TSP-1 deficiency has no effect on platelet activation. (A) (i) Platelet-rich plasma (PRP) from WT (red bars) and TSP-1^−/− (blue bars) mice were stimulated with thrombin (0.01-0.1 U/mL), and platelet aggregation was measured under constant stirring (1000 rpm) at 37°C for 4 minutes. Percentage aggregation is presented as mean ± standard error of the mean (SEM); N = 5). (ii) PRP from WT and TSP-1^−/− mice were stimulated with thrombin (0.001-0.025 U/mL) for 20 minutes, and JonA binding was assessed by using flow cytometry. Data are presented as percent positive platelets (N = 5). (iii) as in (ii), except TLT-1 surface expression (α-granule secretion) was measured. Data are presented as percent positive cells (n = 5). (B) as in (A) except (i), where aggregation was induced by collagen (2.5 or 10 μg/mL), and (ii-iii), where CRP-XL 1 and 5 µg/mL were used (N = 4). (C) PRP from WT (red line) and TSP-1^−/− (blue line) mice (incubated with apyrase [2 U/mL]) were treated with PGI₂ (0-100 nM) for 1 minute before stimulation with collagen (10 μg/mL), and platelet (N = 5) aggregation was recorded over 3 minutes. (i) Percent aggregation is presented as mean ± SEM (P < .05; N = 5). (ii) Representative traces using PGI₂ (5 nM). (D) PRP from WT (red) and TSP-1^−/− (blue) mice (incubated with apyrase [2 U/mL]) were treated with PGI₂ (10 nM) for 1 minute before stimulation with CRP-XL (10 μg/mL) for 20 minutes, and JonA binding was measured. Data are presented as percent positive platelets; mean ± SEM (N = 6; *P < .05, Mann-Whitney U test₂). (E) as in (D), except surface expression of TLT-1 was measured by flow cytometry (N = 6; *P < .02).