Procoagulant activity and active calpain in platelet-derived microparticles.
Microparticles are released during in vitro platelet activation and have been detected in vivo in several pathologies. Their characterization is of interest as they may play a potential role in hemostasis. Here, we report the formation of microparticles as the result of increases in intracellular Ca2+ brought about by inhibition of Ca(2+)-ATPases. They were isolated following centrifugation of the activated platelet suspension over a sucrose layer. Flow cytometric studies using annexin V-FITC as a probe for aminophospholipids, prothrombinase activity measurements and annexin V inhibition experiments enabled us to evaluate the procoagulant activity of microparticles prepared in this way. The efficiency of the annexin V inhibition (IC50 = 10-20 nM) of this activity confirmed significant anticoagulant properties for this protein. Microparticles also contained the glycoprotein IIb-IIIa complex, detected in flow cytometry at a density higher than on the remnant platelets. The activation of calpain, a Ca(2+)-dependent protease, in platelets was shown to be more efficient under conditions of a sudden Ca2+ influx. The microparticles contained only the active form of calpain detected by Western blotting using a monoclonal antibody able to recognize both the unactivated and the activated catalytic subunit of the enzyme. However, flow cytometry failed to find significant amounts of active calpain on the microparticle or on the platelet surface. Our results, while confirming the procoagulant activity of microparticles, also document for the first time the exclusive presence of the activated form of calpain, inferring a possible role for this protease in microparticle-mediated functions.
Pasquet
J M
JM
UMR 5533 CNRS, Hôpital Cardiologique du Haut-Lévèque, Pessac, France.
Toti
F
F
Nurden
A T
AT
Dachary-Prigent
J
J
D017304:Annexin A5 / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002154:Calpain / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D015539:Platelet Activation
D017304:Annexin A5; D002154:Calpain; D000252:Calcium-Transporting ATPases; D002118:Calcium