TSSC6 is expressed on the surface and in an intracellular pool of platelets in complex with integrin α_IIbβ₃. (A-B) Flow cytometric analysis of TSSC6 surface expression on resting and thrombin-stimulated (0-5 U/mL) wild-type murine platelets. P-selectin exposure on thrombin-stimulated (1 U/mL) wild-type murine platelets in the absence and presence of FITC-antimouse P-selectin mAb is included as a positive control marker of alpha granule release. Platelets were stained with the indicated anti–murine 14A6 TSSC6 monoclonal antibody followed by a secondary FITC-conjugated antirat antibody. Data were collected through a live platelet gate based on forward versus side scatter profiles on a FACS Calibur flow cytometer. Results are cumulative data derived from 3 independent experiments and presented as mean fluorescence intensity (MFI) ± SEM (n = 3). (C) 1.5 mg of human platelet lysates solubilized with indicated detergents were immunoprecipitated with antibodies directed against normal mouse IgG₁, PECAM-1, GPIb-IX-V complex, and integrin complex-specific α_IIbβ₃ Ab, P2. Samples were resolved on 12.5% SDS-PAGE under reducing conditions and Western blotted for TSSC6 with a polyclonal anti–human TSSC6 antibody.