CEACAM2 is expressed on the surface and in intracellular pools in murine platelets. (A) Flow cytometric analysis of CEACAM2 surface and total expression on resting murine platelets. Platelets were stained with a polyclonal anti-murine CEACAM2 2052 antibody followed by a secondary PE-conjugated anti-rabbit antibody. Normal rabbit serum was included as a negative control. For total expression, platelets were resuspended in 0.1% (wt/vol) saponin and then washed with a combination of 0.1% (wt/vol) saponin and 0.2% (wt/vol) bovine serum albumin. Data were collected by a live platelet gate based on forward vs side scatter profiles on a FACS Canto II flow cytometer. Results are cumulative data derived from 4 independent experiments and represented as mean fluorescence intensity (MFI) ± SEM (**P < .01; n = 4). (B) CEACAM2 surface expression upon agonist stimulation of murine platelets using thrombin (0.125-1.0 U/mL), PAR-4 agonist peptide (100-300 µM), and collagen-related peptide (CRP; 1.0-4.0 µg/mL) over a dose-dependent range (**P < .01 and ***P < .001; n = 4). CEACAM2 surface expression was determined as described in (A). (C) Platelet lysates from wild-type and Cc2^−/− mice were analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using 1:2000 of rabbit anti-mouse CEACAM2 polyclonal antibody (2052) (upper panel), followed by reprobing with GAPDH antibody to control for protein loading (bottom panel). A ∼52-kDa band representing CEACAM2 and another one (band X) at ∼95kDa representing an unidentified protein were detected. (D) Flow cytometric analysis of PECAM-1, CEACAM1, and CEACAM2 expression on resting wild-type vs Cc2^−/− platelets. Wild-type and Cc2^−/− platelets were stained with a monoclonal anti-murine PECAM-1 antibody, polyclonal anti-murine CEACAM1 2457 antibody, or polyclonal anti-murine CEACAM2 2052 antibody followed by a secondary PE-conjugated anti-rat or anti-rabbit antibody. Normal rabbit serum (NRS) and isotype control antibody CD3 were included as negative controls. Data were collected by a live platelet gate based on forward vs side scatter profiles on a FACS Canto II flow cytometer. Results are cumulative data derived from 4 independent experiments and represented as MFI ± SEM (**P < .01; n = 4). (E) Cell-surface expression of platelet glycoproteins was monitored by flow cytometry using specific monoclonal antibodies for wild-type and Cc2^−/− platelets. Platelets were preincubated with anti-mouse integrin β₃, CD61 (10 µg/mL), anti-mouse integrin α₂β₁, CD49b (15 µg/mL), anti-mouse GPIbα/IX/V, CD42b (10 µg/mL), anti-mouse CD44 (10 µg/mL), anti-mouse GPVI (10 µg/mL), and anti-mouse CD9 (10 µg/mL). MFI was reported with an SEM for at least 4 independent experiments and no significant difference demonstrated. (F) Total expression of platelet glycoproteins on resting wild-type and Cc2^−/− murine platelets was determined as described in (A). Antibodies concentrations were described in (E). (G) Cell-surface expression of CEACAM1 upon agonist stimulation of wild-type vs Cc2^−/− murine platelets was determined as described in (B).