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-Cys(34)SNO; SNALB) in plasma and S-nitrosohemoglobin (Hb-Cys(β93)SNO; 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 (Cys(34)) by nitrosating species such as nitrous acid (HONO) and nitrous anhydride (N(2)O(3)), 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-Cys(34)SH. In the present study, the effects of LMM thiols on the inhibitory potential of ALB-Cys(34)SNO on human washed platelets were investigated. ALB-Cys(34)SNO was prepared by reacting n-butylnitrite with albumin after selective extraction from plasma of a healthy donor on HiTrapBlue Sepharose cartridges. ALB-Cys(34)SNO 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-Cys(34)SNO + R-CysSH ↔ ALB-Cys(34)SH + R-CysSNO. Such S-transnitrosylation reactions may be accompanied by release of NO finally resulting in cGMP-dependent and cGMP-independent mechanisms.