The binding of autotaxin to integrins mediates hyperhomocysteinemia-potentiated platelet activation and thrombosis in mice and humans

Figures

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  Figure 1.. 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…

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  Figure 2.. Hcy amplifies platelet aggregation and degranulation in vitro. Washed platelets harvested from C57BL/6J mice were incubated with or without Hcy (100 μM) for 10 minutes and then stimulated with the indicated concentrations of ADP (A), collagen (B), U46619 (C), and thrombin (D) at 37°C, with constant stirring (1200 rpm). Then, platelet aggregation was monitored with a turbidimetric aggregometer. (E-F) ATP secretion was measured in the presence of luciferin/luciferase reagent. The traces in the figures are representative of at least 3 independent experiments. (G-H) Flow cytometric analysis of P-selectin exposure in platelets with or without Hcy stimulation in the presence of thrombin (0.01 U/mL) (G) or collagen (1.5 μg/mL) (H). MFI, mean fluorescence intensity. (I-J) TXB2 production in control and Hcy-treated platelets stimulated with thrombin (0.01 U/mL) (I) or collagen (1.5 μg/mL) (J) was analyzed by enzyme-linked immunosorbent assay. All data are expressed as the mean ± SEM (n = 3-5). *P < .05 compared with ctrl; #P < .05 compared with thrombin or collagen.

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  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…

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  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…

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  Figure 5.. HHcy activates phospholipid hydrolysis of platelet membrane and ATX secretion. (A-E) HPLC-MS/MS analysis of lipid metabolites in platelets from C57BL/6J mice that were intraperitoneally injected with 100 mg/kg Hcy or vehicle (saline) for 2 hours. (A) Principal component analysis (PCA) scatterplot of lipid metabolites in platelets. (B) VIP scatterplot identified by PCA showing the top 15 lipid metabolites in the different groups. (C) Heat map illustrating the phospholipid metabolic profiles in platelets. (D) HPLC-MS/MS analysis of different levels of LPC species in platelets. (E) cPLA2 activity of platelets from acute HHcy and control mice in vivo. (F-H) C57BL/6J mice were intraperitoneally injected with 100 mg/kg Hcy or vehicle (saline) for 2 hours. Plasma ATX (F) and LPA (G) levels were measured via enzyme-linked immunosorbent assay (ELISA; Cloud-Clone Corp). (H) Western blot analysis of ATX expression in platelets. β-Actin was used as an internal control. (I-K) Washed platelets from C57BL/6J were incubated with 100 μM Hcy for 10 minutes, then stimulated with 1.5 μg/mL collagen. The levels of ATX (I) and LPA (J) in platelet-activated supernatant were measured by ELISA. ATX…

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  Figure 6.. Secreted ATX interacts with integrin β3 and mediates Hcy-induced platelet integrin αIIbβ3 activation. Washed platelets were purified from C57BL/6J mice. (A-B) Supernatant ATX levels were measured by enzyme-linked immunosorbent assay (ELISA). All data are expressed as the mean ± SEM (n = 4). *P < .05 compared with agonists; #P < .05 compared with Hcy+agonists. (C-D) Platelet aggregation after HA130 (0.6 μM) stimulation with collagen (1.5 μg/mL) (C) or thrombin (0.01 U/mL) (D) in the presence/absence of Hcy (100 μM) was monitored with a turbidimetric aggregometer. (E) Washed platelets from C57BL/6J mice were left to adhere and spread on fibrinogen-coated wells with 100 μM Hcy or vehicle in the presence of HA130 (0.6 μM). The spreading area of individual platelets (stainted with phalloidin, original magnification, 200×) was measured with ImageJ software. All data are expressed as the mean ± SEM (n = 3-5). *P < .05 compared with ctrl; #P < .05 compared with Hcy. (F-G) p-AKTSer473 and p-SRCTyr416 protein levels in platelets with or without Hcy incubation were analyzed after treatment with 0.6 μM HA130 for 10 minutes with collagen (1.5 μg/mL) (F) or thrombin (0.01 U/mL) (G) by western…

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  Figure 7.. Pyrro and HA130 alleviate arterial platelet thrombus formation in HHcy ApoE−/− mice. (A-D) ApoE−/− mice were intraperitoneally injected with 100 mg/kg Hcy or vehicle (saline), and after 2 hours, the mice were treated with pyrro (5 μmol/kg) or vehicle (DMSO; ≤1:3000/body weight). (A) ATX level in plasma were measured by ELISA. (B) Tail-bleeding time was determined. (C) Blood loss was assessed. HGB, hemoglobin. (D) Platelets were labeled with calcein-AM. Occlusion time of the mesenteric arteriole (left); representative images of FeCl3-induced mesenteric arteriole thrombosis (right, the dotted line indicates the arterial vessel wall, original magnification, 200×). (E-G) ApoE−/− mice were intraperitoneally injected with 100 mg/kg Hcy or vehicle (saline), and after 2 hours, the mice were treated with HA130 (2 μmol/kg) or vehicle (DMSO). Tail-bleeding time was determined (E) and blood loss was assessed (F). (G) Platelets were labeled with calcein-AM. Occlusion time of the mesenteric arteriole (left) and representative images of FeCl3-induced mesenteric arteriole thrombosis (right, the dotted line indicates the arterial vessel wall, original magnification, 200×). All data are expressed…