Integrin αIIbβ3-mediated outside-in signaling is reduced in EphB2^LacZ platelets. (A) The effect of deletion of EphB2 intracellular domains in clot retraction was analyzed using PRP from control and EphB2^LacZ mice for 2 hours. (B) Clot weight measured after 2 hours. Data represent mean ± SD (n = 3). (C) Integrin β3 was immunoprecipitated from resting and CRP-XL (0.5 µg/mL)–stimulated control and EphB2^LacZ platelets and analyzed for its association with myosin by immunoblot analysis. (D) Phosphorylation of integrin β3 in resting and CRP-XL (0.5 µg/mL)– or thrombin (0.1 U/mL)–activated control and EphB2^LacZ platelets was analyzed using phospho-specific integrin β3 antibody for pY773. The protein 14-3-3ζ was analyzed as a protein loading control by immunoblot analysis. (E) β3 integrin was coimmunoprecipitated with EphB2 in control and EphB2^LacZ platelets, and analyzed for its association by immunoblot. Immunoblots shown are representative of 3 separate experiments. Data represent mean ± SD (n = 3) and the level of phosphorylation obtained in the control (activated) samples was taken as 100%. (F) Washed platelets obtained from control and EphB2^LacZ mice were allowed to spread on fibrinogen-coated cover glass for 45 minutes prior to staining with Alexa Fluor 488 phalloidin and analyzed by confocal microscopy. Images are representative of 3 separate experiments. (G) Multiple images obtained by differential interference contrast microscopy were analyzed by counting numbers of platelets in various stages of spreading using Image J software. P values shown are as calculated by Student t test: *P ≤ .05, **P ≤ .01, and ***P ≤ .001. PRP, platelet-rich plasma.