Calcium Influx is a Determining Factor of Calpain Activation and Microparticle Formation in Platelets

This is the authors' abstract. We don't add key points for this paper.

We have related the release of procoagulant microparticles from platelets to calcium movement and the activation of the Ca(2+)-dependent protease calpain. The effects of the Ca(2+)-ATPase inhibitors thapsigargin, cyclopiazonic acid and 2.5-di-(t-butyl)-1,4-benzohydroquinone were compared with those of the Ca2+ ionophore A23187. Whereas all three Ca(2+)-ATPase inhibitors induced aminophospholipid exposure on platelets, only thapsigargin and cyclopiazonic acid promoted microparticle formation and only when strong Ca2+ influx, calpain activation and proteolysis of cytoskeletal proteins occurred concomitantly. Preincubation with dibutylbenzohydroquinone inhibited the responses to thapsigargin and cyclopiazonic acid but not to A23187. When platelets were suspended in a Ca(2+)-free medium, calpain activation and microparticle formation were not observed, even with maximum mobilisation of internal Ca2+ stores by A23187. Incubation of fluo-3-loaded plateters with A23187 in 0.1 mM EGTA followed by the sequential addition of 25 microM Ca2+ increments to the medium showed that calpain activation occurred when the intraplatelet [Ca2+] reached 3-8 microM. To assess the physiologic significance of these results, the subpopulation of platelets that expressed procoagulant activity after stimulation by a thrombin/collagen mixture was isolated by means of annexin-V-coupled magnetic beads. Subsequent western blotting experiments confirmed that this subpopulation contained activated calpain. Overall, our results provide evidence that microparticle formation and calpain activation require an elevated intraplatelet [Ca2+] that is brought about by influx across the plasma membrane.