Platelet-derived, but not plasma-derived, TSP-1 modulates the platelet response to PGI₂. (A) WT and TSP-1^−/− washed platelets were resuspended in either WT or TSP-1^−/− plasma to a concentration of 2 × 10⁸ platelets/mL in the presence of apyrase (2 U/mL). Platelets, in the presence and absence of PGI₂ (5 nM), were stimulated with collagen (10 μg/mL), and aggregation was measured under constant stirring (1000 rpm) at 37°C for 4 minutes. (i) Representative traces and (ii) percent aggregation are presented as mean ± standard error of the mean (SEM). (N = 5; **P < .01 compared with platelets treated with collagen and PGI₂, Mann-Whitney U test.) (B) Platelet-rich plasma from TSP-1^−/− mice (treated with apyrase) were incubated with releasates from WT platelets or TSP-1^−/− platelets (or platelet poor plasma 1:10 vol/vol), then stimulated with collagen (10 μg/mL) in the presence or absence of PGI₂ (5 nM). (i) Representative traces and (ii) percent aggregation are presented as mean ± SEM (N = 5; *P < .05 compared with platelets treated with collagen and PGI₂, Mann-Whitney U test.) (C) as in (B), except platelets were stimulated with collagen (2 μg/mL) in the absence of PGI₂. (D) Platelet-rich plasma from TSP-1^−/− mice (treated with apyrase) were incubated with human platelet-derived TSP-1 (hTSP-1) before treatment with PGI₂ (5 nM) for 1 minute and stimulation with collagen (10 μg/mL). (i) Representative traces; (ii) percent aggregation is presented by mean ± SEM (N = 4, *P < .05, Mann-Whitney U test). Intracellular cAMP levels are presented as mean ± SEM (N = 5, *P < .05, Mann-Whitney U test). NS, not significant.