Carbon monoxide releasing molecule-2 improves protamine-mediated hypocoagulation/hyperfibrinolysis in human plasma in vitro.

BACKGROUND: Protamine sulfate has been implicated as a possible cause of coagulopathy for over 20 y. Protamine has been demonstrated to decrease thrombin activity and to prolong bleeding. We tested the hypothesis that a new hemostatic agent, carbon monoxide releasing molecule-2 (tricarbonyldichlororuthenium (II) dimer; CORM-2), could attenuate protamine-mediated hypocoagulation/hyperfibrinolysis in plasma. METHODS: Normal plasma was exposed to 0, 12.5, 25, or 50 µg/mL of protamine, with or without addition of 100 µM CORM-2. Tissue factor was used to initiate coagulation, and tissue type plasminogen activator was added in some experiments. Additional experiments were performed wherein plasma was exposed to protamine combined with 0% or 30% dilution with normal saline, with or without CORM-2 addition. Thrombelastography was performed until either stable clot strength or clot lysis occurred. RESULTS: Protamine, in a concentration-dependent fashion, significantly prolonged the onset of coagulation, decreased the velocity of thrombus growth, and decreased clot strength in the absence or presence of tissue type plasminogen activator. Further, protamine significantly decreased the time to onset of fibrinolysis and decreased clot lysis time. CORM-2 exposure significantly diminished all aforementioned protamine-mediated effects on coagulation and fibrinolysis. Lastly, CORM-2 addition significantly increased the velocity of clot growth and strength in diluted, protamine-exposed plasma. CONCLUSIONS: CORM-2 attenuated protamine-mediated hypocoagulation/hyperfibrinolysis at clinically encountered concentrations. Additional preclinical investigation is warranted to determine if CORM-2 administration will be efficacious in diminishing coagulopathy caused by protamine.

Fibrinogen is a heme-associated, carbon monoxide sensing molecule: a preliminary report.

The objective of this study was to determine how carbon monoxide directly modifies fibrinogen utilizing liquid chromatography-mass spectrometry (LC-MS/MS) by examining fibrinogen exposed to carbon monoxide releasing molecule-2 [tricarbonyldichlororuthenium (II) dimer; CORM-2]. Purified fibrinogen was exposed to 0, 25, 50 or 100 µmol/l CORM-2 for 5 min at 37°C and then stored at -80°C before analyses with LC-MS/MS. In a second series of experiments, normal plasma was exposed to 0 or 100 µmol/l CORM-2 in the absence or presence of the nitric oxide donor sodium nitroprusside and hydroquinone (an organic reductant) to compete with carbon monoxide binding to a putative heme group found on fibrinogen. Coagulation was activated with tissue factor (n=8 per condition). Thrombus growth was monitored with thrombelastography for 15 min. LC-MS/MS did not detect any direct modifications of amino acids in fibrinogen, but detection of small regions of both the alpha and gamma chains was lost following exposure to CORM-2 and endoproteinase digestion with trypsin and Glu-C. An ion with the same m/z and expected retention time as heme was found in the purified fibrinogen. Exposure of plasma to nitric oxide/hydroquinone significantly decreased CORM-2-mediated enhancement of coagulation without affecting the coagulation kinetics of plasma not exposed to CORM-2. Carbon monoxide derived from CORM-2 likely modifies fibrinogen via modulation of a fibrinogen-associated heme group(s). Whereas the precise molecular location of heme attachment and three-dimensional conformational change secondary to carbon monoxide exposure remain to be determined, fibrinogen appears to be a carbon monoxide sensing molecule.

Carbon monoxide releasing molecule-2 improves coagulation in patient plasma in vitro following cardiopulmonary bypass.

The objective of the present study was to determine if a new procoagulant molecule, carbon monoxide releasing molecule (tricarbonyldichlororuthenium (II) dimer; CORM-2) would improve coagulation following cardiopulmonary bypass (CPB). Plasma was obtained from patients undergoing elective cardiac surgery requiring CPB. Whole blood was collected and anticoagulated with sodium citrate after induction of anesthesia and again after CPB and heparin neutralization with protamine. Blood samples were centrifuged for 15 min, with plasma collected and stored at -80°C prior to analysis. Samples were subsequently exposed to 0 or 100 µmol/l CORM-2, with coagulation activated with tissue factor. Data were collected with thrombelastography until clot strength stabilized. Patients underwent CPB for 133 ± 61 min (mean ± SD). The velocity of thrombus formation was significantly decreased (52%) by CPB, as was clot strength (53%). Addition of CORM-2 to plasma samples obtained after CPB significantly increased the velocity of clot formation (75%) and strength (52%) compared to matched unexposed samples. The lesion of plasmatic coagulation associated with CPB was significantly improved in vitro by addition of CORM-2. If preclinical assessments of efficacy and safety of CORM-2 are favorable, future clinical trials involving CORM-2 or other CORMs as a hemostatic intervention in the setting of CPB are justified.

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

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.