Abstract
Nitrosylation of sulfhydryl (SH) groups of cysteine (Cys) moieties is an important post-translational modification (PTM), often on a par with phosphorylation. S-Nitrosoalbumin (ALB-Cys34SNO; SNALB) in plasma and S-nitrosohemoglobin (Hb-Cysβ93SNO; HbSNO) in red blood cells are considered the most abundant high-molecular-mass pools of nitric oxide (NO) bioactivity in the human circulation. SNALB per se is not an NO donor. Yet, it acts as a vasodilator and an inhibitor of platelet aggregation. SNALB can be formed by nitrosation of the sole reduced Cys group of albumin (Cys34) by nitrosating species such as nitrous acid (HONO) and nitrous anhydride (N2O3), two unstable intermediates of NO autoxidation. SNALB can also be formed by the transfer (S-transnitrosylation) of the nitrosyl group (NO+) of a low-molecular-mass (LMM) S-nitrosothiol (RSNO) to ALB-Cys34SH. In the present study, the effects of LMM thiols on the inhibitory potential of ALB-Cys34SNO on human washed platelets were investigated. ALB-Cys34SNO was prepared by reacting n-butylnitrite with albumin after selective extraction from plasma of a healthy donor on HiTrapBlue Sepharose cartridges. ALB-Cys34SNO was used in platelet aggregation measurements after extended purification on HiTrapBlue Sepharose and enrichment by ultrafiltration (cutoff, 20 kDa). All tested LMM cysteinyl thiols (R-CysSH) including l-cysteine and L-homocysteine (at 10 µM) were found to mediate the collagen-induced (1 µg/mL) aggregation of human washed platelets by SNALB (range, 0–10 µM) by cGMP-dependent and cGMP-independent mechanisms. The LMM thiols themselves did not affect platelet aggregation. It is assumed that the underlying mechanism involves S-transnitrosylation of SH groups of the platelet surface by LMM RSNO formed through the reaction of SNALB with the thiols: ALB-Cys34SNO + R-CysSH ↔ ALB-Cys34SH + R-CysSNO. Such S-transnitrosylation reactions may be accompanied by release of NO finally resulting in cGMP-dependent and cGMP-independent mechanisms.
Highlights
Human serum albumin (HSA) or human plasma albumin (ALB) is one of the most abundant circulating proteins with numerous physiological functions and roles in health and various diseases including cardiovascular disease (Chien et al 2017; Argues 2020)
CysSH, CysSH-Et, hCysSH, CysGly and GSH each at 10 μM did not change considerably the collagen-induced aggregation of washed human platelets in the absence of SNALB or in the absence of ODQ as compared to physiological saline which served as a control; maximum aggregation: 69 ± 4, 61 ± 5, 68 ± 2, 67 ± 3, 69 ± 4 and 73 ± 6%, respectively
The soluble guanylyl cyclase (sGC) inhibitor ODQ is commonly used at a concentration of 10 μM in platelet aggregation and vasodilation experiments to demonstrate involvement of nitric oxide (NO)
Summary
Human serum albumin (HSA) or human plasma albumin (ALB) is one of the most abundant circulating proteins with numerous physiological functions and roles in health and various diseases including cardiovascular disease (Chien et al 2017; Argues 2020). Based on the nM-concentrations of SNALB (< 200 nM; Tsikas et al 1999a, 2002; Tsikas 2008) and NTALB (< 20 nM) (Tsikas and Duncan 2014) in plasma of healthy and ill subjects, S-nitrosylation of Cys and nitration of Tyr in albumin are considered rather minor from a quantitative point of view. The pathophysiological roles of NTALB remain still unexplored; circulating NTALB is considered a biomarker of oxidative and nitrosative stress (Tsikas and Duncan 2014). In contrast to NTALB, circulating SNALB is considered a major reservoir of nitric oxide (NO)-related bioactivity due to its potential to release NO from its S-nitroso group on Cys under certain conditions possibly leading to vasodilation and platelet anti-aggregation (Giustarini et al 2007). Using ODQ, which is considered a relatively specific inhibitor of the soluble guanylyl cyclase (sGC), SNALB and S-nitroso-cysteine (CysSNO) were found to inhibit the aggregation of human platelets in platelet-rich plasma and of washed platelets in part by activating intra-platelet sGC and in part by inhibiting intraplatelet synthesis of thromboxane A2 (TxA2) (Tsikas et al 1999a, b, c), one of the most potent endogenous activators of platelet aggregation, an antagonist of NO and prostacyclin (PGI2)
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