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 o f the present study was to determine the possibility of involvement of calpain (a Ca2+-dependent thiol protease) in MP formation. Washed pla telets 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 antibodie s against GP IIb-IIIa or GP IIb and fluorescein 5-isothiocyanate label ed goat anti-mouse IgG. MP formation stimulated either by thrombin plu s collagen or by A23187 was inhibited by calpeptin in a dose dependent manner. The microparticle formation from platelets activated by A2318 7 reached a plateau in approximately 5 min after activation, whereas t hat from platelets activated by thrombin plus collagen reached a plate au at 30 min following the stimulation. These time sequences correspon ded well with those of degradation of actin-binding protein (ABP), a w ell known substrate of calpain, of platelets activated by these two st imulations. However, the inhibition of MP formation by calpeptin was m ore marked in the early stage (within 10 min) than in the late stage ( after 30 min) of platelet activation. At 30 min after platelet activat ion by either two stimulations, a significant amount of microparticle formation was observed in the presence of 30 muM calpeptin, which inhi bited hydrolysis of ABP almost completely. Our data suggest the involv ement of calpain in the early stage (especially within 10 min) of micr oparticle formation.