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.

• Calpain
• plasma membrane Ca²⁺-ATPase
• platelet activation
• proteolysis
• signal transduction