The effects of calpeptin (a calpain specific inhibitor) on agonist induced microparticle formation from the platelet plasma membrane.

Platelets activated by various agonists produce formation of vesicles shed from the plasma membrane (microparticles). However, the mechanism of microparticle (MP) formation has not been clarified yet. The aim of the present study was to determine the possibility of involvement of calpain (a Ca(2+)-dependent thiol protease) in MP formation. Washed platelets preincubated with calpeptin, a cell permeable calpain specific inhibitor, or with a vehicle were activated by thrombin plus collagen or by calcium ionophore A23187. Flow cytometry was used to detect the amount of microparticle formation by using murine monoclonal antibodies against GP IIb-IIIa or GP IIb and fluorescein 5-isothiocyanate labeled goat anti-mouse IgG. MP formation stimulated either by thrombin plus collagen or by A23187 was inhibited by calpeptin in a dose dependent manner. The microparticle formation from platelets activated by A23187 reached a plateau in approximately 5 min after activation, whereas that from platelets activated by thrombin plus collagen reached a plateau at 30 min following the stimulation. These time sequences corresponded well with those of degradation of actin-binding protein (ABP), a well known substrate of calpain, of platelets activated by these two stimulations. However, the inhibition of MP formation by calpeptin was more marked in the early stage (within 10 min) than in the late stage (after 30 min) of platelet activation. At 30 min after platelet activation by either two stimulations, a significant amount of microparticle formation was observed in the presence of 30 microM calpeptin, which inhibited hydrolysis of ABP almost completely. Our data suggest the involvement of calpain in the early stage (especially within 10 min) of microparticle formation.