The Antiplatelet Action of S-Nitroso Human Serum Albumin in Whole Blood

Abstract

Nitric oxide donors (NO-donors) have been shown to have therapeutic potential (e.g., ischemia/reperfusion injury). However, due to their release rate/antiplatelet properties, they may cause bleeding in patients. We therefore studied the antiplatelet effects of the two different NO-donors, i.e., S-NO-Human Serum Albumin (S-NO-HSA) and Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate) in whole blood (WB) samples. WB samples were spiked with S-NO-HSA or DEA-NONOate (100 µmol/L or 200 µmol/L), and the NO release rate (nitrite/nitrate levels via HPLC) and antiplatelet efficacy (impedance aggregometry, platelet function analyzer, Cone-and-platelet analyzer, thrombelastometry) were assessed. S-NO-HSA had a significantly lower NO release compared to equimolar concentrations of DEA-NONOate. Virtually no antiplatelet action of S-NO-HSA was observed in WB samples, whereas DEA-NONOate significantly attenuated platelet function in WB. Impedance aggregometry measurements revealed that Amplitudes (slope: -0.04022 ± 0.01045 ohm/µmol/L, p = 0.008) and Lag times (slope: 0.6389 ± 0.2075 s/µmol/L, p = 0.0051) were dose-dependently decreased and prolonged by DEA-NONOate. Closure times (Cone-and-platelet analyzer) were dose-dependently prolonged (slope: 0.3738 ± 0.1403 s/µmol/L, p = 0.0174 with collagen/ADP coating; slope: -0.5340 ± 0.1473 s/µmol/L, p = 0.0019 with collagen/epinephrine coating) by DEA-NONOate. These results in WB further support the pharmacological potential of S-NO-HSA as an NO-donor due to its ability to presumably prevent bleeding events even at high concentrations up to 200 µmol/L.

Keywords: impedance aggregometry; nitric oxide donors; platelet function.

Figure 1

Nitrite/nitrate amounts in the presence of increasing concentrations of S-NO-HSA or DEA-NONOate in WB. The increase in the sum of nitrite and nitrate amounts in µmol/L served as a measure for NO release from the NO-donors in WB. Significantly higher amounts of NO were released by DEA-NONOate (100 and 200 µmol/L final concentration) than by equimolar amounts of S-NO-HAS after 6 min aggregation. ANOVA and Bonferroni post-test were used to evaluate differences in plasma levels of nitrite/nitrate. The results are presented as median with range (n = 6). *** p ≤ 0.001.

Figure 2

Effects of increasing concentrations of S-NO-HSA or DEA-NONOate on impedance aggregometry values in WB. Panel (A): Addition of S-NO-HSA had no effect on amplitudes, while the addition of equimolar amounts of DEA-NONOate led to decreased amplitudes. Linear regression analysis showed that slopes differed significantly (p = 0.0009. Panel (B): Addition of S-NO-HSA had no influence on Slopes while the addition of equimolar amounts of DEA-NONOate caused a dose-dependent decrease of Slopes. Linear regression analysis showed that slopes differ significantly (p = 0.0009). Panel (C): Addition of S-NO-HSA had no effect on Lag times, while the addition of equimolar amounts of DEA-NONOate led to prolonged lag times. Linear regression analysis showed that the slopes differ significantly (p = 0.0061). The results are presented as mean ± SD; n = 16 (S-NO-HSA), n = 13 (DEA-NONOate). The data at 0 µmol/L represent the values of the appropriate controls (no NO-donor added).

Figure 3

Effects of increasing concentrations of S-NO-HSA or DEA-NONOate on PFA 200 values in WB. Panel (A): Utilizing cartridges coated with collagen/ADP, addition of S-NO-HSA (n = 12) had no effect on CloTs, while the addition of equimolar concentrations of DEA-NONOate (n = 8) led to dose-dependently prolonged CloTs. Linear regression analysis showed that slopes differ significantly (p = 0.02639). Panel (B): Utilizing cartridges coated with collagen/epinephrine, addition of S-NO-HSA (n = 12) had no effect on CloTs, while the addition of equimolar amounts of DEA-NONOate (n = 10) led to prolonged CloTs. Linear regression analysis showed that the slopes differ significantly (p = 0.006435). The results are presented as mean ± SD. The data at 0 µmol/L represent the values of the appropriate controls (no NO-donor added).

Figure 4

Effects of increasing concentrations of S-NO-HSA or DEA-NONOate on nitrite levels and on impedance aggregometry amplitudes in PRP. Panel (A): Nitrite levels dose-dependently increased in the presence of increasing concentrations of S-NO-HSA (n = 6). At 20 µmol/L DEA-NONOate, nitrite levels were 21.59 (16.51–32.75) µmol/L (p < 0.001 vs. physiological sodium chloride; data not illustrated in the figure). ANOVA and Bonferroni post-test were used to evaluate differences in plasma nitrite levels. The results are presented as median with range. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Panel (B): Both S-NO-HSA (n = 17) and DEA-NONOate (n = 8) efficiently inhibited platelet aggregation. Virtually no platelet aggregation occurred in the presence of 20 µmol/L S-NO-HSA and 10 µmol/L DEA-NONOate (n = 8), respectively. The results are presented as mean ± SD. The data at 0 µmol/L represent the values of the appropriate controls (no NO-donor added).