Specific phosphatidylserine exposure and microparticules shedding induced by a synthetic peptide

Abstract number: P1079

Adam^* F., Huisse^* M. G., Verbeuren† T. J., Fauchere† J. L., Guillin^* M. C., Jandrot-Perrus^* M.

^*INSERM E9907, France; †Institut de Recherches Servier, France

Platelets activated by strong agonists expose phosphatidylserine (PS) on the outer leaflet of their membrane and provide a surface for the assembly of the tenase and prothrombinase complexes that are responsible for the production of FXa and thrombin, respectively. Loss of membrane asymmetry is accompanied by the shedding of microparticules which increases the procoagulant surface. Intracellular events leading to redistribution of phospholipids remain unclear but are associated to signaling pathways as different as those coupled to thrombin or collagen receptors. We observed that a synthetic peptide (Bta-Cha-Cha-RY-NH2) specifically induced phospholipid redistribution. BCCRY is a PAR1 antagonist that blocks platelet activation induced by thrombin and SFLLRN. However, platelet incubation with 100 mM BCCRY rapidly induced a strong staining by FITC-annexin V (88% positive platelets) and the formation of microparticules. Using a prothrombinase assay, we observed that thrombin formation was catalyzed by BCCRY-treated platelets with a rate of 0.2 ± 0.01 pool min^-1 compared to about 0 on nonstimulated platelets. A similar effect of BCCRY was observed on red blood cells and RBL-2H3 cells indicating that PS exposure was not cell specific and was independent of PAR1. Changes in BCCRY-triggered intracellular calcium concentration measured using Fura2-am loaded platelets were limited to a very slow entry of external calcium in BCCRY-treated platelets. However, BCCRY triggered calpain activation as indicated by the generation of activation products detected by immunoblotting and by the hydrolysis of cytoskeletal proteins. Calpeptin inhibited BCCRY-induced calpain activation and procoagulant activity and decreased microparticules shedding. Further investigations using inhibitors of signaling enzymes showed that PAO and Y27632 reduced the procoagulant activity of BCCRY-treated platelets indicating the participation of phosphatases and of Rho-kinase in this process. BCCRY may thus be a useful tool to identify the mechanisms leading to the loss of membrane asymmetry.