Figure 2.

Epac1^−/− mice have fewer, but more reticulated platelets and more agonist-responsive blood platelets than WT mice. (A) Scanning electron micrographs of resting and thrombin-activated platelets from WT and Epac1^−/− mice. The platelets had been incubated for 10 min with vehicle (control) or 0.07 U/ml thrombin. The bars represent 1 μm. OCS: open canalicular system. (B) The average platelet diameter of resting platelets from WT and Epac1^−/− mice (WT: n = 3 mice with 28 platelet diameters measured, Epac1^−/− : n = 3 mice with 26 platelet diameters measured.) (C) Platelets from WT (n = 9) and Epac1^−/− (n = 10) mice were identified and counted by flow cytometry using the platelet specific marker CD41 and forward- and side scatter. (D,E,F) Reticulated platelet count in platelet-rich plasma from WT and Epac1^−/− mice. Platelets were stained with thiazole orange without (D) or with (E) RNAse treatment prior to staining. The platelets were then analyzed by flow cytometry, and the percent of reticulated platelets determined as described in the methods section. (F): Box-plot showing the reticulated platelet fraction from WT- and Epac1^−/− mice. The data are average of three mice from each group. P = 0.068, Student’s t-test. Platelets from Epac1^−/− or WT mice were exposed to various concentrations of thrombin (G), ADP (H) or collagen (I), and analyzed for P-selectin externalization by flow cytometry. Data shown are average + /− SEM from three independent experiments. (J) The gating strategy for the flow cytometric analyses, including histogram (right panel) showing a shift in mean fluorescence intensity after treatment with 0.1 U/ml Thrombin (light grey). Dark grey: unsitmulated platelets (K) Whole blood from WT (n = 8) and Epac^−/− (n = 8) mice were used in an ADP-induced aggregation assay. Data shown are average + /− SEM. OCS; open canalicular system. P; platelets. P-sel. + ; P-selectin positive. *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t-test.