Figure 3.
A high-affinity state of integrin αIIbβ3 mediates HHcy-induced platelet activation and thrombosis in mice. (A) Flow cytometric analysis of PE-JON/A binding to control and Hcy-incubated platelets from C57BL/6J mice stimulated with thrombin (0.01 U/mL). (B) Flow cytometric analysis of fluorescein isothiocyanate-labeled fibrinogen binding to control and Hcy-incubated platelets from C57BL/6J mice stimulated with thrombin (0.01 U/mL). MFI, mean fluorescence intensity. (C) 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 or without thrombin (Th), at 37°C for 2 hours. Representative images (left, stained with phalloidin, original magnification, 400×); surface areas of single platelets (right). (D) Western blot analysis of p-AKTSer473 and p-SRCTyr416 protein expression and quantification. β-Actin was used as an internal control. (E) Washed platelets from C57BL/6J mice were solubilized at the indicated time points and the amount of β3 that coimmunoprecipitated with Gα13 was analyzed by western blot (n = 3-5). *P < .05 compared with ctrl; #P < .05 compared with thrombin. (F) p-AKTSer473 and p-SRCTyr416 protein levels in platelets with or without Hcy stimulation and treatment with 10 μM mP6 for 10 minutes with thrombin (0.01 U/mL) were analyzed by western blot (n = 3). *P < .05 compared with agonists; #P < .05 compared with HHcy+agonists. OD, optical density. (G) Platelet aggregation with 10 μM mP6 or control peptide in the presence/absence of Hcy was monitored with a turbidimetric aggregometer. (H) Washed platelets from C57BL/6J mice were allowed to adhere and spread on fibrinogen-coated wells with 100 μM Hcy in the presence of mP6 (20 μM). The spreading area of individual platelets (stainted with phalloidin, original magnification, 200×) was measured with ImageJ software (n = 3-5). *P < .05 compared with ctrl; #P < .05 compared with Hcy. (I-K) ApoE−/− mice were intraperitoneally injected with 100 mg/kg Hcy or vehicle (saline), and after 2 hours, the mice were treated with mP6 liposomes (10 μmol/kg) or vehicle (control peptide liposomes). (I) 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). Tail-bleeding time was determined (J), and blood loss was assessed (K). HGB, hemoglobin. All data are expressed as the mean ± SEM (n = 5). *P < .05 compared with ApoE−/−; #P < .05 compared with ApoE−/−+HHcy. PLT, platelets.

A high-affinity state of integrin αIIbβ3 mediates HHcy-induced platelet activation and thrombosis in mice. (A) Flow cytometric analysis of PE-JON/A binding to control and Hcy-incubated platelets from C57BL/6J mice stimulated with thrombin (0.01 U/mL). (B) Flow cytometric analysis of fluorescein isothiocyanate-labeled fibrinogen binding to control and Hcy-incubated platelets from C57BL/6J mice stimulated with thrombin (0.01 U/mL). MFI, mean fluorescence intensity. (C) 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 or without thrombin (Th), at 37°C for 2 hours. Representative images (left, stained with phalloidin, original magnification, 400×); surface areas of single platelets (right). (D) Western blot analysis of p-AKTSer473 and p-SRCTyr416 protein expression and quantification. β-Actin was used as an internal control. (E) Washed platelets from C57BL/6J mice were solubilized at the indicated time points and the amount of β3 that coimmunoprecipitated with Gα13 was analyzed by western blot (n = 3-5). *P < .05 compared with ctrl; #P < .05 compared with thrombin. (F) p-AKTSer473 and p-SRCTyr416 protein levels in platelets with or without Hcy stimulation and treatment with 10 μM mP6 for 10 minutes with thrombin (0.01 U/mL) were analyzed by western blot (n = 3). *P < .05 compared with agonists; #P < .05 compared with HHcy+agonists. OD, optical density. (G) Platelet aggregation with 10 μM mP6 or control peptide in the presence/absence of Hcy was monitored with a turbidimetric aggregometer. (H) Washed platelets from C57BL/6J mice were allowed to adhere and spread on fibrinogen-coated wells with 100 μM Hcy in the presence of mP6 (20 μM). The spreading area of individual platelets (stainted with phalloidin, original magnification, 200×) was measured with ImageJ software (n = 3-5). *P < .05 compared with ctrl; #P < .05 compared with Hcy. (I-K) ApoE−/− mice were intraperitoneally injected with 100 mg/kg Hcy or vehicle (saline), and after 2 hours, the mice were treated with mP6 liposomes (10 μmol/kg) or vehicle (control peptide liposomes). (I) 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). Tail-bleeding time was determined (J), and blood loss was assessed (K). HGB, hemoglobin. All data are expressed as the mean ± SEM (n = 5). *P < .05 compared with ApoE−/−; #P < .05 compared with ApoE−/−+HHcy. PLT, platelets.

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