Fig. 4: TgC3G and tgC3GΔCat platelets show differential sensitivities to inhibition of the P2Y12, PI3K, ERK and p38 MAPK signaling pathways.

a Platelets from mice of the different genotypes under study were pretreated with 100 μM clopidogrel for 5 min and then stimulated for 15 min with 1 U/ml thrombin or 10 μM ADP, as indicated. The histograms represent the mean ± SD of the percentage of inhibition of activated integrin αIIbβ3 in platelets treated with agonist + inhibitor compared to platelets treated with agonist. b Platelets were pretreated with 100 nM wortmannin for 5 min and then stimulated with 1 U/ml thrombin for 15 min. The histograms represent the mean ± SD of the percentage of inhibition of activated integrin αIIbβ3 in platelets treated with agonist + inhibitor compared to platelets treated with agonist. c Platelets were pretreated with 100 nM wortmannin or 50 μM 2-MeSAMP for 5 min and then stimulated with 0.5 U/ml thrombin for 5 min. Rap1-GTP was isolated by pulldown with GST-RalGDS-RBD and detected by immunoblotting with anti-Rap1 antibodies. Left panel: representative western blots. Right panel: line/scatter plots of Rap1-GTP levels (n = 2). Values (mean ± SEM) are relative to those in unstimulated wild-type platelets and were normalized to total Rap1 levels. d–f Platelets were pretreated with 20 μM U0126 or SB203580 for 5 min and then stimulated with 1 U/ml thrombin (15 min for activation, 10 min for aggregation), as indicated. The histograms represent the mean ± SD of the percentages of inhibition of the expression of P-selectin on the surface (d), activation of integrin αIIbβ3 (e) or aggregation (f) in platelets treated with thrombin + inhibitor, compared to thrombin-treated platelets. *p < 0.05, **p < 0.01, ***p < 0.001. tg: transgenic; wt: wild-type; T: thrombin; W: wortmannin; MeS: 2-MeSAMP.