Anticoagulation with unfractionated heparin or direct thrombin inhibitors such as argatroban is frequently used for thromboprophylaxis in several perioperative settings, including mechanical circulatory support either as bridging anticoagulation or, in the case of argatroban, as antithrombotic therapy in cases of heparin-induced thrombocytopenia.1 Unfortunately, despite meticulous efforts to maintain clinical hemostasis with careful hematological monitoring of anticoagulation, important bleeding complications still occur. It may be difficult or clinically undesirable (e.g., avoiding pump thrombosis) to reverse the effects of heparin or direct thrombin inhibitors and restore normal thrombin generation. Thus, it would be of interest to identify an alternative method to enhance coagulation and diminish fibrinolysis in such settings without enhancing thrombin generation.
One such intervention may be the use of a newly identified hemostatic agent, carbon monoxide releasing molecule-2 (CORM-2; tricarbonyldichlororuthenium [II] dimer). It has been recently documented that exposure of human plasma to CORM-2 enhanced the velocity of thrombus formation and strength2–4 and diminished vulnerability to tissue-type plasminogen activator (tPA)-mediated fibrinolysis.3 Also, CORM-2 enhanced the velocity of thrombus formation and strength in factor VII-, VIII-, and IX-deficient plasma, even when the onset of coagulation occurred after 20 minutes.4 The apparent mechanism of the aforementioned CORM-2–mediated effects seemed to be an enhancement of thrombin-fibrinogen interactions during clot formation.2,3 Similarly, given that the half-time of carbon monoxide release from CORMs is 1 minute after placement in aqueous solution with pH = 7.4 at a temperature of 37°C,5 it seemed most likely that CORM-2 was modifying fibrinogen rather than prothrombin, because essentially all released carbon monoxide had likely diffused from plasma samples before thrombin formation.4 In further support, electron microscopic examination of plasma thrombi demonstrated that CORM-2 exposure resulted in a marked decrease in thick fibrin fiber formation.6 Lastly, it has recently been demonstrated that exposure of prothrombin to CORM-2 in isolation does not enhance coagulation kinetics in prothrombin-deficient plasma; instead, CORM-2 improved coagulation in a fibrinogen concentration-dependent manner in fibrinogen-depleted plasma.7 Therefore, several indirect lines of evidence support the concept that carbon monoxide derived from CORM-2 most likely modifies fibrinogen to enhance coagulation kinetics and diminish fibrinolytic vulnerability.
The purpose of this investigation was to determine whether plasma anticoagulated with heparin or argatroban would have increased coagulation and decreased vulnerability to fibrinolysis after exposure to CORM-2.
Pooled normal plasma (George King Bio-Medical, Overland Park, KS) anticoagulated with sodium citrate was used for experimentation. This lot (#1223) of plasma had a prothrombin time of 12.8 seconds, international normalized ratio value of 1.0, an activated partial thromboplastin time of 29.4 seconds, and a fibrinogen concentration of 310 mg/dL. This plasma was exposed to 0 or 100 U/mL tPA (580 U/μg; Genentech, Inc., San Francisco, CA), in the subsequently described experiments.
Plasma Sample Composition of Experiments Without tPA Addition
The final volume for all subsequently described plasma sample mixtures was 359.6 μL. Plasma was exposed to 0 or 0.1 U/mL heparin (APP Pharmaceuticals, LLC, Schaumburg, IL) or argatroban (GlaxoSmithKline, Research Triangle Park, NC) just before experimentation. The volume of added heparin or argatroban was <1% of total plasma stock volume before thrombelastographic analyses. These concentrations of anticoagulant were used as pilot data and demonstrated that larger concentrations would result in an essential loss of measurable thrombus formation in the presence of tPA in our system. Sample composition consisted of 326 μL plasma; 10 μL tissue factor (TF) reagent (0.1% final concentration in dH2O; Instrumentation Laboratory, Lexington, MA); 3.6 μL dimethyl sulfoxide (DMSO) or DMSO with CORM-2 (100 μM final concentration; Sigma-Aldrich, St. Louis, MO); and 20 μL of 200 mM CaCl2. TF stock was kept on ice before use and was remade every 2 hours. The concentration of CORM-2 used was that associated with maximal effect on coagulation and fibrinolysis in this system.3 DMSO was added to dry CORM-2 just before placement into the plasma mixture. Six replicate experiments per condition were performed.
Plasma Sample Composition of Experiments with tPA Addition
As in the first series of experiments, the final volume of plasma sample mixtures was 359.6 μL. Sample composition consisted of 316 μL plasma; 10 μL TF reagent (0.1% final concentration); 3.6 μL DMSO without or with CORM-2 (100 μM final concentration); 10 μL tPA diluted with 10 mM potassium phosphate buffer (pH 7.4) for a final activity of 100 IU/mL; and 20 μL of 200 mM CaCl2. As with TF reagent, tPA stock was kept on ice before use and was remade every 2 hours. Six replicate experiments per condition were performed.
Clot Lifespan Model Analyses
Plasma sample mixtures were placed in a disposable cup in a computer-controlled thrombelastograph® hemostasis system (Model 5000; Haemoscope Corp., Niles, IL), with addition of CaCl2 as the last step to initiate clotting. In the first series of experiments, no tPA was added, so data were collected until clot strength (maximum amplitude) was stable. In the second series of experiments in which tPA was added, data were collected until clot lysis time (CLT) occurred. The following variables were determined at 37°C depending on whether tPA was present in the reaction mixture: clot growth time (CGT, time from clot amplitude of 2 mm [102 dynes/cm2] until maximum strength is achieved, in seconds), CLT (time from when maximum strength was observed to 2-mm amplitude, in seconds), and clot lifespan (CLS, the sum of CGT and CLT). Additional elastic modulus-based parameters previously described were determined2–4,8 and are displayed in Figure 1. The nomenclature used to describe these phenomena is as follows. Time to maximum rate of thrombus generation (TMRTG): This is the time interval (seconds) observed before maximum speed of clot growth. Maximum rate of thrombus generation (MRTG): This is the maximum velocity of clot growth observed (dynes/cm2/s). Total thrombus generation (TTG): This is the total area under the velocity curve during clot growth (dynes/cm2), representing the amount of clot strength generated during clot growth. Time to maximum rate of lysis (TMRL): This is the time interval (seconds) measured from the time of maximum strength to the time when the velocity of clot disintegration is maximal. Maximum rate of lysis (MRL): This is the maximum velocity of clot disintegration observed (−dynes/cm2/s). Area of clot lysis (ACL): This is the total area under the velocity curve during clot disintegration (−dynes/cm2), representing the amount of clot strength lost during clot disintegration. Because this model involves complete fibrinolysis, TTG is equivalent to ACL; therefore, ACL was not presented because it provides no additional information.
Data obtained from pooled normal plasma are presented as mean ± SD. Analyses of the effects of CORM-2 on thrombelastographic variables obtained from pooled normal plasma were conducted using 1-way analysis of variance with Student-Newman-Keuls post hoc test. A P value <0.05 was considered significant. The graphical representation of the thrombelastographic data derived from experimentation was generated with commercially available software (SigmaPlot 11.0; Systat Software, Inc., San Jose, CA).
Experiments Without tPA Addition
The anticoagulant effects of heparin and argatroban on coagulation kinetics are displayed in Table 1, with representative individual plasma sample data depicted in Figure 2. Heparin anticoagulation resulted in a significant 271% increase in TMRTG and 78% decrease in MRTG values compared with normal plasma values. Argatroban anticoagulation resulted in a significant 196% increase in TMRTG and 57% decrease in MRTG compared with normal plasma values. Neither anticoagulant significantly decreased TTG values compared with normal values. The addition of CORM-2 to heparin-exposed plasma resulted in a significant 22% decrease in TMRTG and 75% increase in MRTG values compared with anticoagulated samples without CORM-2 exposure. In contrast, addition of CORM-2 to argatroban-exposed plasma resulted in a significant 11% increase in TMRTG that was nevertheless associated with a 41% increase in MRTG values compared with anticoagulated samples without CORM-2 exposure. Under normal and anticoagulated conditions, TTG was increased 69% to 72% by CORM-2 exposure, without any significant difference between conditions. Lastly, despite improving coagulation kinetics in samples exposed to heparin or argatroban, CORM-2 addition still resulted in TMRTG and MRTG values significantly different from normal plasma samples, indicative of persistent suppression of coagulation kinetics.
Experiments with tPA Addition
The anticoagulant effects of heparin and argatroban on coagulation/fibrinolytic kinetics are displayed in Table 2, with representative individual plasma sample data depicted in Figure 3. Heparin anticoagulation resulted in a significant 591% increase in TMRTG, 96% decrease in MRTG, and 91% decrease in TTG values compared with normal plasma values. Argatroban anticoagulation resulted in a significant 195% increase in TMRTG, 53% decrease in MRTG, and 12% decrease in TTG values compared with normal plasma values. The addition of CORM-2 to heparin-exposed plasma resulted in a significant 43% decrease in TMRTG, 367% increase in MRTG, and 731% increase in TTG values compared with anticoagulated samples without CORM-2 exposure. Similarly, addition of CORM-2 to argatroban-exposed plasma resulted in a significant 5% decrease in TMRTG, 94% increase in MRTG, and 150% increase in TTG values compared with anticoagulated samples without CORM-2 exposure. When compared with normal values, CORM-2 exposure did not restore TMRTG values to normal values in either heparin or argatroban anticoagulated samples. However, whereas CORM-2 exposure only partially restored MRTG values in heparin anticoagulated samples, samples anticoagulated with argatroban demonstrated normal MRTG values after addition of CORM-2. Similarly, CORM-2–exposed, heparin anticoagulated samples demonstrated partial restoration of TTG values, whereas CORM-2–exposed, argatroban anticoagulated samples demonstrated supernormal TTG values.
With regard to fibrinolysis, both heparin and argatroban significantly decreased TMRL by 68% compared with normal sample values. In contrast, whereas MRL was decreased by 86% by heparin anticoagulation, MRL was increased by 86% in samples with argatroban. Addition of CORM-2 significantly increased TMRL by 31% in heparin-treated plasma, whereas argatroban-exposed samples demonstrated a 147% increase compared with anticoagulated samples not exposed to CORM-2. Heparin-exposed samples with CORM-2 addition demonstrated a 650% increase in MRL, and argatroban-exposed samples with CORM-2 demonstrated an 82% increase in MRL compared with anticoagulated samples not exposed to CORM-2. Lastly, CORM-2 exposure did not restore heparin anticoagulated sample TMRL or MRL values to normal plasma values; however, in argatroban-treated samples, CORM-2 addition did result in TMRL values not different from normal plasma values, and associated MRL values were significantly greater than normal plasma values.
The summation of the aforementioned thrombus growth and fibrinolysis variables can be observed in the changes in CGT, CLT, and CLS. Heparin anticoagulation resulted in a significant 68% decrease in CGT, 72% decrease in CLT, and 72% decrease in CLS values compared with normal plasma values. Argatroban anticoagulation resulted in no change in CGT, a significant 58% decrease in CLT, and a 48% decrease in CLS compared with normal plasma values. The addition of CORM-2 to heparin-exposed plasma resulted in a significant 421% increase in CGT, 100% increase in CLT, and 178% increase in CLS values compared with anticoagulated samples without CORM-2 exposure. Similarly, addition of CORM-2 to argatroban-exposed plasma resulted in a significant 116% increase in CGT, 103% increase in CLT, and 106% increase in CLS values compared with anticoagulated samples without CORM-2 exposure. Of interest, CORM-2 treatment significantly increased CGT in both heparin and argatroban anticoagulated samples compared with normal plasma without CORM-2 exposure. In contrast, heparin-exposed samples with CORM-2 addition had significantly smaller CLT than normal plasma, whereas argatroban anticoagulated samples with CORM-2 exposure had CLT restored to normal values. The effects of CORM-2 on CLS in heparin and argatroban anticoagulated samples compared with normal plasma essentially mirrored those observed with CLT.
The primary finding of this investigation was that CORM-2 exposure improved most aspects of coagulation kinetics and diminished fibrinolytic vulnerability in heparin and argatroban anticoagulated plasma. In the absence of tPA, CORM-2 exposure resulted in partial restoration of TMRTG and MRTG with supernormal clot strength in heparin anticoagulated samples. Similarly, argatroban-exposed plasma with CORM-2 addition had partial restoration of MRTG coupled with supernormal clot strength. In sum, in plasma anticoagulated with heparin or argatroban, CORM-2 exposure resulted in improved but persistently slower-forming thrombi with supernormal strength in the absence of tPA.
The interpretation of data derived from the fibrinolytic environment is complex and requires additional explanation of some differences between heparin and argatroban. Given equivalent anticoagulant concentrations, argatroban is more efficient at facilitating fibrinolysis than heparin secondary to complete inhibition of thrombin activatable fibrinolysis inhibitor activation coupled with partial inhibition of factor XIII activation as we have previously described.9 This biochemical phenomena results in an orderly clot matrix that is more vulnerable to fibrinolytic attack, displayed as enhanced clot MRL and decreased CLT.9 In contrast, heparin anticoagulation results in either unchanged9 or decreased (Table 2) MRL secondary to loss of both factor XIII and thrombin activatable fibrinolysis inhibitor activation. It is important to note that the concentration of heparin used in the present investigation resulted in greater anticoagulation than that previously reported.9 We suspect that differences in heparin vendor (and biological activity) account for this enhanced anticoagulation. Furthermore, the activator used in previous experiments (TF/kaolin)9 likely resulted in increased thrombin generation compared with TF alone. These aforementioned biochemical explanations for the differences between the 2 anticoagulants in the fibrinolytic environment should facilitate interpretation of the phenomena produced by CORM-2 exposure.
The data in Table 2 and Figure 3 demonstrate that, in the presence of tPA, CORM-2 exposure partially restores all aspects of thrombus growth in heparin anticoagulated plasma, with some prolongation in onset of fibrinolysis noted as well. The result of these effects is complete restoration of CGT, a return to somewhat over half-normal CLT, with a consequent CLS nearly 80% of normal values. Nevertheless, despite treatment with CORM-2, heparin anticoagulated clots still must be characterized as slower growing and more quickly lysed compared with normal plasma. In contrast, argatroban anticoagulated plasma exposed to CORM-2 demonstrates normal velocity of clot formation and supranormal clot strength in the fibrinolytic environment. Despite a nearly 3-fold–greater MRL, argatroban anticoagulated plasma exposed to CORM-2 displayed a time to onset of lysis that was nearly 75% of normal plasma values. The summation of these kinetic events in argatroban anticoagulated plasma was a near doubling of CGT, a 14% decrease in CLT compared with normal plasma, with consequent CLS not different from normal plasma. Based on previous findings,2 the most likely mechanism for the CORM-2–mediated attenuation of fibrinolytic vulnerability in both heparin and argatroban anticoagulated plasma is enhancement of plasminogen activator inhibitor-1 and α2-antiplasmin activity, enzymes not dependent on thrombin activity for activation.
In conclusion, CORM-2 exposure partially restores coagulation kinetics and antifibrinolytic defenses in plasma exposed to heparin or argatroban anticoagulation. It will be critical to investigate the efficacy of CORM-2 in attenuating such anticoagulation in vivo, most likely in a rabbit model because rabbit plasma responds to CORM-2 exposure in a manner similar to that observed with human plasma.10 Thus, we plan further study of the route of administration, safety, and efficacy of CORM-2 and other CORMs to treat coagulopathic bleeding complications in the setting of heparin or direct thrombin inhibitor–mediated anticoagulation.
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2. Nielsen VG, Kirklin JK, George JF. Carbon monoxide releasing molecule-2 increases the velocity of thrombus growth and strength in human plasma. Blood Coagul Fibrinolysis 2009;20:377–80
3. Nielsen VG, Kirklin JK, George JF. Carbon monoxide releasing molecule-2 decreases fibrinolysis in human plasma. Blood Coagul Fibrinolysis 2009;20:448–55
4. Nielsen VG, Kirklin JK, George JF. Carbon monoxide releasing molecule-2 increases the velocity of thrombus growth and strength in hemophilia A, hemophilia B and factor VII deficient plasmas. Blood Coagul Fibrinolysis 2010;21:41–5
5. Motterlini R, Mann BE, Foresti R. Therapeutic applications of carbon monoxide-releasing molecules. Expert Opin Investig Drugs 2005;14:1305–18
6. Nielsen VG, Kirklin JK, George JF. Carbon monoxide releasing molecule-2 decreases thick diameter fibrin fibre formation in normal and factor XIII deficient plasma. Blood Coagul Fibrinolysis 2010;21:101–5
7. Nielsen VG, Malayaman SN, Khan ES, Kirklin JK, George JF. Carbon monoxide releasing molecule-2 increases fibrinogen-dependent coagulation kinetics but does not enhance prothrombin activity. Blood Coagul Fibrinolysis 2010;21:349–53
8. Nielsen VG, Cohen BM, Cohen E. Effects of coagulation factor deficiency on plasma coagulation kinetics determined via thrombelastography: critical roles of fibrinogen and factors II, VII, X and XII. Acta Anaesthesiol Scand 2005;49:222–31
9. Nielsen VG, Kirklin JK. Argatroban enhances fibrinolysis by differential inhibition of thrombin-mediated activation of thrombin activatable fibrinolysis inhibitor and factor XIII. Blood Coagul Fibrinolysis 2008;19:793–800
10. Nielsen VG, Khan ES, Huneke RB. Carbon monoxide releasing molecule-2 enhances coagulation in rat and rabbit plasma. Blood Coagul Fibrinolysis 2010;21:298–9
VGN helped conduct the study, analyze the data, and write the manuscript; ESK helped conduct the study; and JKK and JFG helped analyze the data and write the manuscript. All authors approved the final manuscript.
VGN, JKK, and JFG have applied for a use patent wherein CORM-2 would be utilized as a procoagulant/antifibrinolytic agent. ESK reports no conflicts of interest.