Calcium influx is a determining factor of calpain activation and microparticle formation in platelets.

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.