Nitrite circumvents platelet resistance to nitric oxide in patients with heart failure preserved ejection fraction and chronic atrial fibrillation

Figures

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  Figure 1. Staining of RBCs and leukocytes in a preparation of washed platelets. RBCs and platelets were stained with 0.2 µg anti-CD235a PE-conjugated antibody or with IgG isotype control. Representative overlay plots are shown (RBCs on the left and platelets on the right) (A). A gate was drawn to include 97–99% of RBCs and applied to the washed platelet sample; cells in the gate were counted as RBCs contaminating the preparation (B, n = 8). RBCs were lysed in whole blood and the remaining cells or a suspension of washed platelets were stained with APC-conjugated anti-CD45 or with IgG isotype control. Representative overlay plots are shown (blood on the left and platelets on the right) (C) A gate was drawn around the leukocyte population (D, left panel) and applied to the washed platelet sample stained with CD45 or isotype control (D, centre and right panel, respectively) (n = 3). (E) Assessment of plasma protein contamination was determined in washed platelets (first and second wash) and compared with PRP (n = 3).

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  Figure 2. Platelet aggregation is inhibited by NaNO2. 2 × 108/ml washed platelets from HVs, HFpEF-AF and chronic AF alone were incubated with increasing concentrations of NaNO2 or SNP for 5 min, activated with 3 µg/ml collagen (A and B) and studied by light transmission aggregometry. Statistical differences were determined by repeated measures two-way ANOVA with Dunnett’s test for multiple comparisons with the control dose values (**P < 0.01, ****P < 0.0001; n = 8–10). (C) Platelet responses to nitrite and SNP in the HFpEF-AF group. The difference between nitrite (1 mM) and SNP (10 µM) responses to collagen with their respective control HFpEF were analysed. *P < 0.05 unpaired t test with Welch’s correction (n = 8–10).

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  Figure 3. Representative platelet aggregation traces for experiments performed from Figure 2 are shown. Platelet responses to nitrite and SNP from HVs (A and B, HV), HF with preserved ejection fraction with chronic AF (C and D, HFpEF-AF) and chronic AF (E and F, chronic AF).

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  Figure 4. NaNO2 effects on aggregation depend on sGC. 2 × 108/ml washed platelets from HVs (n = 8–11) were incubated with NO scavengers (A) 100 µM PTIO or (B) 10 µM OxyHb immediately before addition of 1 mM NaNO2 or 100 nM SNP, or (C) sGC inhibitor 10 µM ODQ 15 min. 2 × 108/ml washed platelets from HFpEF-AF patients (n = 8–9) were incubated with (D) PTIO, or (E) OxyHB or (F) ODQ. Aggregation was triggered by 3 µg/ml collagen. Representative traces for all the performed experiments are shown in Supplementary material online, Figure S2. Repeated measures one-way ANOVA with Dunnett’s test for multiple comparisons was performed to compare SNP and nitrite to control. Differences between control scavengers/inhibitors to nitrite was evaluated by Wilcoxon matched-pairs signed rank test (*P < 0.05, **P < 0.01, ***P < 0.001).

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  Figure 5. NaNO2 triggers phosphorylation of VASP. 5 × 108/ml washed platelets from healthy young volunteers were incubated with increasing concentrations of NaNO2 or SNP and lysed by addition of SDS containing 5× sample buffer at the indicated time point. Aliquots of the lysates were used for SDS-PAGE (10%). Blots were probed with anti p-VASP Serine 239 (1:1000) or α-tubulin (1:5000) and the appropriate secondary antibodies (anti-rabbit or anti-mouse 1:10000). Representative blots (A) and densitometrical analysis of all the experiments performed (B, n = 4–5) are shown.

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  Figure 6. NaNO2 upregulates VASP serine-239 phosphorylation NO-independently. 5 × 108/ml washed platelets from HVs (A and B; n = 8–9) or HFpEF-AF patients (C and D; n = 10) were incubated with ODQ, PTIO, or OxyHb before addition of NaNO2 for 10 min and lysed by addition of 5× sample buffer. Aliquots of the lysates were used for SDS-PAGE (10%) and western blot as in Figure 5. Representative blots (A and C) and densitometrical analysis of all the experiments performed (B and D) are shown. Statistical differences were determined by one-way ANOVA and Dunnet’s post-test. Differences within groups (control NO scavengers/sGC inhibitors vs. nitrite) were evaluated by Wilcoxon matched-pairs signed rank test (*P < 0.05; **P < 0.01; ***P < 0.001).

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  Figure 7. NaNO3 has no inhibitory effects in washed platelets 5 × 108 /ml washed platelets from HVs were incubated with increasing concentrations of NaNO3 (A, n = 5) for 5 min, activated with 3 µg/ml collagen. 5 × 108/ml washed platelets from healthy subjects were incubated with 1 mM NaNO3, lysed after 10 min and used for SDS-PAGE and western blot as above. A representative blot is shown (B, n = 4).