Extra-platelet low-molecular-mass thiols mediate the inhibitory action of S-nitrosoalbumin on human platelet aggregation via S-transnitrosylation of the platelet surface

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-Cys^β93SNO; 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.

Keywords: Aggregation; PTM; Platelets; S-Nitrosothiols; Thiols.