Increased bleeding time and aggregation defects in platelets from E2-treated mice. (A) Tail bleeding times of ovariectomized (-E2), sham-operated (Sham), and ovariectomized E2-treated mice (+E2) were monitored. The experiment was stopped after 30 minutes if no cessation of blood flow occurred (> 30). Each point represents 1 individual. (B) Aggregation of washed platelets from ovariectomized mice treated or not with E2 was initiated by the addition of thrombin, the thromboxane A2 analog U46619, collagen, or the GPVI agonist convulxin and assessed with a chrono-log dual-channel aggregometer with stirring at 900 rev/min. Tracings are representative of at least 5 independent experiments. (C) Scanning electron micrographs of platelets from ovariectomized mice treated or not with E2 and stimulated by thrombin in suspension and under nonaggregating conditions (scale bar: 1 μm). Filopodia length was measured using ImageJ software from scanning electron micrographs. Platelets from E2-treated mice stimulated by thrombin (0.3 IU/mL) or U46619 (3μM) exhibit longer filopodia than platelets from ovariectomized mice. Results are mean values ± SEM from 3 independent experiments and at least 75 platelets. ***Significant difference (P < .001). (D) The binding of labeled fibrinogen to platelets from ovariectomized mice treated or not with E2 and activated by thrombin (0.1 and 0.3 IU/mL) for 10 minutes was measured by flow cytometry. A representative example of 3 independent experiments is shown. R indicates resting. (E) Platelets were activated as described in panel B then fixed with 1.5% paraformaldehyde for 30 minutes at room temperature and observed using differential interference contrast with a Nikon Eclipse TE 2000-U equipped with a 10× objective and a DXM 1200 digital camera (scale bar: 40 μm).