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.

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 enhances coagulation and attenuates fibrinolysis by two mechanisms: insights gained with colloid dilution.

Carbon monoxide releasing molecule-2 (CORM-2, tricarbonyldichlororuthenium (II) dimer) enhances coagulation and attenuates vulnerability to fibrinolysis. Our goal was to further define the CORM-2-mediated mechanisms using colloids with known effects on coagulation/fibrinolytic proteins. Plasma diluted 0 or 30% with 0.9% NaCl, 5% human albumin, low molecular weight hydroxyethyl starch (130 kDa) or high molecular weight hydroxyethyl starch (450 kDa) was exposed to 0 or 100 µmol/l CORM-2 before activation with tissue factor (n = 8 per condition). A second identically diluted series of experiments were performed with the addition of tissue-type plasminogen activator. Thrombelastographic data were collected until clot strength stabilized or clot lysis occurred. Dilution resulted in fluid-specific decreases in velocity of clot growth, strength and clot growth time with progressive increases in macromolecule size. CORM-2 exposure significantly increased the velocity of clot formation and strength (thin fibrin fiber promoting), but not clot growth time, under all conditions. Fibrinolysis was enhanced to the greatest extent by hydroxyethyl starch (antiα2-antiplasmin effect), and CORM-2 addition attenuated fibrinolysis in all conditions (α2-antiplasmin enhancement). CORM-2 exposure attenuated the decrease in coagulation kinetics mediated by hemodilution by two different mechanisms based on kinetic profile differences between the diluents tested. Further laboratory-based investigation is warranted to further define the molecular mechanisms responsible for CORM-2-mediated effects on coagulation and fibrinolysis.