Ruthenium, Not Carbon Monoxide, Inhibits the Procoagulant Activity of Atheris, Echis, and Pseudonaja Venoms

International Journal of Molecular Sciences

Article Ruthenium, Not Carbon Monoxide, Inhibits the Procoagulant Activity of Atheris, Echis, and Pseudonaja Venoms

Vance G. Nielsen

Department of Anesthesiology, University of Arizona College of Medicine, Tucson, AZ 85719, USA; [email protected]

Received: 5 March 2020; Accepted: 22 April 2020; Published: 23 April 2020

Abstract: The demonstration that carbon monoxide releasing molecules (CORMs) affect experimental systems by the release of carbon monoxide, and not via the interaction of the inactivated CORM, has been an accepted paradigm for decades. However, it has recently been documented that a radical intermediate formed during carbon monoxide release from ruthenium (Ru)-based CORM (CORM-2) interacts with histidine and can inactivate bee phospholipase A2 activity. Using a thrombelastographic based paradigm to assess procoagulant activity in human plasma, this study tested the hypothesis that a Ru-based radical and not carbon monoxide was responsible for CORM-2 mediated inhibition of Atheris, Echis, and Pseudonaja species snake venoms. Assessment of the inhibitory effects of ruthenium chloride (RuCl3) on snake venom activity was also determined. CORM-2 mediated inhibition of the three venoms was found to be independent of carbon monoxide release, as the presence of histidine-rich albumin abrogated CORM-2 inhibition. Exposure to RuCl3 had little effect on Atheris venom activity, but Echis and Pseudonaja venom had procoagulant activity significantly reduced. In conclusion, a Ru-based radical and ion inhibited procoagulant snake venoms, not carbon monoxide. These data continue to add to our mechanistic understanding of how Ru-based molecules can modulate hemotoxic venoms, and these results can serve as a rationale to focus on perhaps other, complementary compounds containing Ru as antivenom agents in vitro and, ultimately, in vivo.

Keywords: hemotoxin; procoagulant; ruthenium; carbon monoxide releasing molecule; thrombelastography

1. Introduction The use of carbon monoxide releasing molecules (CORMs) to deliver carbon monoxide (CO) in a site-directed fashion to presumably alter heme-modulated systems has been part of experimental designs for decades, with hundreds of manuscripts incorporating this methodology. The key element of the paradigm that implicates CO as the mechanism behind the effects of CORMs is the determination that the inactivated releasing molecule (iRM), the portion of the CORM that remains after CO release, has no effect or a different effect on the system tested with the CORM compared to the anticipated CO effect. This laboratory has used this CORM-based paradigm for the past few years to demonstrate that CO inhibited the various procoagulant and anticoagulant activities of hemotoxic venoms and enzymes collected from dozens of snake and lizard species [1–13]. The particular CORM used was CORM-2 (tricarbonyldichlororuthenium (II) dimer) [1–13]. The presumed mechanism was that CO must be interacting with a cryptic heme group attached to the various venoms and enzymes or in some other way interacting with these diverse enzymes and venoms [1–13]. Thus, this potentially heme-based, CO-modulated paradigm of snake venom activity seemed plausible with the aforementioned paradigm of CORM-CO release-inert iRM interactions with target molecules.

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However, cracks in the edifice of this paradigm began to appear in the year 2017 with the publication of a work that demonstrated CO-independent inhibition of K+ channels with a putative Ru-based radical formed from CORM-2 during CO release and likely prior to formation of its iRM [14]. To substantiate this claim, the authors demonstrated that free histidine or albumin, which is resplendent with histidine residues, quenched the inhibition of potassium channels by CORM-2. Furthermore, using mass spectroscopy, the authors demonstrated histidine-Ru-based radical formation following exposure of free histidine with CORM-2 [14]. Lastly, using other various CORMs with other metal centers, the authors demonstrated no CO effects on the channel assessed [14]. This laboratory became aware of this work recently, and using a similar approach, demonstrated an identical outcome wherein the anticoagulant activity of the purified phospholipase A2 (PLA2) of Apis mellifera venom was inhibited by CORM-2 in a CO-independent, albumin-inhibitable fashion [15]. Furthermore, we recently demonstrated that the anticoagulant metalloproteinases of mamba venoms are inhibited by CORM-2 in a CO-independent, albumin-inhibitable manner [16]. Taken as a whole, it was entirely possible that Ru-based interactions with venom proteins could be responsible for the inhibition noted in our previous works [13]; and critically, if Ru-based modifications were the underpinning of such inhibition rather than the interaction of CO with a heme group, then Ru-based CORMs could well serve as permeant antivenom agents. The importance of these line of investigation involving ion channels [14], phospholipase A2 [15], and metalloproteinases [16] is that they lay the foundation to seriously reconsider the paradigm that Ru-based CORMs affect systems as simple as enzymes to as complex as whole animal models of disease in CO-independent ways—potentially affecting the interpretation of data contained in several hundred manuscripts. While it is unreasonable to reassess all previous venoms inhibited by CORM-2 to determine if a Ru-based radical rather than CO was mediating the inhibition [1–13], assessing a few representative venoms would be of benefit. To this end, three procoagulant venoms derived from diverse species from Africa and Australia were selected that have already been characterized as inhibited by CORM-2 but not by its iRM by this laboratory [8,10]. The species chosen are displayed in Table1, and the venom proteomes of these particular and snakes within the same genus are similar in terms of presence of snake venom serine proteases (SVSP), snake venom metalloproteinases (SVMP), and PLA2 [17–21]. Fortuitously, archived aliquots of these three venoms that were never thawed or used in the original studies [8,10] were maintained at 80 C and were available for the present investigation to test the − ◦ hypothesis that inhibition by ruthenium molecular species and not carbon monoxide may be the mechanism by which these procoagulant venoms were inhibited by CORM-2.

Table 1. Properties of procoagulant snake venoms investigated.

Species Common Name Proteome CORM-2/iRM Inhibition

Atheris nitschei [16] Great Lakes Bush Viper SVSP, SVMP, PLA2 Yes/No Echis leucogaster [17–19] White-Bellied Carpet Viper SVSP, SVMP, PLA2 Yes/No Pseudonaja textilis [20] Eastern Brown Snake SVSP, SVMP, PLA2 Yes/No

Considering the aforementioned, the present investigation had the following goals. First, determination of inhibition of the procoagulant activities of these venoms by their exposure in isolation to CORM-2 in the absence or presence of albumin was to be performed as previously described with bee venom PLA2 [15] and mamba venom [16]. Second, to further assess if Ru-based molecules may aﬀect venom procoagulant activity, the three venoms were exposed to equimolar concentrations +3 +2 of ruthenium chloride (RuCl3) which contains a Ru state compared to the Ru state of CORM-2. +3 Compounds incorporating Ru more complex than RuCl3 have been demonstrated to covalently bond to histidine residues in several proteins [22,23], thus oﬀering the possibility that RuCl3 could interact with histidine-bearing venom enzymes. Critically, the use of RuCl3 in this investigation was purely to provide mechanistic insight, and as there is no clinical indication to administer it to humans or any other species, I am not advocating it as a therapeutic option. As previously described [1–13,15,16], Int. J. Mol. Sci. 2020, 21, 2970 3 of 12

Int.changes J. Mol. Sci. in procoagulant 2019, 20, x FOR PEER activity REVIEW were assessed with human plasma via changes in coagulation kinetics3 of 12 determined with thrombelastography. 2. Results 2. Results 2.1. Assessment of the CO-Independent, Ru-Dependent Inhibition of CORM-2 on Procoagulant Activity of 2.1. Assessment of the CO-Independent, Ru-Dependent Inhibition of CORM-2 on Procoagulant Activity of A. nitschei , E. leucoga leucogasterster, and P. textilistextilis VenomsVenoms AssessedAssessed withwith ThrombelastographyThrombelastography TheThe subsequent results results were obtained using concentrations of the aforementioned venoms previously publishedpublished [ 8,[8,10];10]; specifically, specifically,A. nitschei A. nitscheiand E. leucogasterand E. leucogastervenoms had venoms a final concentrationhad a final concentrationof 1 µg/mL in theof plasma1 µg/mL mixtures in the whereas plasmaP. mixtures textilis venom whereas was atP. a finaltextilis concentration venom was of 100at a ng final/mL. concentrationVenom concentrations of 100 ng were/mL.originally Venom concentrations chosen based onwere a performance originally chosen basis whereinbased on the a performance activation of basiscoagulation wherein by the the venomactivation statistically of coagulation exceeded by thethe activation venom statistically observed by exceeded contact activationthe activation with observedthrombelastographic by contact cupactivation and pin with contact thrombelastographic with plasma as previouslycup and pin described contact [ 8with,10]. Allplasma venom as previouslysolutions without described or with [8,10]. chemical All venom additions solutions in isolation without were or with added chemical as a 1% additions addition toin theisolation plasma were mix addedused in as our a thrombelastographic1% addition to the plasma system [mix1–13 used]. This in dilution our thrombelastographic is critical, as it reduces system the concentration [1–13]. This dilutionof CORM-2 is critical, to 1 µM, as ait concentrationreduces the concentration at which this of compound CORM-2 doesto 1 µM, not aff concentrationect coagulation at kineticswhich th [9is]. compoundThe thrombelastographic does not affect model coagulation describes coagulationkinetics [9]. kinetics The thrombelastographic with the following three model variables: describes time coagulationto maximum kinetics thrombus with generation the following (TMRTG, three minutes—a variables: measuretime to maximum of time to thrombus onset of coagulation), generation (TMRTG,maximum minutes rate of thrombus—a measure generation of time (MRTG, to onset dynes of/cm coagulation),2/s—a measure maximum of the velocity rate ofof clot thrombus growth) 2 generationand total thrombus (MRTG, generationdynes/cm (TTG,/sec—a dynes measure/cm2 —aof the measure velocity of clotof clot strength). growth) The and results total of thrombus exposing 2 generationthe three venoms (TTG, todynes/cm CORM-2— ina themeasure absence of orclot presence strength) of. 5% The human results albumin of exposing (n = 6 the for three all conditions) venoms toare CORM depicted-2 in in the Figures absence1–3 .or presence of 5% human albumin (n = 6 for all conditions) are depicted in Figures 1–3.

Figure 1. Procoagulant activity of A. nitschei venom in plasma after exposure to CORM-2 without or Figure 1. Procoagulant activity of A. nitschei venom in plasma after exposure to CORM-2 without or with albumin in isolation. Data is presented as mean SD. Control = no additives; V = venom; VC = V with albumin in isolation. Data is presented as mean± ± SD. Control = no additives; V = venom; VC = with 100 µM CORM-2 in PBS; VC + A = V with 100 µM CORM-2 in albumin. * p < 0.05 vs. control; V with 100 µM CORM-2 in PBS; VC + A = V with 100 µM CORM-2 in albumin. *p < 0.05 vs. control; p < 0.05 vs. V; p < 0.05 vs. VC via one-way analysis of variance (ANOVA) with Holm–Sidak post ††p < 0.05 vs. V; ‡‡p < 0.05 vs. VC via one-way analysis of variance (ANOVA) with Holm–Sidak post hoc test. hoc test.

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FigureFigureFigure 2. 2. Procoagulant ProcoagulantProcoagulant activity activityactivity of ofof E. E.E. leuc leucogasterleucogasterogaster venom venomvenom in inin plasma plasmaplasma after afterafter exposure exposure to to CORM CORM-2CORM--22 without without oror with with albumin albumin in in isolation. isolation. Data Data are are presented presented as as mea meann ± SD.SD. Control Control = no= additives;no additives; V = Vvenom;= venom; VC or with albumin in isolation. Data are presented as mean ± ±SD. Control = no additives; V = venom; VC =VC= V V =with withV with 100 100 100µM µMµ CORM MCORM CORM-2--22 in in PBS; inPBS; PBS; V VC VCC + + A +A A= = V =V with Vwith with 100 100 100 µM µMµM CORM CORM CORM-2--22 in in in albumin. albumin. albumin. * *ppp < << 0 0.050.05.05 vs. vs. vs. control control control;; ; †pp << 0.050.05 vs. vs. V; V; ‡p

FigureFigureFigure 3.3. ProcoagulantProcoagulant activity activityactivity of ofofP. P.P. textilis textilistextilisvenom venomvenom in inplasmain plasmaplasma after afterafter exposure exposureexposure to CORM-2 toto CORMCORM without--22 withoutwithout or with oror withalbuminwith albumin albumin in isolation. in in isolation. isolation. Data Data Data are presentedare are presented presented as mean as as mea meaSD.nn ± ± S ControlSD.D. Control Control= no = = no additives;no additives; additives; V = V Vvenom; = = venom; venom; VC VC VC= V= = ± VwithV with with 100 100 100µM µM µM CORM-2 CORM CORM- in-22 in PBS;in PBS; PBS; VC V VC+C + A+ A A= = V= V V with with with 100 100 100µ µM MµM CORM-2 CORM CORM--2 in2 in in albumin. albumin. albumin. ** *pp < < 0 0.050.05.05 vs. vs. control control;control; ; ††pp < < 0 0.050.05.05 vs. vs. vs. V; V; V; ‡ ‡pp < p< 0 <0.05.050.05 vs. vs. vs. VC VC VC via via via one one one-way--wayway ANOVA ANOVA ANOVA with with with Holm Holm Holm–Sidak––SidakSidak post post post hoc hoc hoc test test test.. . † ‡ AllAllAll three three venomsvenoms behaved behaved kinetically kinetically as asas procoagulants procoagulantsprocoagulants as asas previously previouslypreviously noted notednoted [8,10], [[8,10],8,10], significantly signiﬁcantlysignificantly decreasingdecreasingdecreasing TMRTG TMRTGTMRTG and andand increasing increasingincreasing MRTG MRTGMRTG values. values. Similarly, Similarly, exposure exposureexposure of ofof the the venoms venoms to toto CORM CORM-2CORM--22 in in PBSPBSPBS significantly signiﬁcantlysignificantly attenuated attenuated procoagulant procoagulantprocoagulant activity activityactivity [8,10]. [[8,10].8,10]. However, However, when whenwhen the thethe venoms venomsvenoms were werewere exposedexposed tototo CORM CORM-2CORM--22 d dissolveddissolvedissolved in inin 5% 5% humanhuman albumin,albumin, procoagulantprocoagulant activityactivity waswas uninhibited.uninhibited. These TheseThese results resultsresults stronglystronglystrongly support supportsupport a aa CO CO-independent,CO--independent,independent, Ru Ru-dependentRu--dependentdependent mechanism mechanismmechanism of ofof inhibition inhibitioninhibition of ofof venom venomvenom procoagulant procoagulantprocoagulant activityactivity byby CORMCORM--22. . ResultsResults concerningconcerning thethe effectseffects ofof RuClRuCl33 onon plasmaticplasmatic coagulcoagulationation andand venomvenom procoagulantprocoagulant activity activity are are subsequently subsequently presented. presented.

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Int. J. Mol. Sci. , 20, x FOR PEER REVIEW 5 of 12 activity by CORM-2.2019 Results concerning the effects of RuCl3 on plasmatic coagulation and venom procoagulant activity are subsequently presented. 2.2. Assessment of the Effects of RuCl3 on Human Plasmatic Coagulation—Roles of Concentration and 2.2.Vehicle Assessment of the Effects of RuCl3 on Human Plasmatic Coagulation—Roles of Concentration and Vehicle PriorPrior to experimentation with venom, preliminary investigation demonstrated that the expected residual 1 µµMM concentration of RuCl33 dissolved in PBS increased MRTG values compared to control conditions. It It has has already already been been demonstrated demonstrated that that up up to to 10 10 µMµM CORM CORM-2-2 in PBS has no effect effect on plasmatic coagulationcoagulation kinetics kinetics [9], so[9], a moreso a formalmore determinationformal determination of why RuCl of3 displayedwhy RuCl procoagulant3 displayed propertiesprocoagulant was properties indicated. was Given indicated. that the Given only that compounds the only presentcompounds with present anions with different anions from different RuCl3 potentiallyfrom RuCl3 availablepotentially to available displace Clto displace in the PBS Cl usedin the was PBS KH used2PO was4 (1.5 KH mM)2PO4 and (1.5 NamM)2HPO and4 Na(8.12HPO mM),4 a(8.1 comparison mM), a comparison of the effects of th ofe effects 1 and 10of µ1 Mand RuCl 10 µM3 (final RuCl concentration)3 (final concentration) dissolved dissolved in dH2O in or dH PBS2O wasor PBS performed was p inerformed plasma asin a 1%plasma addition as a (v /v1%) with addition coagulation (v/v) assessedwith coagulation via thrombelastography. assessed via Thethrombelastography. results of these experiments The results of are these displayed experiments in Figure are4 displayed( n = 6 per in condition).Figure 4 (n = As 6 per can condition). be readily discerned,As can be therereadily appears discerned, to be there a Ru-dependent, appears to be vehicle-independent a Ru-dependent, vehicle significant-independent decrease insignificant TMRTG anddecrease increase in TMRTG in MRTG and values increase when in consideringMRTG values the when two-way considering analysis the of variance two-way (ANOVA) analysis of results variance and post(ANOVA) hoc comparison results and of post the two hoc concentrations comparison of of the RuCl two3 dissolved concentrati inons dH 2ofO. RuCl Similarly,3 dissolved increased in dH RuCl2O.3 concentrationsSimilarly, increased significantly RuCl3 concentrations decrease TMRTG significantly and increase decrease MRTG TMRTG values and when increase PBS MRTG is the vehicle. values However,when PBS andis the critically, vehicle. the However, coagulation and critically, kinetic diff theerences coagulation caused bykinetic RuCl differences3 are significantly caused enhancedby RuCl3 byare thesignificantly fluid it is dissolved enhanced in by as indicatedthe fluid byit theis dissolved two-way ANOVAin as indicated significance by the values two in-way each ANOVA panel of Figuresignificance4. Of interest,values in while each TMRTG panel of and Figure MRTG 4. Of values interest, change while in aTMRTG manner and indicative MRTG of values procoagulation, change in TTGa manner is decreased indicative by interactionsof procoagulation, of RuCl 3TTGand fluid.is decreased This thrombelastographic by interactions of patternRuCl3 and is indicative fluid. This of enhancedthrombelastographic thrombin–fibrinogen pattern is interactions indicative withoutof enhanced enhanced thrombin activation–fibrinogen of factor interactions XIII (FXIII) without [24,25]. enhanced activation of factor XIII (FXIII) [24,25].

Figure 4.4. Interactions ofof RuCl RuCl3concentration concentration and and ﬂuid fluid within within which which it is it dissolved. is dissolved. Data Data are are mean meaSD.n ± 3 ± 1-WSD. 1=-W1 µ= M1 µM RuCl RuCl3 in3 dH in dH2O;2 1-PBSO; 1-PBS= 1 =µ 1M µM RuCl RuCl3 in3 in PBS; PBS; 10-W 10-W= =10 10µ µMM RuCl RuCl3 3in in dH dH22O;O; 10-PBS10-PBS == 10 µµMM RuClRuCl 3 inin PBS.PBS. **p << 0.050.05 vs. 1-W;1-W; †pp < 00.05.05 vs. vs. 1 1-PBS;-PBS; ‡pp << 00.05.05 vs. vs. 10 10-W-W via via two two-way-way ANOVA ANOVA 3 † ‡ with Holm–SidakHolm–Sidak post hoc test. Two Two-way-way ANOVA resultsresults forfor interactioninteraction ofof RuClRuCl33 concentration and ﬂuidfluid are indicated withinwithin eacheach panel.panel.

As allall of of the the venoms venoms investigated investigated over over the pastthe fewpast years few years have been have suspended been suspended in PBS for in the PBS purposes for the ofpurposes preserving of preserving enzymatic enzymatic function within function a physiological within a physiological pH, storage, pH, and storage experimentation, and experimentation [1–13,15,16], it[1 seemed–13,15,16 prudent], it seemed to continue prudent with to thecontinue use of PBSwith in the the use subsequently of PBS in described the subsequently experimental described series experimental series assessing the effects of RuCl3 on the three venoms of the present work. The caveat that should be kept in mind was that small enhancements of MRTG in human plasma could be secondary to a RuCl3/PBS interaction when determining if RuCl3 inhibited venom procoagulant activity.

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assessing the eﬀects of RuCl3 on the three venoms of the present work. The caveat that should be kept in mind was that small enhancements of MRTG in human plasma could be secondary to a RuCl3/PBS Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 12 interactionInt. J. Mol. Sci. when2019, 20 determining, x FOR PEER REVIEW if RuCl 3 inhibited venom procoagulant activity. 6 of 12

2.3. Assessment of RuCl3-Dependent Modulation of CORM-2 on the Procoagulant Activity of A. nitschei, 2.3. Assessment of RuCl3-Dependent Modulation of CORM-2 on the Procoagulant Activity of A. nitschei, 2.3. Assessment of RuCl3-Dependent Modulation of CORM-2 on the Procoagulant Activity of A. nitschei, E. leucogaster, and P. textilistextilis VenomsVenoms AssessedAssessed withwith ThrombelastographyThrombelastography E. leucogaster, and P. textilis Venoms Assessed with Thrombelastography Utilizing thethe same same general general experimental experimental approach approach and speciﬁcand specific concentrations concentrations of the threeof the venoms three Utilizing the same general experimental approach and specific concentrations of the three venomstested, additional tested, additional aliquots aliquots of each venomof each wasvenom exposed was exposed to 100 µ Mto concentrations100 µM concentrations of RuCl 3ofin RuCl PBS3 for in venoms tested, additional aliquots of each venom was exposed to 100 µM concentrations of RuCl3 in PBS5 min for prior 5 min to prior being to placed being placed into the into plasma the plasma mixture mixture as a 1% as additiona 1% addition (v/v). ( Thev/v). rationaleThe rationale for this for PBS for 5 min prior to being placed into the plasma mixture as a 1% addition (v/v). The rationale for thisconcentration concentration of RuCl of RuCl3 was3 was that that it wouldit would be be similar similar to to that that of of the the CORM-2 CORM-2 exposureexposure experiments this concentration of RuCl3 was that it would be similar to that of the CORM-2 exposure experiments described in Section 2.12.1.. The results of these experi experimentsments are displayed in Figures5 5––77.. described in Section 2.1. The results of these experiments are displayed in Figures 5–7.

Figure 5. EffectEﬀect of exposure ofofA. A. nitschei,nitschei, E. E. leucogaster, leucogasterand, andP. P. textilis textilisvenom venom to to 100 100µM µM RuCl RuCl3 in3 PBSin PBS on Figure 5. Effect of exposure of A. nitschei, E. leucogaster, and P. textilis venom to 100 µM RuCl3 in PBS TMRTGon TMRTG values values in human in human plasma. plasma. Data areData presented are presented as mean as meaSD.n White ± SD. bars White= no bars RuCl =3 noexposure; RuCl3 on TMRTG values in human plasma. Data are presented as± mean ± SD. White bars = no RuCl3 blackexposure; bars black= 100 barsµM = RuCl 100 3µMin PBSRuCl exposure.3 in PBS exposure. * p < 0.05 *p vs. < 0 No.05 vs. RuCl No3 RuClin PBS3 in exposure PBS exposure via two-tailed, via two- exposure; black bars = 100 µM RuCl3 in PBS exposure. *p < 0.05 vs. No RuCl 3 in PBS exposure via two- tailed,unpaired unpairedt-test. t-test. tailed, unpaired t-test.

Figure 6. EffectEﬀect of exposure ofofA. A. nitschei,nitschei, E. E. leucogaster, leucogasterand, andP. P. textilis textilisvenom venom to to 100 100µM µM RuCl RuCl3 in3 PBSin PBS on Figure 6. Effect of exposure of A. nitschei, E. leucogaster, and P. textilis venom to 100 µM RuCl3 in PBS onMRTG MRTG values values in humanin human plasma. plasma. Data Data are are presented presented as as mean mean ±SD. SD. White White bars bars= = no RuCl3 exposure;exposure; on MRTG values in human plasma. Data are presented as mean± ± SD. White bars = no RuCl3 exposure; black bars = 100 µµMM RuCl33 in PBSPBS exposure.exposure. *pp << 00.05.05 vs. vs. no no RuCl RuCl33 inin PBS PBS exposure exposure via two two-tailed,-tailed, unpairedblack bars t -test.-=test 100. µM RuCl3 in PBS exposure. *p < 0.05 vs. no RuCl3 in PBS exposure via two-tailed, unpaired t-test.

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µ Figure 7. EffectEﬀect of exposure ofof A.A. nitschei,nitschei, E.E. leucogaster, leucogasterand, andP. P. textilis textilisvenom venom to to 100 100M µM RuCl RuCl3 in3 PBSin PBS on MRTG values in human plasma. Data are presented as mean SD. White bars = no RuCl exposure; on MRTG values in human plasma. Data are presented as mean± ± SD. White bars = no RuCl3 exposure; µ black bars = 100100 µMM RuCl3 inin PBS exposure. * *pp << 00.05.05 vs. vs. no no RuCl RuCl33 inin PBS PBS exposure exposure via via two two-tailed,-tailed, unpaired t--test.test.

Unlike in the series with CORM CORM-2-2 wherein wherein the pattern of inhibition of procoagulation was was very very similar amongamong the the three three venoms venoms tested, tested, there wasthere remarkable was remarkable diversity indiversity modulation in ofmodulation procoagulant of activity when the venoms were exposed to RuCl3. A. nitschei venom had a very diminutive increase in procoagulant activity when the venoms were exposed to RuCl3. A. nitschei venom had a very diminutiveTMRTG which increase is indicative in TMRTG of decreasedwhich is indicative procoagulant of decreased activity inprocoagulant response to activity RuCl3 exposure, in response but to a significant increase in both MRTG and TTG is consistent with an enhancement of procoagulation RuCl3 exposure, but a significant increase in both MRTG and TTG is consistent with an enhancement following RuCl3 exposure. In contrast, E. leucogaster venom demonstrated a significant increase in of procoagulation following RuCl3 exposure. In contrast, E. leucogaster venom demonstrated a TMRTG, decrease in MRTG and decrease in TTG values following RuCl3 exposure. Lastly, P. textilis significant increase in TMRTG, decrease in MRTG and decrease in TTG values following RuCl3 venom demonstrated significant loss of procoagulant activity after RuCl exposure to a qualitatively exposure. Lastly, P. textilis venom demonstrated significant loss of procoagulant3 activity after RuCl3 exposuregreater extent to a thanqualitatively the other greater two venoms. extent Whenthan comparedthe other two to the venoms. relatively When consistent compared pattern to andthe relativelydegree of consistent procoagulant pattern activity and degree among of the procoagulant three venoms activity provided among by the CORM-2 three venoms via apresumed provided byCO-independent CORM-2 via a/ Ru-dependentpresumed CO- mechanism,independent/Ru modulation-dependent of the mechanism venoms by, modulation RuCl3 resulted of the in venoms diverse changes in venom activity. by RuCl 3 resulted in diverse changes in venom activity. 3. Discussion 3. Discussion This investigation accomplished its objectives. The first series of experiments demonstrated that This investigation accomplished its objectives. The first series of experiments demonstrated that the procoagulant activity of A. nitschei, E. leucogaster, and P. textilis venom was not inhibited by CO the procoagulant activity of A. nitschei, E. leucogaster, and P. textilis venom was not inhibited by CO but instead by a presumed Ru-based CORM-2 radical that likely binds to venom enzyme histidine but instead by a presumed Ru-based CORM-2 radical that likely binds to venom enzyme histidine residues, evidenced by the loss of CORM-2 mediated inhibition in the presence of histidine-rich human residues, evidenced by the loss of CORM-2 mediated inhibition in the presence of histidine-rich albumin. These studies provided excess histidines to bind with reactive Ru species formed during CO human albumin. These studies provided excess histidines to bind with reactive Ru species formed release from CORM-2 by using 5% albumin (752 µM) as the solution for isolated exposures of venom during CO release from CORM-2 by using 5% albumin (752 µM) as the solution for isolated exposures to 100 µM CORM-2. Given that CORM-2 forms 70 µM of reactive Ru species during CO release from of venom to 100 µM CORM-2. Given that CORM-2 forms 70 µM of reactive Ru species during CO 100 µM CORM-2 [26], and that albumin has 16 histidine residues [27], in the first series of experiments release from 100 µM CORM-2 [26], and that albumin has 16 histidine residues [27], in the first series there was a 160:1 molar excess of histidine to react with Ru-based species. Thus, as was recently of experiments there was a 160:1 molar excess of histidine to react with Ru-based species. Thus, as demonstrated with the same experimental approach with PLA2 derived from Apis mellifera venom [15] was recently demonstrated with the same experimental approach with PLA2 derived from Apis and SVMP contained within mamba venom [16], it appears that procoagulant enzymes derived from mellifera venom [15] and SVMP contained within mamba venom [16], it appears that procoagulant the three venoms tested are vulnerable to inhibition by a Ru-based species formed from CORM-2. enzymes derived from the three venoms tested are vulnerable to inhibition by a Ru-based species The second series of experiments provided further evidence that Ru-based molecules could formed from CORM-2. modulate procoagulant snake venom enzymes, but with more variability of response to exposure to The second series of experiments provided further evidence that Ru-based molecules could RuCl compared to the CORM-2 experiments. There were three very different degrees in increase in modulate3 procoagulant snake venom enzymes, but with more variability of response to exposure to TMRTG values in response to RuCl3 exposure as seen in Figure5. In contrast to TMRTG, in the case RuCl3 compared to the CORM-2 experiments. There were three very different degrees in increase in of MRTG values it appeared that RuCl3 exposure enhanced A. nitschei venom procoagulant activity, TMRTG values in response to RuCl3 exposure as seen in Figure 5. In contrast to TMRTG, in the case whereas in the cases of E. leucogaster and P. textilis venoms there was inhibition of procoagulant activity of MRTG values it appeared that RuCl3 exposure enhanced A. nitschei venom procoagulant activity, whereas in the cases of E. leucogaster and P. textilis venoms there was inhibition of procoagulant activity by RuCl3 as displayed in Figure 6. Again, as with MRTG, changes in TTG values generated

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by RuCl3 as displayed in Figure6. Again, as with MRTG, changes in TTG values generated by the three venoms to RuCl3 followed the same species-specific pattern of enhanced procoagulant activity by A. nitschei venom and inhibited activity of E. leucogaster and P. textilis venoms as noted in Figure7. Given that RuCl3 in PBS does enhance MRTG to a small but significant extent, the MRTG results obtained with A. nitschei venom may be indicative of venom-independent enhancement of coagulation; however, the mixed finding of small increases in TMRTG values and increase in TTG values are likely secondary to direct modulation of A. nitschei venom as there were no venom-independent effects on TMRTG and TTG by RuCl3 (Figure4) consistent with these venom-mediated changes. The mechanisms +2 +3 responsible for differential effects on the three venoms by CORM-2 (Ru ) compared to RuCl3 (Ru ) remain to be defined, but when considered as a whole, the data of the present work strongly support the concept that Ru-based molecules, and not CO, are likely responsible for the CORM-2 mediated inhibition of diverse snake and lizard venoms [1–13]. An unexpected finding was the procoagulant effects of RuCl3 on human plasma that was enhanced by having the compound dissolved in PBS as seen in Figure4. This laboratory, using thrombelastographic methods, has already documented the effects of Fe+3 [28] and Cu+2 [29] as procoagulant and anticoagulant metals, respectively, via modulation of fibrinogen. However, neither Hg+2 nor Pb+2 at lethal concentrations were found to affect human plasmatic coagulation +3 with the same thrombelastographic methods [30]. Thus, the discovery that a Ru compound (RuCl3) at concentrations of a 1 µM affects plasmatic coagulation while a Ru+2 compound (inactivated CORM-2 [31,32]) needs to be at 100 µM concentrations to increase MRTG was unanticipated and interesting. The precise mechanism responsible for RuCl3 and its PBS induced ionic species was not defined by this investigation as it is beyond its scope. The utilization of thrombelastography to ask and answer molecular biology questions has been occurring for well over two decades, with numerous articles addressing hematological matters in several fields of investigation. It is critical to note that it is not the technique in the mechanical sense, but rather the parameters assessed and the composition of the sample analyzed that transform descriptive data that is phenomenological to parametric data that provides mechanistic insight into a focused experimental approach to testing molecular biological hypotheses. For example, the venoms assessed in this work are thrombin-generators that either activate prothrombin directly or indirectly by activating immediate precursor serine proteases in human plasmatic coagulation pathways. This feature of the venoms is best tested in a system with a relatively weak endogenous thrombin-generating scenario such as that associated with contact protein activation via interaction with the plastic surfaces of the thrombelastographic cup and pin. This allows the venom to outcompete such comparatively weak contact protein activation, permitting one the ability to assess the procoagulant activity in the presence or absence of prior isolated exposure to inhibitors or other relevant modulators. Use of the parameters TMRTG, MRTG, and TTG permits the use of parametric statistics as these expressions of clot initiation, velocity of growth, and final strength are not relatively qualitative as the unprocessed thrombelastographic tracing or nonparametric parameters such as the angle (◦) or maximum amplitude (mm) [33]. Furthermore, the use of plasma, and not whole blood with intact platelet activity, simplifies the output of the experiment wherein the coagulation kinetics are dependent on the invariant fibrinogen concentration and FXIII activity that are critical as previously published [24,25]. When summated, this experimental system will provide unambiguous data that is highly reproducible. The introduction of the variation of platelet concentration, variability in platelet glycoprotein IIb/IIIa receptor content, red blood cell concentration, artificially created blood flow models, etc., provide no additional mechanistic insight and instead introduce multiple confounding effects that may preclude testing the hypothesis of the present work. Similarly, utilizing standard coagulation assessments such as activated partial thromboplastin time (contact protein activation) or prothrombin time (tissue factor activated) simply introduce increased thrombin generation via activation of plasmatic contact protein and factor VII pathways, respectively—which would only compete with the venoms tested and provide no mechanistic insight. As was mentioned previously in this passage, the goal was to create an Int. J. Mol. Sci. 2020, 21, 2970 9 of 12 environment wherein the thrombin-generating activity of the venoms would not be confounded by any other coagulation activation. Taken as a whole, the experimental approach taken by the present investigation was designed to produce the unambiguous data presented to vigorously and conclusively test the hypothesis espoused. In conclusion, the present work determined that CORM-2 inhibited three already characterized procoagulant venoms via a CO-independent, likely Ru-based radical-dependent mechanism. Furthermore, a Ru+3-based ion also differentially affected the procoagulant activity of the venoms tested. With regard to utilization of Ru-based compounds as antivenoms, while the inhibitory effects on venom hemotoxic activity was inhibited by albumin in vitro at concentrations observed in vivo in the circulation, it is planned to administer such Ru-based compounds at concentrations at least 10-fold greater at the bite wound. Venom exposure to such concentrations has already been performed with neutralization in vivo in rabbits as recently noted [34]. Furthermore, these results continue to broaden the questioning of the effects of CORM-2 being CO-based, supporting the concept that the several hundred investigations conducted over the past few decades may include situations wherein Ru-based radical activity may be responsible for the effects of CORM-2. Lastly, these data continue to add to our mechanistic understanding of how Ru-based molecules can modulate hemotoxic venoms, and these results can serve as a rationale to perhaps focus on other complementary compounds containing Ru as antivenom agents in vitro and ultimately in vivo.

4. Materials and Methods

4.1. Chemicals and Human Plasma Lyophilized A. nitschei, E. leucogaster, and P. textilis venoms were originally obtained from Mtoxins (Oshkosh, WI, USA). Venomswere dissolved into calcium-free phosphate buﬀered saline (PBS, Millipore Sigma, Saint Louis, MO, USA) to a ﬁnal 50 mg/mL concentration, aliquoted, and maintained at 80 C. − ◦ The aliquots used came from the same lot published previously [8,10]. Dimethyl sulfoxide (DMSO), tricarbonyldichlororuthenium (II) dimer (CORM-2), and RuCl3 were obtained from Millipore Sigma (Saint Louis, MO, USA). Human albumin solution (5% in 0.9% NaCl) was obtained from Grisfols Biologicals Inc. (Los Angeles, CA, USA). Calcium chloride (200 mM) was obtained from Haemonetics Inc. (Braintree, MA, USA). Pooled normal human plasma that was sodium citrate anticoagulated and maintained at 80 C was obtained from George King Bio-Medical (Overland Park, KS, USA). − ◦ This plasma is a commercial product collected from consented, anonymous, and compensated healthy donors by the vendor, so no further consent is needed to be obtained by end users.

4.2. Thrombelastographic Analyses The volumes of subsequently described plasmatic and other additives summed to a final volume of 360 µL. Samples were composed of 320 µL of plasma; 16.4 µL of PBS, 20 µL of 200 mM CaCl2, and 3.6 µL of PBS, RuCl3, or venom solution mixture, which were pipetted into a disposable cup in a thrombelastograph® hemostasis system (Model 5000, Haemonetics Inc., Braintree, MA, USA) at 37 ◦C, and then rapidly mixed by moving the cup up against and then away from the plastic pin five times. The PBS, RuCl3, or venom solution mixtures was always the last constituent added prior to mixing and data collection. The following viscoelastic parameters described previously [1–13] were measured: time to maximum rate of thrombus generation (TMRTG): this is the time interval (minutes) 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/s); and total thrombus generation (TTG, dynes/cm2), the final viscoelastic resistance observed after clot formation. Data were collected until the clot strength reached its final plateau (maximum amplitude) that was stable for 3 min. The concentrations of venom that were used were as previously indicated in results and past manuscripts [8,10]. Int. J. Mol. Sci. 2020, 21, 2970 10 of 12

4.3. CORM-2 Addition Experiments In experiments with CORM-2 the conditions utilized were: (1) control condition—no venom, DMSO 1% addition (v/v) in PBS; (2) V condition—venom at the concentration determined in preliminary studies, DMSO 1% addition (v/v) in PBS; (3) VC condition—venom at the concentration as in condition 2, CORM-2 1% addition in DMSO in PBS (100 µM); (4) VC+A condition—venom and CORM-2 1% addition in DMSO in 5% human albumin (100 µM ﬁnal concentration). Solutions were incubated for 5 min at room temperature, and then 3.6 µL of one of these solutions was added to the plasma sample in the plastic cup.

4.4. RuCl3 Addition Experiments

In preliminary experiments with RuCl3, it was determined that the ﬁnal concentration of 1 µM increased MRTG. Thus, experiments wherein RuCl3 was dissolved in either dH2O or PBS were conducted with the ﬁnal concentration of RuCl3 in plasma being 1 or 10 µM. Data were collected until maximum amplitude was observed. In venom exposure experiments, aliquots of venom dissolved in PBS were exposed to 100 µM RuCl3 previously dissolved in PBS for 5 min at room temperature prior to being placed in the plasma mixture in the thrombelastographic cup as a 1% (v/v) addition. Data were collected until maximum amplitude was observed.

4.5. Graphics and Statistical Analyses Data are presented as mean SD. Graphics were generated with a commercially available ± program (Origen2020, OrigenLab Corporation, Northampton, MA, USA). Experimental conditions were composed of n = 6 replicates per condition as this provides a statistical power >0.8 with p < 0.05 utilizing these techniques [1–13]. A statistical program was used for one-way analyses of variance (ANOVA) comparisons between conditions, followed by Holm–Sidak post hoc analysis (SigmaPlot 14, Systat Software, Inc., San Jose, CA, USA). p < 0.05 was considered signiﬁcant.

Funding: This project received no external funding. Conﬂicts of Interest: The author does not have any conﬂict of interest.

Abbreviations

CO carbon monoxide CORM carbon monoxide releasing molecule; can have Ru, or other metal centers CORM-2 tricarbonyldichlororuthenium (II) dimer; Ru-based CORM Cu copper DMSO dimethyl sulfoxide Fe iron Hg mercury MRTG maximum rate of thrombus generation (dynes/cm2/s) Pb lead PBS phosphate buﬀered saline, calcium-free, pH = 7.1–7.4 PLA2 phospholipase A2 Ru ruthenium RuCl3 ruthenium chloride SD standard deviation SVMP snake venom metalloproteinase SVSP snake venom serine protease TMRTG time to maximum rate of thrombus generation (minutes) TTG total thrombus generation (dynes/cm2) Int. J. Mol. Sci. 2020, 21, 2970 11 of 12

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