PROCOAGULANT ACTIVITY AND ACTIVE CALPAIN IN PLATELET-DERIVED MICROPARTICLES

Microparticles are released during in vitro platelet activation and ha ve been detected in vivo in several pathologies. Their characterizatio n 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 Ca2+-ATPases. Th ey were isolated following centrifugation of the activated platelet su spension over a sucrose layer. Flow cytometric studies using annexin V -FITC as a probe for aminophospholipids, prothrombinase activity measu rements and annexin V inhibition experiments enabled us to evaluate th e procoagulant activity of microparticles prepared in this way. The ef ficiency of the annexin V inhibition (IC50 = 10-20 nM) of this activit y confirmed significant anticoagulant properties for this protein. Mic roparticles 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 Ca2+-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. Ho wever, flow cytometry failed to find significant amounts of active cal pain on the microparticle or on the platelet surface. Our results, whi le confirming the procoagulant activity of microparticles, also docume nt for the first time the exclusive presence of the activated form of calpain, inferring a possible role for this protease in microparticle- mediated functions.