Gas6 signaling through its receptors Tyro3, Axl, and Mer. (A) Gas6 promotes phosphorylation of its receptors (shown for Axl), PI3K, Akt, and β₃ integrin in WT but not in Tyro3^–/–, Axl^–/– (not shown), or Mer^–/– (not shown) platelets. Platelets were incubated with 400 ng/ml human recombinant Gas6 (hrGas6) for 3 minutes. For detection of Axl or PI3K phosphorylation, platelets were lysed and phosphotyrosine-containing proteins were immunoprecipitated. The precipitates were then separated by SDS-PAGE and Western-blotted (WB) with anti-Axl or anti-PI3K antibodies. For Akt and β₃ integrin phosphorylation studies, lysed platelets in sample buffer were subjected to SDS-PAGE and Western-blotted with anti–phospho-Akt antibody, anti–total Akt antibody, anti–β₃ integrin [pY⁷⁷³] phosphospecific antibody, or anti–β₃ integrin antibody. (B) β₃ Integrin levels measured by flow cytometry were comparable on the surface of WT and Tyro3^–/–, Axl^–/–, or Mer^–/– platelets (only Tyro3^–/– data are shown). Black lines denote controls; red shading denotes platelets stained with PE-conjugated anti–β₃ integrin antibody. A representative example of 3 independent experiments is shown. (C) β₃ Integrin tyrosine phosphorylation in response to thrombin in WT and Tyro3^–/– platelets. Platelets were stimulated with increasing concentrations of thrombin for 3 minutes. A representative example of 3 independent experiments is shown.