Annexin V distribution on the surface of activated platelets. Platelets (5 × 104 μL−1) were activated with thrombin at 100 nM for 15 minutes, incubated with Annexin V-FITC for 5 minutes, and analyzed using flow cytometry and confocal microscopy. (A) Typical histograms of Annexin V binding to activate platelets in the absence (red line) or presence of EDTA (black line). Region marked M1 indicates the PS-positive platelets. (B) Distribution of Annexin V on the surface of activated platelets. Representative confocal images of DIC, FITC (green) fluorescence of Annexin V, and PE (red) fluorescence of CD61 are shown. Upper row is a typical nonprocoagulant platelet, and lower row is a typical procoagulant “ballooned” platelet. See also supplemental Video 1. Scale bars, 5 μm. (С) Quantitation of Annexin V-FITC binding to the “cap” and to the “balloon” for 3 different donors. The numbers below the bar graphs here and in the remaining figures are donor numbers. (D) Comparison between the sizes of the “caps” and the “balloons” for 20 donors. Results in (C-D) are mean ± standard deviation (SD), determined for at least 50 platelets in each experiment. PE, phycoerythrin.
