Elevated plasma Hcy levels are associated with a prothrombotic state in both humans and ApoE^−/− mice. (A-B) Correlations between human plasma Hcy concentrations and the APTT or PT. (C-D) Human plasma TXB2 and sP-selectin levels were measured by enzyme-linked immunosorbent assay (n = 88; TXB2; Cloud-Clone Corp; sP-selectin; ABclonal Technology). (E-G) ApoE^−/− mice were intraperitoneally injected with Hcy (100 mg/kg) or vehicle (saline), and, after 2 hours, tail-bleeding time was determined (E), and blood loss was assessed (F). HGB, hemoglobin. (G) Platelets were labeled with calcein-AM. Representative images of FeCl₃-induced mesenteric arteriole thrombosis (left, original magnification, 200×; the dotted line indicates the arterial vessel wall). Occlusion time of the mesenteric arteriole (right). (H-K) Washed platelets harvested from C57BL/6J mice, with or without Hcy (100 mg/kg) injection for 2 hours were stimulated with ADP (0.01 g/mL) (H), U46619 (400 nM) (I), collagen (1.5 μg/mL) (J), and thrombin (0.01 U/mL) (K), at 37°C with constant stirring (1200 rpm). Then, platelet aggregation was monitored, using a turbidimetric aggregometer. Histograms of maximal platelet aggregation under the indicated conditions. (L-M) Flow cytometric analysis of P-selectin expression in platelets from control and HHcy C57BL/6J mice in the presence of collagen (1.5 μg/mL) (L) or thrombin (0.01 U/mL) (M). All data are expressed as the mean ± SEM (n = 5). *P < .05, compared with ctrl (control); #P < .05, compared with thrombin or collagen. MFI, mean fluorescence intensity.