Thrombospondin triggers P-selectin expression and phosphatidyl serine exposure on platelets

Abstract number: P0732

Le Courvoisier-Flaujac C., Adam F., Guillin M.C., Jandrot-Perrus M.

INSERM E9907, France

Thrombospondin-1 (TSP1), a major a-granule protein of platelets, promotes platelet aggregation and stabilizes aggregates (Dixit VM et al. Proc Natl Acad Sci 1985; 82: 3472; Tuszynski GP et al. Blood 1988; 72: 109). A peptide from the C-terminal domain of TSP1, 4N1-1 (RFYVVMWK), has been reported to induce platelet aggregation by a signaling pathway close to that coupled to the collagen receptor glycoprotein (GP)VI (Tulasnes D et al. Blood 2001; 98: 3346). In the present study, we observed that 4N1-1-triggered platelet activation was independent of the engagement of GPVI for it was not prevented by a soluble form of GPVI or blocking anti-GPVI Fab fragments. 4N1-1-induced alpha and dense granules release as measured by P-selectin expression and serotonin secretion. Using Fura-2 loaded platelets, we observed that 4N1-1 triggered a transient peak in intracellular calcium concentration. Moreover, platelet membrane asymmetry was lost upon platelet activation by 4N1-1: phosphatidylserine (PS) exposure was indicated by an intense staining with annexin V-FITC (72% positive platelets) and by the increased procoagulant activity of the activated platelets measured in a prothrombinase assay. To determine if the observations made with 4N1-1 may have a physiologic relevance, we checked whether TSP could reproduce all or part of 4N1-1-induced responses. Purified TSP1 at a concentration up to 120 mg mL^-1 did not induce platelet aggregation or intracellular calcium mobilization. Nevertheless, TSP1 induced P-selectin expression with ~20% positive platelet detected in flow cytometry after incubation with 80 mg mL^-1 TSP. P-Selectin expression was further pronounced when TSP was coincubated with fibrinogen and ADP, positive platelets reaching 40%. In addition, we observed that TSP in the presence of ADP and fibrinogen triggered PS exposure at the platelet surface as evidenced by positive staining with annexin V and prothrombinase activity. These results suggest that TSP1 released by activated platelets, via its C-terminal domain, could promote adhesive properties and procoagulant activity of platelets, amplifying thrombin generation and thrombi formation.