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.
Pasquet
J M
JM
URA 1464 CNRS, Hôpital Cardiologique du Haut-Lëvèque, Pessac, France.
Dachary-Prigent
J
J
Nurden
A T
AT
D001692:Biological Transport; D001777:Blood Coagulation / Q000502:physiology*; D001792:Blood Platelets / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002154:Calpain / Q000378:metabolism*; D003094:Collagen / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D010316:Particle Size; D015539:Platelet Activation / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology
D000001:Calcimycin; D003094:Collagen; D013917:Thrombin; D002154:Calpain; D000252:Calcium-Transporting ATPases; D002118:Calcium