Figure 5. RAP1 and integrin activation in Rasa3 mutant platelets is insensitive to inhibitors of the P2Y12/PI3K signaling pathway.

(A) Aggregation of WT, Caldaggef1^–/–, or Caldaggef1^–/– Rasa3^hlb/hlb platelets stimulated with 250 μM Par4-activating peptide in the presence or absence or 100 μM 2-MeSAMP or 200 nM wortmannin. Aggregation was monitored for 3 minutes. (B) Levels of activated RAP1 (RAP1-GTP) and AKT (phosphorylated AKT [AKT-P]) in WT, Caldaggef1^–/–, or Caldaggef1^–/– Rasa3^hlb/hlb platelets stimulated for 3 minutes with Par4p in the presence of vehicle, 2-MeSAMP, or wortmannin. Total RAP1 and AKT are shown for loading controls. (C) Real-time flow cytometry analysis of αIIbβ3 activation in WT, Caldaggef1^–/–, or Caldaggef1^–/– Rasa3^hlb/hlb platelets stimulated with Par4p. The blue box highlights delayed αIIbβ3 activation in Caldaggef1^–/– and Caldaggef1^–/– Rasa3^hlb/hlb platelets. (D) Effect of ADP or 2-MeSAMP on Par4p-induced αIIbβ3 activation kinetics in Caldaggef1^–/– Rasa3^+/+ or Caldaggef1^–/– Rasa3^hlb/hlb platelets. (E) Effect of P2Y12 inhibition on αIIbβ3 activation in Rasa3^hlb/hlb platelets stimulated for 10 minutes with the indicated concentrations of Par4p. Data represent MFI ± SD in activated blood samples after subtraction of MFI measured in nonactivated cells. ***P < 0.0001, 2-way ANOVA with Bonferroni post-test (n = 6). (F) Aggregation of Caldaggef1^+/– Rasa3^+/hlb or Caldaggef1^+/– Rasa3^hlb/hlb platelets (in PRP at 1.5 × 10⁸ platelets/ml) stimulated with 3 μM ADP, 0.1 μM MRS-2365 (P2Y1 agonists), or MRS-2365 plus 1 μM epinephrine (α_2A-R agonist). Results in A–D and F are representative of 4 independent experiments.