Carbon monoxide releasing molecule-2 increases fibrinogen-dependent coagulation kinetics but does not enhance prothrombin activity

Abstract

We have previously determined that tricarbonyldichlororuthenium (II) dimer (CORM-2) increases plasma clot velocity of formation and strength by enhancing thrombin-fibrinogen interactions as determined by thrombelastography. The purpose of the present investigation was to further define the nature of CORM-2 interaction with prothrombin and fibrinogen by exposing purified proteins to CORM-2 or generating protein concentration-response curves in the absence or presence of CORM-2. Purified prothrombin was exposed to 0 or 100 micromol/l CORM-2 prior to being added to prothrombin-deficient plasma (n=7-8 per condition). Fibrinogen-deficient plasma had fibrinogen added for a final concentration of 100-800 mg/dl and was exposed to 0 or 100 micromol/l CORM-2 (n=4 per condition). Following tissue factor activation, thrombelastographic data were collected until clot strength stabilized. Exposure of prothrombin to CORM-2 did not significantly enhance coagulation kinetics. In sharp contrast, CORM-2 exposure enhanced fibrinogen coagulation kinetics in a concentration-dependent fashion, with peak effect seen at a fibrinogen concentration of 300 mg/dl that then progressively decreased throughout the range tested. Our data demonstrate that CORM-2 does not enhance plasma coagulation kinetics by modifying prothrombin; instead, the concept that CORM-2 modifies fibrinogen is the most likely explanation for the enhanced thrombin-fibrinogen interactions observed.

Antithrombotic Properties of Water-Soluble Carbon Monoxide-Releasing Molecules

“…This is also confirmed by previous studies by Soni et al 13 Those authors showed that CORM-3 decreased plasma PAI-1 levels, but in their model CORM-3 was administered by guest on May 11, 2018 http://atvb.ahajournals.org/ Downloaded from during a 10-minute infusion at 3 mg/kg (which is equivalent to 100 µmol/kg of CORM-3), thus the CO, which was liberated, could have been in contact with the released from platelets PAI-1. However, Nielsen et al 11,12,44 have shown that CORM-2 inhibits fibrynolysis. Nevertheless, their experiments were preformed only in vitro using this lipid-soluble and fast metalcontaining CO releaser, which makes it difficult to compare with our results obtained with water-soluble CORMs in vivo.…”

“…Indeed, CO paradoxically increases the formation of fibrin 11 and inhibits fibrinolysis 12 in in vitro studies, whereas it exerts antithrombotic effect in vivo, which involves activation of fibrinolysis. 13 Notably, a direct role of CO gas in mitigating the process of thrombosis was assessed by True et al 14 These authors found that Hmox-1 knockout mice display a prothrombotic phenotype characterized by damage of endothelial cells and apoptosis, platelet activation, elevation in both tissue factor and von Willebrand factor, and reactive oxygen species.…”

Carbon monoxide: Anticoagulant or procoagulant?

“…were exposed to CORM-2 [51][52][53][54][55][56][57] In order to further characterize the mechanism by which CO enhanced coagulation, experiments involving isolated exposure of prothrombin [58] or fibrinogen [59] to CORM-2 were performed, with the purified protein function assessed in prothrombin or fibrinogen deficient plasmas, respectively. These works lead to the determination that only fibrinogen was affected by CO [58,59], and additional work utilizing mass spectrometry [60] demonstrated that protease digestion of fibrinogen was modified by exposure to CO. More importantly, it was noted that a heme group(s) was present in the fibrinogen digest samples, and following exposure of human plasma to compounds that either produced a carboxyheme or metheme state, plasma strength was increased or decreased respectively, indicative of heme-based modulation of fibrinogen function [60].…”

“…These works lead to the determination that only fibrinogen was affected by CO [58,59], and additional work utilizing mass spectrometry [60] demonstrated that protease digestion of fibrinogen was modified by exposure to CO. More importantly, it was noted that a heme group(s) was present in the fibrinogen digest samples, and following exposure of human plasma to compounds that either produced a carboxyheme or metheme state, plasma strength was increased or decreased respectively, indicative of heme-based modulation of fibrinogen function [60]. Exploiting this newfound redox chemistry of fibrinogen, an assay was devised to determine the presence of CO bound to fibrinogen, named carboxyhemefibrinogen (COHF), as a modification of thrombelastographic methodology [61].…”

“…Platelets appear to respond to CO in a similar fashion among human and rodent studies [10][11][12]16,21,22], but plasmatic coagulation also appears to be enhanced in humans, rats and rabbits [48,[51][52][53][54][55][56][57][58][67][68][69]. In an effort to reveal potential sources of specie specific differences, one may consider the differences in thrombus ultrastructure between humans, rabbits, mice and rats [94,95].…”