Figure 4.
PS exposure mediates platelet integrin αIIbβ3 activation in response to Hcy stimulation. (A) Calcein-AM–labeled platelets and iFluor 555-annexin V antibodies were injected into C57BL/6J mice with or without Hcy (100 mg/kg) injection (left). Representative images of FeCl3-induced mesenteric arteriole thrombosis (green indicates platelets, red indicates annexin V right, the dotted line indicates the arterial vessel wall, original magnification, 400×). The relative quantitation for PS exposure (HHcy-MFI/control-MFI) in vivo. (B-C) Washed platelets from C57BL/6J mice were incubated with or without Hcy (100 μM) for 20 minutes and then stimulated with thrombin (0.01 U/mL) or collagen (1.5 μg/mL) at 37°C for 30 minutes. Representative immunofluorescence staining (left) and quantitation (right) of PS exposure were analyzed by image-capture flow cytometry. All data are expressed as the mean ± SEM (n = 3). *P < .05 compared with ctrl; #P < .05 compared with thrombin or collagen. (D-E) JON/A binding after 20 μM A01 or vehicle (DMSO, no greater than 0.1%) treatment of 10 minutes, followed by stimulation with (D) thrombin (Th, 0.01 U/mL) or collagen (Co, 1.5 μg/mL) (E) in the presence/absence of Hcy (100 μM) was assessed by flow cytometry. All data are expressed as the mean ± SEM (n = 5). *P < .05 compared with agonists; #P < .05 compared with Hcy+agonists. (F-G) Platelets from C57BL/6J mice were aggregated after stimulation with 20 μM A01 or vehicle, followed by collagen (1.5 μg/mL) (F) or thrombin (0.01 U/mL) (G) stimulation in the presence/absence of Hcy on a turbidimetric aggregometer. (H) Washed platelets from C57BL/6J mice were allowed to adhere and spread on fibrinogen-coated wells in the presence of 100 μM Hcy or vehicle with A01 (20 μM). The spreading area of individual platelets (stainted with phalloidin, original magnification, 200×) was measured using ImageJ software. All data are expressed as the mean ± SEM (n = 5). *P < .05 compared with ctrl; #P < .05 compared with Hcy. (I-J) p-AKTSer473 and p-SRCTyr416 protein levels in platelets, with or without Hcy, were analyzed after treatment with 20 μM A01 for 10 minutes with collagen (1.5 μg/mL) (I) or thrombin (0.01 U/mL) (J) by western blot analysis. All data are expressed as the mean ± SEM (n = 3-5). *P < .05 compared with agonists; #P < .05 compared with Hcy+agonists. MFI, mean fluorescence intensity.

PS exposure mediates platelet integrin αIIbβ3 activation in response to Hcy stimulation. (A) Calcein-AM–labeled platelets and iFluor 555-annexin V antibodies were injected into C57BL/6J mice with or without Hcy (100 mg/kg) injection (left). Representative images of FeCl3-induced mesenteric arteriole thrombosis (green indicates platelets, red indicates annexin V right, the dotted line indicates the arterial vessel wall, original magnification, 400×). The relative quantitation for PS exposure (HHcy-MFI/control-MFI) in vivo. (B-C) Washed platelets from C57BL/6J mice were incubated with or without Hcy (100 μM) for 20 minutes and then stimulated with thrombin (0.01 U/mL) or collagen (1.5 μg/mL) at 37°C for 30 minutes. Representative immunofluorescence staining (left) and quantitation (right) of PS exposure were analyzed by image-capture flow cytometry. All data are expressed as the mean ± SEM (n = 3). *P < .05 compared with ctrl; #P < .05 compared with thrombin or collagen. (D-E) JON/A binding after 20 μM A01 or vehicle (DMSO, no greater than 0.1%) treatment of 10 minutes, followed by stimulation with (D) thrombin (Th, 0.01 U/mL) or collagen (Co, 1.5 μg/mL) (E) in the presence/absence of Hcy (100 μM) was assessed by flow cytometry. All data are expressed as the mean ± SEM (n = 5). *P < .05 compared with agonists; #P < .05 compared with Hcy+agonists. (F-G) Platelets from C57BL/6J mice were aggregated after stimulation with 20 μM A01 or vehicle, followed by collagen (1.5 μg/mL) (F) or thrombin (0.01 U/mL) (G) stimulation in the presence/absence of Hcy on a turbidimetric aggregometer. (H) Washed platelets from C57BL/6J mice were allowed to adhere and spread on fibrinogen-coated wells in the presence of 100 μM Hcy or vehicle with A01 (20 μM). The spreading area of individual platelets (stainted with phalloidin, original magnification, 200×) was measured using ImageJ software. All data are expressed as the mean ± SEM (n = 5). *P < .05 compared with ctrl; #P < .05 compared with Hcy. (I-J) p-AKTSer473 and p-SRCTyr416 protein levels in platelets, with or without Hcy, were analyzed after treatment with 20 μM A01 for 10 minutes with collagen (1.5 μg/mL) (I) or thrombin (0.01 U/mL) (J) by western blot analysis. All data are expressed as the mean ± SEM (n = 3-5). *P < .05 compared with agonists; #P < .05 compared with Hcy+agonists. MFI, mean fluorescence intensity.

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