Direct Inhibitory Effects of Carbon Monoxide on ... - Wiley Online Library

Basic & Clinical Pharmacology & Toxicology, 2017, 120, 207–212

Doi: 10.1111/bcpt.12654

Direct Inhibitory Effects of Carbon Monoxide on Six Venoms Containing Fibrinogenolytic Metalloproteinases Vance G. Nielsen and Philip A. Losada The Department of Anesthesiology, University of Arizona College of Medicine, Tucson, AZ, USA (Received 12 July 2016; Accepted 16 August 2016) Abstract: Since the introduction of antivenom administration over a century ago to treat venomous snake bite, it has been the most effective therapy for saving life and limb. However, this treatment is not always effective and not without potential lifethreatening side effects. We tested a new paradigm to abrogate the plasmatic anticoagulant effects of fibrinogenolytic snake venom metalloproteinases (SVMP) by inhibiting these Zn+2-dependent enzymes directly with carbon monoxide (CO) exposure. Assessment of the fibrinogenolytic effects of venoms collected from the Arizona black rattlesnake, Northern Pacific rattlesnake, Western cottonmouth, Eastern cottonmouth, Broad-banded copperhead and Southern copperhead on human plasmatic coagulation kinetics was performed with thrombelastography in vitro. Isolated exposure of all but one venom (Southern copperhead) to CO significantly decreased the ability of the venoms to compromise coagulation. These results demonstrated that direct inhibition of transition metal-containing venom enzymes by yet to be elucidated mechanisms (e.g. CO, binding to Zn+2 or displacing Zn+2 from the catalytic site, CO binding to histidine residues) can in many instances significantly decrease fibrinogenolytic activity. This new paradigm of CO-based inhibition of the anticoagulant effects of SVMP could potentially diminish haemostatic compromise in envenomed patients until antivenom can be administered.

Since the original description over a century ago of the treatment of venomous snake envenomation by injection of ‘antivenomous serum’ to rabbits and human beings [1], the gold standard for saving life and limb has been the administration of antivenom. This therapeutic intervention has saved numerous lives over the decades; however, even the most recently manufactured antibody fragment-based antivenom is not always effective and is associated with infrequent lifethreatening complications [2]. Also of interest, patients can suffer venom ‘rebound’ following antivenom therapy, manifested by recurrent coagulopathy when venom from the bite site is released as the antivenom antibodies begin to reach their nadir [3,4]. Considered as a whole, antivenom therapy is crucial to optimal patient care after snake envenomation, but it is not without important limitations and side effects. A new approach to attenuating some of the fibrinogenolytic effects of snake venom has been developed over the past year via modification of fibrinogen in our laboratory. By exposing human plasma to carbon monoxide (CO) which binds to fibrinogen-bound haem groups [5,6], and to ferric iron, which appears to modulate the alpha chain of fibrinogen [7], it has been determined that the catalysis of fibrinogen by fibrinogenolytic snake venom metalloproteinases (SVMP) found in North American vipers can be significantly attenuated [8–10]. Also of interest, preliminary experiments to establish negative control values revealed possible inhibition of Author for correspondence: Vance G. Nielsen, Department of Anesthesiology, The University of Arizona College of Medicine, P.O. Box 245114, 1501 North Campbell Avenue, Tucson, AZ 85724-5114, USA (fax +(520) 626 6943, e-mail [email protected]).

fibrinogenolytic, Zn+2-containing SVMP by isolated exposure to CO. This finding is consistent with inhibition of Zn+2-containing human metalloproteinases by CO released from COreleasing molecule-2 [11]. When combined both these findings, modification of fibrinogen with iron/CO and possible direct inhibition of fibrinogenolytic SVMP could lead to a potent, therapeutic approach that could complement traditional antivenom administration. Thus, the hypothesis of this investigation was that exposure of fibrinogenolytic SVMP to CO in isolation would decrease coagulation kinetics in human plasma. This hypothesis was tested in a thrombelastographic model previously presented [8–10]. Materials and Methods Plasma, venom and chemicals. Pooled normal human plasma (George King Bio-Medical, Overland Park, KS, USA) anticoagulated with sodium citrate (nine parts blood to one part 0.105 M sodium citrate) stored at 80°C was utilized in all subsequently described experiments. Fifty milligrams of lyophilized venom from six North American pit vipers with documented fibrinogenolytic metalloproteinase activity [12–16] listed in table 1 was obtained from the National Natural Toxins Research Center at Texas A&M University, Kingsville, TX, USA. All of these venoms are known to contain fibrinogenolytic metalloproteinases in isolation, with the exception of the Southern copperhead venom, which also possesses two serine proteases that are fibrin clot-promoting [17]. The venom was reconstituted in calcium-free phosphate-buffered saline (PBS; Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 50 mg/ ml, aliquoted and stored at 80°C until experimentation. CORM-2 [tricarbonyldichlororuthenium (II) dimer, a CO-releasing molecule], calcium-free PBS and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich.

© 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)

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Table 1. Fibrinogenolytic metalloprotease containing snake venoms assessed. Common name Arizona black rattlesnake Northern Pacific rattlesnake Western cottonmouth Eastern cottonmouth Broad-banded copperhead Southern copperhead

Species

commercially available program (OrigenPro 7.5; OrigenLab Corporation, Northampton, MA, USA). p < 0.05 was considered significant.

References

Crotalus oreganus cerberus

[12]

Crotalus oreganus oreganus

[13]

Agkistrodon piscivorus leucostoma Agkistrodon piscivorus piscivorus Agkistrodon contortrix laticinctus Agkistrodon contortrix contortrix

[14]

Results Isolated venom exposure to CO experiments. The results of these experiments are shown in figs 1–6. For clarity, the results obtained with each venom will be individually described.

[14] [15] [16,17]

Thrombelastographic analyses. The final plasma sample mixture volume for subsequently described experiments was 359.6 ll. Sample composition consisted of 326 ll of plasma, 3.6 ll of PBS with DMSO or venom, 10 ll of tissue factor reagent (0.1% final concentration in distilled water; Diagnostica Stago S.A.S., Asnieres sur Seine, France) and 20 ll of 200 mM CaCl2 (Haemonetics Inc., Braintree, MA, USA), which were rapidly mixed with immediate data collection commenced. The aforementioned plasma and venom mixtures were placed in a disposable cup in a computer-controlled Thrombelastographâ Hemostasis System (Model 5000; Haemonetics Inc.) at 37°C. The following elastic modulus-based parameters previously described [8– 10] were determined: time to maximum rate of thrombus generation (TMRTG) – this is the time interval (min.) observed prior to maximum speed of clot growth; maximum rate of thrombus generation (MRTG) – this is the maximum velocity of clot growth observed (dynes/cm2/ sec.); and total thrombus generation (TTG, dynes/cm2) – the final viscoelastic resistance observed after clot formation. Data were collected for 15 min. Lastly, in preliminary experiments, the concentration used of each of the six venoms was determined by a marked decrease in coagulation kinetics, based in part from similar work with these same venoms in the past [8–10]. Isolated venom exposure to CO experiments. Exposure of the venoms in isolation to CO was accomplished by adding CORM-2 to subsequently described solutions of venom in PBS. To isolate the effects of CO from the carrier molecule of CORM-2, a 100-lM solution of CORM-2 was placed in a sealed plastic tube and incubated in a to a 37°C water bath for 18 hr in order to release CO and become inactive (iCORM-2) as has been previously described [18]. To test the effects of CO on venom activity, a 1-ml quantity of venom in PBS was prepared for each condition subsequently presented. The conditions were as follows: (i) 1% addition (v/v) of PBS and DMSO to PBS without venom, (ii) 1% addition (v/v) of PBS and DMSO to PBS with venom, (iii) 1% addition of 100 lM CORM-2 to PBS with venom and lastly (iv) 1% addition of PBS and 100 lM iCORM-2 to PBS with venom. After 5 min. of incubation at room temperature, 3.6 ll of solution from one of these four solutions was placed into the aforementioned plasma mixture in a disposable cup in a computercontrolled Thrombelastographâ Hemostasis System (Model 5000; Haemonetics Inc.), with CaCl2 immediately added. Statistical analyses. Data are presented as mean S.D. All conditions were represented by n = 6 replicates, as this provides a statistical power >0.8 with p < 0.05 using this thrombelastographic methodology [8–10]. A commercially available statistical program was used for one-way analysis of variance followed by Student–Newman–Keuls post hoc analyses (SigmaStat 3.1; Systat Software, Inc., San Jose, CA, USA). Graphics depicting coagulation kinetic data were generated with a

Arizona black rattlesnake. The concentration of venom used in these experiments was 2 lg/ml final in the plasma mixture, and data are shown in fig. 1. Compared to samples with DMSO addition alone (Control), plasma with venom addition (V) demonstrated significantly greater TMRTG values, significantly smaller MRTG values and significantly smaller TTG values. When venom was exposed to CORM-2 (V+CO) and placed in plasma, the resultant TMRTG, MRTG and TTG values were not significantly different from Control samples, but all V+CO coagulation kinetic values were significantly different from V samples. Lastly, venom exposed to iCORM-2 (V+iCO)

Fig. 1. Effects of carbon monoxide (CO) exposure on venom activity of the Arizona black rattlesnake. Data are presented as mean S.D. TMRTG, maximum rate of thrombus generation (min.); MRTG, maximum rate of thrombus generation (dynes/cm2/sec.); TTG, total thrombus generation (dynes/cm2); DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline. Control = 1% addition of DMSO/PBS to PBS solution; V = DMSO/PBS+venom (2 lg/ml final concentration in plasma); V+CO = venom exposed to 100 lM CORM-2; V+iCO = venom exposed to 100 lM iCORM-2. *p < 0.05 versus Control; †p < 0.05 versus V; ‡p < 0.05 versus V+CO.


CARBON MONOXIDE INACTIVATION OF SNAKE VENOM METALLOPROTEINASES

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Fig. 2. Effects of carbon monoxide (CO) exposure on venom activity of the Northern Pacific Rattlesnake. Data are presented as mean S.D. TMRTG, maximum rate of thrombus generation (min.); MRTG, maximum rate of thrombus generation (dynes/cm2/sec.); TTG, total thrombus generation (dynes/cm2); DMSO, dimethyl sulfoxide; PBS, phosphatebuffered saline. Control = 1% addition of DMSO/PBS to PBS solution; V = DMSO/PBS+venom (2 lg/ml final concentration in plasma); V+CO = venom exposed to 100 lM CORM-2; V+iCO = venom exposed to 100 lM iCORM-2. *p < 0.05 versus Control; †p < 0.05 versus V; ‡p < 0.05 versus V+CO.

Fig. 3. Effects of carbon monoxide (CO) exposure on venom activity of the Western cottonmouth. Data are presented as mean S.D. TMRTG, maximum rate of thrombus generation (min.); MRTG, maximum rate of thrombus generation (dynes/cm2/sec.); TTG, total thrombus generation (dynes/cm2); DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline. Control = 1% addition of DMSO/PBS to PBS solution; V = DMSO/PBS+venom (5 lg/ml final concentration in plasma); V+CO = venom exposed to 100 lM CORM-2; V+iCO = venom exposed to 100 lM iCORM-2. *p < 0.05 versus Control; †p < 0.05 versus V; ‡p < 0.05 versus V+CO.

demonstrated significantly greater TMRTG values than the other three conditions; however, V+iCO MRTG values and TTG values were only significantly different from Control and V+CO values.

results are shown in fig. 3. Compared with Control sample values, V samples demonstrated significantly greater TMRTG values, significantly smaller MRTG values and significantly smaller TTG values. Plasma with V+CO demonstrated TMRTG, MRTG and TTG values that were not significantly different from Control samples, but all V+CO coagulation kinetic values were significantly different from V samples. Lastly, samples with V+iCO addition demonstrated significantly greater TMRTG values, significantly smaller MRTG values and significantly smaller TTG values than the other three conditions.

Northern Pacific rattlesnake. The concentration of venom used in these experiments was 2 lg/ml final in the plasma mixture, and data are depicted in fig. 2. Compared with Control sample values, V samples demonstrated significantly greater TMRTG values, significantly smaller MRTG values and significantly smaller TTG values. Plasma with V+CO demonstrated TMRTG, MRTG and TTG values that were not significantly different from Control samples, but all V+CO coagulation kinetic values were significantly different from V samples. Lastly, samples with V+iCO addition demonstrated significantly greater TMRTG values, significantly smaller MRTG values and significantly smaller TTG values than the other three conditions. Western cottonmouth. The concentration of venom used in these experiments was 5 lg/ml final in the plasma mixture, and

Eastern cottonmouth. The concentration of venom used in these experiments was 5 lg/ml final in the plasma mixture, and these data are shown in fig. 4. Compared with Control sample values, V samples demonstrated significantly greater TMRTG values, significantly smaller MRTG values and significantly smaller TTG values. Plasma with V+CO demonstrated TMRTG, MRTG and TTG values that were not significantly different from Control samples, but all V+CO coagulation kinetic values were significantly different from V


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Fig. 4. Effects of carbon monoxide (CO) exposure on venom activity of the Eastern cottonmouth. Data are presented as mean S.D. TMRTG, maximum rate of thrombus generation (min.); MRTG, maximum rate of thrombus generation (dynes/cm2/sec.); TTG, total thrombus generation (dynes/cm2); DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline. Control = 1% addition of DMSO/PBS to PBS solution; V = DMSO/PBS+venom (5 lg/ml final concentration in plasma); V+CO = venom exposed to 100 lM CORM-2; V+iCO = venom exposed to 100 lM iCORM-2. *p < 0.05 versus Control; †p < 0.05 versus V; ‡p < 0.05 versus V+CO.

samples. Lastly, samples with V+iCO addition demonstrated significantly greater TMRTG values and significantly smaller MRTG values than Control and V+CO samples; further, TTG values of samples with V+iCO addition were significantly smaller than the other three conditions. Broad-banded copperhead. The concentration of venom used in these experiments was 30 lg/ml final in the plasma mixture, and results are depicted in fig. 5. Compared with Control sample values, V samples demonstrated significantly greater TMRTG values and significantly smaller MRTG values but not significantly smaller TTG values. Plasma with V+CO demonstrated TMRTG values not different from either Control or V sample values, MRTG values significantly greater than V sample values but not different from Control sample values and TTG values that were significantly greater than those of both Control and V sample values. Lastly, samples with V+iCO addition had TMRTG values not significantly different from the other three conditions, MRTG values significantly smaller than

Fig. 5. Effects of carbon monoxide (CO) exposure on venom activity of the Broad-banded copperhead. Data are presented as mean S.D. TMRTG, maximum rate of thrombus generation (min.); MRTG, maximum rate of thrombus generation (dynes/cm2/sec.); TTG, total thrombus generation (dynes/cm2); DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline. Control = 1% addition of DMSO/PBS to PBS solution; V = DMSO/PBS+venom (30 lg/ml final concentration in plasma); V+CO = venom exposed to 100 lM CORM-2; V+iCO = venom exposed to 100 lM iCORM-2. *p < 0.05 versus Control; †p < 0.05 versus V; ‡p < 0.05 versus V+CO.

Control and V+CO sample values and TTG values greater than Control sample values. Southern copperhead. The concentration of venom used in these experiments was 10 lg/ml final in the plasma mixture, and data are shown in fig. 6. Compared with Control samples, all three conditions with venom addition had significantly greater TMRTG values; further, V+CO samples had significantly smaller TMRTG values than V+iCO samples. Similarly, Control samples had significantly greater MRTG values compared with all three conditions with venom addition; additionally, V+CO samples had significantly greater MRTG values than V+iCO samples. Lastly, Control samples had significantly greater TTG values compared with all three conditions with venom addition; however, V+iCO samples had significantly smaller TTG values than V or V+CO samples. Discussion The primary finding of the present investigation was that some, though not all, fibrinogenolytic SVMP were inhibited in


CARBON MONOXIDE INACTIVATION OF SNAKE VENOM METALLOPROTEINASES

Fig. 6. Effects of carbon monoxide (CO) exposure on venom activity of the Southern copperhead. Data are presented as mean S.D. TMRTG, maximum rate of thrombus generation (min.); MRTG, maximum rate of thrombus generation (dynes/cm2/sec.); TTG, total thrombus generation (dynes/cm2); DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline. Control = 1% addition of DMSO/PBS to PBS solution; V = DMSO/PBS+venom (10 lg/ml final concentration in plasma); V+CO = venom exposed to 100 lM CORM-2; V+iCO = venom exposed to 100 lM iCORM-2. *p < 0.05 versus Control; †p < 0.05 versus V; ‡p < 0.05 versus V+CO.

isolation by CO. The hierarchy of inhibitory effect of CO on specific venoms was Arizona black rattlesnake = Northern Pacific rattlesnake = Western cottonmouth = Eastern cottonmouth > Broad-banded copperhead >>> Southern copperhead (no CO effect). While unknown for the Broad-banded copperhead venom, Southern copperhead venom is known to have serine proteases that can polymerize fibrinogen [17], which could compete with the fibrinogenolytic metalloproteinase it coexists with [16] – a competition of which in raw venom may result in dominance of serine protease effects, making inhibition of the metalloproteinase unimportant haemostatically. Our experiments were not designed to sort out this enzymatic balance in Southern copperhead venom, and such an enterprise is beyond the scope of the present work. Nevertheless, our primary goal of ascertaining a CO-mediated inhibition of fibrinogenolytic SVMP was successful, and variation in biological response was both expected and reassuring, given the diversity of species studied. The mechanism underlying CO-mediated inhibition of SVMP remains to be elucidated on the molecular level. CORM-2-derived CO has only once been shown to directly

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inhibit the activity of metalloproteinases 1 and 2 in a human alveolar epithelial cell line [11]. These authors posited that CO, being known to bind to transition metals [19,20], could have bound to the Zn+2 metal located in the catalytic centre of the enzyme as the mechanism of inhibition [11]. However, Zn+2 binds to CO with less avidity than other transition metals [21]. Another possible mechanism includes CO interaction with a histidine residue within the SVMP, as CO has been demonstrated to activate [22,23] or inactivate [24] ion channels by interacting with histidine residues within these channels in a haem-independent fashion. Thus, these potential mechanisms and perhaps others are at play in the COmediated inhibition of fibrinogenolytic SVMP, but their elucidation is beyond the scope of the present work. It should be noted that iCORM-2, the inactive carrier molecule, appeared to enhance to a small degree the action of venom on coagulation in all but one venom (Broad-banded copperhead). Unlike CO, in the case of which one can at least postulate that interactions with Zn+2 or histidine residues may play a role in modulation of the SVMP tested in our investigation, a mechanism by which iCORM-2 affects activity remains elusive. Importantly, iCORM-2 has been demonstrated to mediate effects similar to or opposite of CO in vitro and ex vivo [25,26]. Nevertheless, given that the carrier molecule at worst appears to affect venom activity in an opposite manner compared with CORM-2, one is assured that CO interactions with the fibrinogenolytic SVMP are responsible for inhibition of the enzymes. In sum, these phenomena must be kept in mind when interpreting the effects of CO on fibrinogenolytic SVMP activity and serve as a limitation given that the mechanisms responsible for iCORM-2 effects are not known. In conclusion, this investigation demonstrated that CO inhibited the fibrinogenolytic action in human plasma of five of six venoms known to contain metalloproteinases [12–16]. Possible mechanisms by which CO inhibited fibrinogenolytic SVMP activity may be by interacting with Zn+2 in the catalytic site of the enzyme or interacting with a critical histidine residue(s) in these enzymes as has occurred in other biomolecules. Our findings serve as the rational basis for our planned continued preclinical evaluations of CO-mediated inhibition of fibrinogenolytic SVMP in vivo, likely using mouse and rabbit models. Funding This investigation was supported by the Department of Anesthesiology. Conflict of Interest All authors have no conflict of interests to disclose. References 1 Calmette A. The treatment of animals poisoned with snake venom by the injection of antivenomous serum. Br Med J 1896;2:399– 400. 2 Dart RC, McNally J. Efficacy, safety, and use of snake antivenoms in the United States. Ann Emerg Med 2001;37:181–8. 3 Ruha AM, Curry SC, Albrecht C, Riley B, Pizon A. Late hematologic toxicity following treatment of rattlesnake envenomation with


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