Phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets is increased by nitrite and partially deoxygenated erythrocytes

Figures

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  Fig 1. Increased P-VASPSer239 induced by DEANONOate but not nitrite in PRP. Representative Western blot bands of P-VASPSer239 and VASP expression in PRP treated with 10 μM nitrite (A) or 1 μM DEANONOate (B) and quantification of P-VASPSer239/VASP (C) at 37 °C for 5, 10, 15, 20, 40 and 60 minutes are shown. Data are mean ± SEM (n = 3). *P < 0.05 tested by Student’s t-test.

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  Fig 2. Nitrite increased P-VASPSer239 but not P-VASPSer157 in the presence of deoxygenated erythrocytes. Quantification of baseline P-VASPSer239/ VASP (A) and P-VASPSer157/ VASP (B) in platelets from PRP, PRP + erythrocytes (20% hematocrit) with or without deoxygenation, representative Western blot bands of P-VASPSer239 (C), P-VASPSer157 (D), VASP expression and quantification of P-VASPSer239/VASP (E) and P-VASPSer157/VASP (F) in control and nitrite treated deoxygenated PRP + erythrocytes (20% hematocrit) at different time points are shown in this figure. PRP + erythrocytes (20% hematocrit) were deoxygenated by helium for 10 minutes (pO2 ~ 25 mmHg). Nitrite (10 μM) was added in deoxygenated samples and incubated for 5, 10, 15, 20, 30, 40 and 60 minutes. Data are mean ± SEM (n = 7). *P < 0.05 compared with PRP and tested by one-way ANOVA with Tukey’s multiple comparison. #P < 0.05 compared with control and tested by paired Student’s t-test.

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  Fig 3. Nitrite effects on P-VASPSer239 in platelets in the presence of deoxygenated erythrocytes at different hematocrit values. Quantification of P-VASPSer239/VASP (A) and fold increase (B) of P-VASPSer239 expression in control and nitrite treated platelets at different hematocrit values are shown. PRP alone or PRP + erythrocytes (10, 15, 20, 30 and 40% hematocrit) were deoxygenated by helium. Nitrite (10 μM) was added to deoxygenated samples and incubated at 37 °C for 10 minutes. Data are mean ± SEM (n = 5). *P < 0.05 compared with control and tested by paired Student’s t-test. #P < 0.05 compared with 0% hematocrit and tested by one-way ANOVA with Tukey’s multiple.

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  Fig 4. Erythrocytes diminished P-VASPSer239 induced by NO donors. Representative Western blot bands of P-VASPSer239 and VASP expression in platelets (A) and quantification of P-VASPSer239/VASP (B) and fold increase of P-VASPSer239 expression (C) in platelets in PRP and whole blood treated with DEANONOate and sodium nitroprusside are shown. DEANONOate (1 μM) and sodium nitroprusside (10 μM) were incubated in PRP or venous whole blood (WB) at 37 °C for 5 minutes. Data are mean ± SEM (n = 4). *P < 0.05 compared with baseline (BL) and tested by one-way ANOVA with Tukey’s multiple comparison. #P < 0.05 compared with PRP and tested by paired Student’s t-test. DEA/NO = DEANONOate, SNP = Sodium nitroprusside.

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  Fig 5. Comparative effects of nitrite and NO donors in deoxygenated whole blood. Quantification of P-VASPSer239/VASP at baseline (A) and after treatment of deoxygenated whole blood with nitrite, DEANONOate and sodium nitroprusside (B) are shown. Whole blood was deoxygenated by helium for 10 minutes. Nitrite (10 μM) was incubated in deoxygenated whole blood for 15 minutes while DEANONOate (1 μM) and sodium nitroprusside (10 μM) were incubated for 5 minutes, respectively. Data are mean ± SEM (n = 5). *P < 0.05 compared with PRP and tested by one-way ANOVA with Tukey’s multiple comparison. #P < 0.05 compared with control and tested by one-way ANOVA with Tukey’s multiple comparison.