Activated platelets expose FXIII-A on their membrane surface. (A-B) Washed platelets were incubated with FITC-labeled anti-FXIII-A antibody and were left unstimulated (unstim) or stimulated with CVX (100 ng/mL) and TRAP-6 (20 μM) or thrombin (100 nM) for 45 minutes at room temperature before analyzing the number of positive cells by flow cytometry. (A) Representative contour plot showing side scatter (SSC-A) against FXIII-A-intensity or (B) mean data with standard error of the mean (SEM) from 4 experiments. *P < .05; **P < .01 vs unstimulated platelets. (C) Prior to fractionation, platelets were left unstimulated or were stimulated for 45 minutes at 37°C with 200 μM TRAP-6/200 μg/mL collagen. FXIII-A antigen was detected in the membrane and cytoplasm fractions by western blotting. Data are representative of 2 experiments. (D) Washed platelets (5 × 10⁷/mL) were left unstimulated or were activated with 20 μg/mL collagen/20 μM TRAP-6 and stained using FITC-labeled anti-FXIII-A antibody (green) and Alexa-fluor647 Annexin-V to detect phosphatidylserine (red). Scale bar represents 10 μm. (E) PS and FXIII-A co-expressing platelets stimulated by 20 μg/mL collagen/20 μM TRAP-6 or 20 μg/mL collagen/100 nM thrombin. Differential interference contrast (DIC) image is shown and the scale bar represents 5 μm. Results are representative of 4 experiments. (F) PS-negative platelets stimulated with 20 μg/mL collagen/20 μM TRAP-6 or 20 μg/mL collagen/100 nM thrombin. Scale bar represents 5 μm. Results are representative of 4 experiments. (D-F) Images were obtained with a Zeiss LSM710 confocal microscope with a 63 × 1.40 oil immersion objective and were analyzed using Zen 2012 software.