Downregulation of platelet signaling induced by Cvx. Washed P1 platelets in suspension were activated by Cvx (200 or 800 pM) for 3 minutes in the absence of stirring and in the presence of apyrase (2 U/mL) and indomethacin (10 μM). (A) Ca²⁺ mobilization was assessed in unstirred platelets preincubated with the cytosolic Ca²⁺ fluorescent probe Oregon green BAPTA-AM after stimulation by Cvx (200-800 pM) by flow cytometry in conditions of no external Ca²⁺ (1 mM EGTA). (B-C) Tyrosine phosphorylation of Akt (p-S473), Syk (p-Y525/526), PLCγ2 (p-Y1217), and FcRγ was assessed by immunoblotting using anti-Akt-P, anti-Syk-P, anti-PLCγ2-P, and anti-phosphotyrosine antibodies, respectively. Results are representative of 2 experiments. (D) Lyn phosphorylation on Y396 was assessed by immunoblotting using an anti-Lyn-P. Data represent mean ± SEM of 4 independent experiments (*P < .05, **P < .01, unpaired Student t test).