ca2+ efflux in platelets, it is a key point for regulation of platelet Ca2+ metabolism. PMCA has been shown to be an excellent substrate for the Ca2+-activated cysteine protease calpain, a major platelet protein that is turned on during platelet activation. The objectives of the present work were to determine if PMCA is degraded during thrombin- and collagen-mediated platelet activation, and if calpain is responsible. The kinetics of PMCA degradation during platelet activation were analysed using SDS polyacrylamide gel electrophoresis and immunoblotting. The role of calpain was tested using the calpain inhibitors calpeptin and ALLN. Platelet activation mediated by both collagen and thrombin resulted in degradation of 60% of platelet PMCA within 18 minutes. Calpeptin and ALLN significantly inhibited the rate and extent of PMCA degradation. We conclude that calpain-mediated degradation of PMCA during platelet activation likely contributes significantly to Ca2+ regulation and, therefore, to platelet function.