EphB2 influences calcium mobilization and PI3K signaling. (A) Fluo4-NW–labeled control and EphB2^LacZ mouse whole blood was stimulated with 1 µg/mL CRP-XL and the intracellular calcium levels measured by flow cytometry. Data represent mean ± SD (n = 4). CRP-XL (0.5 µg/mL)–induced phosphorylation of Src (B), Lyn (B), Syk (B), LAT (B), PLCγ2 (C), and AKT (E) was analyzed by immunoblot analysis using phospho-specific antibodies or following immunoprecipitation. Similarly, thrombin (0.1 U/mL)–induced phosphorylation of PLCβ3 (D) and AKT (F) was measured using immunoblot analysis. The protein 14-3-3ζ was detected as a loading control in all of the samples. Blots are representative of 3 separate experiments. The level of phosphorylation obtained with control platelets at 90 seconds was taken as 100%. Ionomycin (5 µM [G] and 10 µM [H])–induced fibrinogen binding was measured in whole blood using flow cytometry. Data represent mean (of median fluorescence intensity) ± SD (n = 3) (Student t test: *P ≤ .01, **P ≤ .01, and ***P ≤ .001). AKT, protein kinase B.