Dataset Information

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.

SUBMITTER: Tsikas D

PROVIDER: S-EPMC8107154 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundThere has been much interest in targeting intracellular redox pathways as a therapeutic approach for cancer. Given recent data to suggest that the redox status of extracellular protein thiol groups (i.e. exofacial thiols) effects cell behavior, we hypothesized that redox active anti-cancer agents would modulate exofacial protein thiols.Methodology/principal findingsTo test this hypothesis, we used the sesquiterpene lactone parthenolide, a known anti-cancer agent. Using flow cytometry, and western blotting to label free thiols with Alexa Fluor 633 C(5) maleimide dye and N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM), respectively, we show that parthenolide decreases the level of free exofacial thiols on Granta mantle lymphoma cells. In addition, we used immuno-precipitation techniques to identify the central redox regulator thioredoxin, as one of the surface protein thiol targets modified by parthenolide. To examine the functional role of parthenolide induced surface protein thiol modification, we pretreated Granta cells with cell impermeable glutathione (GSH), prior to exposure to parthenolide, and showed that GSH pretreatment; (a) inhibited the interaction of parthenolide with exofacial thiols; (b) inhibited parthenolide mediated activation of JNK and inhibition of NFkappaB, two well established mechanisms of parthenolide activity and; (c) blocked the cytotoxic activity of parthenolide. That GSH had no effect on the parthenolide induced generation of intracellular reactive oxygen species supports the fact that GSH had no effect on intracellular redox. Together these data support the likelihood that GSH inhibits the effect of parthenolide on JNK, NFkappaB and cell death through its direct inhibition of parthenolide's modulation of exofacial thiols.Conclusions/significanceBased on these data, we postulate that one component of parthenolide's anti-lymphoma activity derives from its ability to modify the redox state of critical exofacial thiols. Further, we propose that cancer cell exofacial thiols may be important and novel targets for therapy.

Project description:Nonsurgical bleeding is the most frequent complication of left ventricular assist device (LVAD) support. Supraphysiologic shear rates generated in LVAD causes impaired platelet aggregation, which increases the risk of bleeding. The effect of shear rate on the formation size of platelet aggregates has never been reported experimentally, although platelet aggregation size can be considered to be directly relevant to bleeding complications. Therefore, this study investigated the impact of shear rate and exposure time on the formation size of platelet aggregates, which is vital in predicting bleeding in patients with an LVAD. Human platelet-poor plasma (containing von Willebrand factor, vWF) and fluorochrome-labeled platelets were subjected to a range of shear rates (0-10 000 s-1 ) for 0, 5, 10, and 15 minutes using a custom-built blood-shearing device. Formed sizes of platelet aggregates under a range of shear-controlled environment were visualized and measured using microscopy. The loss of high molecular weight (HMW) vWF multimers was quantified using gel electrophoresis and immunoblotting. An inhibition study was also performed to investigate the reduction in platelet aggregation size and HMW vWF multimers caused by either mechanical shear or enzymatic (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13-ADAMTS13, the von Willebrand factor protease) mechanism under low and high shear conditions (360 and 10 000 s-1 ). We found that the average size of platelet aggregates formed under physiological shear rates of 360-3000 s-1 (200-300 μm2 ) was significantly larger compared to those sheared at >6000 s-1 (50-100 μm2 ). Furthermore, HMW vWF multimers were reduced with increased shear rates. The inhibition study revealed that the reduction in platelet aggregation size and HWM vWF multimers were mainly associated with ADAMTS13. In conclusion, the threshold of shear rate must not exceed >6000 s-1 in order to maintain the optimal size of platelet aggregates to "plug off" the injury site and stop bleeding.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets