The role of CORM-2 as a modulator of oxidative stress and hemostatic parameters of human plasma in vitro

Weronika Adach; Beata Olas

doi:10.1371/journal.pone.0184787

Abstract

Purpose

The main aim of the experiment is to examine the effect of CORM-2, a donor of carbon monoxide (CO), on oxidative stress in human plasma in vitro. In addition, it examines the effects of CORM-2 on the hemostatic parameters of plasma: the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT).

Methods

Human plasma was incubated for 5–60 min with different concentrations of CORM-2: 0.1–100 μM. Following this, various hemostatic factors and biomarkers of oxidative stress were studied. Lipid peroxidation was measured as thiobarbituric acid reactive substance (TBARS) concentration, and the oxidation of amino acid residues in proteins was measured by determining the amounts of carbonyl and thiol groups.

Results

Two oxidative stress inducers: hydrogen peroxide (H₂O₂) and the donor of hydroxyl radical (H₂O₂/Fe) were used. Decrease in protein carbonylation, thiol group oxidation and lipid peroxidation were detected at tested concentrations of CORM-2.

Conclusion

Our results indicate that CORM-2 may have antioxidant properties in human plasma treated with H₂O₂ or H₂O₂/Fe. In addition, our results indicate the anti-coagulant activities of CORM-2 in vitro.

Citation: Adach W, Olas B (2017) The role of CORM-2 as a modulator of oxidative stress and hemostatic parameters of human plasma in vitro. PLoS ONE 12(9): e0184787. https://doi.org/10.1371/journal.pone.0184787

Editor: Ferenc Gallyas Jr., University of PECS Medical School, HUNGARY

Received: June 11, 2017; Accepted: August 30, 2017; Published: September 26, 2017

Copyright: © 2017 Adach, Olas. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by grant 506/1136 from University of Lodz.

Competing interests: The authors have declared that no competing interests exist.

Introduction

For decades, scientists have wondered if the biological relevance of toxic carbon monoxide (CO) could be isolated. The fact that uncontrolled quantities of inhaled gas lead to serious system complications and neuronal disturbances cannot be ignored. CO is a colorless, odorless and non-irritating gas. It is produced endogenously in the body, however, at elevated concentrations is highly toxic to organism [1]. During heme degradation, CO (by heme oxygenase (OH)) is synthetized. CO is also produced as a result of photooxidation, metabolism of xenobiotics and lipid peroxidation [2–5]. Nowadays, CO is becoming an important and versatile physiological mediator that has a beneficial effect on many models of vascular and inflammatory diseases [6–8]. CO regulates blood pressure and inhibits blood platelet aggregation [9–11]. Nielsen and Pretorius [12] suggest that CO may not only act as an anticoagulant, but also as a procoagulant. In addition, results of Nielsen and Garza et al. [13] demonstrated that CO—releasing molecules (CORMs): CORM-2 and CORM-3 enhanced thrombus strength and the velocity of clot formation in human plasma. Therefore, the question is whether there is a possibility to achieve therapeutic and safe concentrations of CO in tissues and organs, without providing a toxic dose through the lungs [1].

Because, mechanism(s) involved the relationship between the action of CO and various components of hemostasis (i.e. blood platelets, the coagulation and fibrinolysis) are still unknown, the aim of our experiments was to explain the effects of CORM-2 (the donor of CO; tricarbonyldichlororuthenium (II) dimer; Ru₂Cl₄(CO)₆; at tested concentrations: 0.1–100 μM) on the three hemostatic parameters: the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) of human plasma in vitro. In addition, as the influence of donors of CO on oxidative stress remains also unknown, the aim of our experiment was to also determine the effects of CORM-2 on oxidative stress in human plasma when administered at concentrations of 0.1 to 100 μM in vitro. The study tests the activity of CORM-2 against the effect of a strong biological oxidant, either hydrogen peroxide (H₂O₂) or H₂O₂/Fe (a hydroxyl radical donor), on plasma lipids and proteins. Oxidative modifications of these components, especially plasma proteins are very important for modulating of hemostasis. They have been reported in various conditions, including cardiovascular disorders. Oxidative stress was measured by examining the concentrations of well-known oxidative biomarkers such as thiobarbituric acid reactive substance (TBARS), a marker of lipid peroxidation, the concentration of carbonyl groups and the concentration of thiol groups, which are markers of oxidative damage in proteins.

Materials and methods

CORM-2, 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB), thiobarbituric acid (TBA) and H₂O₂ were purchased from Sigma Chemical Co. (Germany). The coagulation reagents were obtained from Diagon Ltd. (Hungary) and Behringer Ingelheim. All other chemicals were reagent-grade products purchased from POCh (Gliwice, Poland).

A stock solution of CORM-2 was made in DMSO (the final concentration of DMSO in a stock solution of CORM-2 was 50%). The final concentration of DMSO in samples was lower than 0.05% and its effects were determined in all experiments. Inactive CORM-2 (i-CORM-2) was prepared by keeping the stock solution in 50% DMSO over night at 37°C [13,14].

Exposure of human plasma to CORM-2

Fresh human plasma was obtained from medication-free, regular donors at the blood bank (Lodz, Poland). Samples of human plasma were exposed to 1) CORM-2 at a final concentration between 0.1 and 100 μM plus 2 mM H₂O₂; 2) CORM-2 at a final concentration between 0.1 and 100 μM plus 4.7 mM H₂O₂/3.8 mM Fe₂SO₄/2.5 mM EDTA. Samples were incubated for 5, 15, 30 and 60 minutes at 37°C. In addition, plasma was exposed to iCORM-2 (at a final concentration 100 μM) or CORM-2 (at a final concentration 100 μM) and albumin (at a final concentration 10%) for 30 minutes at 37°C. Moreover, samples of plasma were also exposed to iCORM-2 (at a final concentration 100 μM) or CORM-2 (at a final concentration 100 μM) and albumin (at a final concentration 10%) plus 4.7 mM H₂O₂/3.8 mM Fe₂SO₄/2.5 mM EDTA for 30 minutes at 37°C.

The protein concentration by measuring absorbance at 280 nm (in tested samples) was calculated according to the procedure of Whitaker and Granum [15].

Lipid peroxidation measurement

Lipid peroxidation was quantified by measuring the concentration of TBARS. The incubation of plasma (controls, tested compounds) was stopped by cooling in an ice bath. The plasma samples were transferred to an equal volume of 15% (v/v) cold trichloroacetic acid in 0.25 M HCl and centrifuged at 8000 x g for 15 minutes. One volume of clear supernatant was mixed with 0.2 volume of 0.12 M thiobarbituric acid in 0.25 M HCl and immersed in a boiling water bath for 15 minutes. Then the cooled samples was centrifuged at 8000 x g for 5 min and the absorbance was measured at 535 nm using the SPECTROstar Nano Microplate Reader- BMG LABTECH (Germany) [16,17]. The TBARS concentration was calculated using the molar extinction coefficient (ε = 156,000 M^-1cm^-1). The level of TBARS (as nmol TBARS/ml of plasma) was expressed as % of control.

Carbonyl group measurement

The detection of carbonyl groups in proteins was carried out according to Levine et al. [18] and Bartosz [17]. Carbonyl content was measured by absorbance at 375 nm (the SPECTROstar Nano Microplate Reader- BMG LABTECH, Germany). The carbonyl group concentration was calculated using a molar extinction coefficient (ε = 22,000 M^-1cm^-1), and the level of carbonyl groups (as nmol carbonyl groups/mg of plasma protein) was expressed as % of control.

Thiol group measurement

The thiol group content in plasma proteins was measured spectrophotometrically (the SPECTROstar Nano Microplate Reader- BMG LABTECH, Germany) by absorbance at 412 nm with Ellman’s reagent. The thiol group concentration was calculated using the molar extinction coefficient (ε = 13,600 M^-1cm^-1) [17,19,20]. The level of thiol groups was expressed as μmol thiol groups/ml of plasma.

The measurement of PT

Human plasma (50 μl) was incubated for 2 min. at 37°C on block heater. After incubation, the measuring cuvette was transferred to the measuring holes and 100 μl of Dia-PT liquid (commercial preparation) was added. The PT was determined coagulometrically (Optic Coagulation Analyser model K-3002; Kselmed, Grudziadz, Poland) [21].

The measurement of TT

Human plasma (50 μl) was added to the measuring cuvette and incubated for 1 minute at 37°C on a block heater. The measuring cuvette was transferred to the measuring holes and 100 μl of thrombin was added (final concentration—1 U/ml). The TT was determined coagulometrically using a K-3002 Optic Coagulation Analyser (Kselmed, Grudziadz, Poland) [21].

The measurement of APTT

Human plasma (50 μl) was added to measuring cuvette and incubated with Dia-PTT liquid (commercial thromboplastin) for 3 minutes at 37°C on a block heater. The measuring cuvette was transferred to the measuring holes and 50 μl of 25 mM CaCl₂ was added. The APTT was determined coagulometrically using a K-3002 Optic Coagulation Analyser (Kselmed, Grudziadz, Poland) [21].

Data analysis

In order to eliminate uncertain data, the Q-Dixon test was performed. All the values in this study were expressed as mean ± SD. Obtained results firstly were analysed under the account of normality with Shapiro-Wilk test and equality of variance with Levine test. Statistically significant differences were assessed by applying the ANOVA test, followed by Tukey multiple comparisons test or Kruskal-Wallis test.

Results

CORM-2 (Fig 1) added to human plasma (treated with H₂O₂ or H₂O₂/Fe) in vitro at concentrations of 0.1–100 μM had different effect on lipid peroxidation (measured by TBARS level) after 5, 15, 30 and 60 minutes of incubation (Figs 2 and 3). The lipid peroxidation induced by H₂O₂ was not influenced by CORM-2 at short incubation time (5 and 15 minutes) (Fig 2). However, CORM-2 (at all tested concentrations: 0.1, 1, 10, 50 and 100 μM, and at longer incubation time: 30 and 60 minutes) reduced lipid peroxidation induced by H₂O₂ (Fig 2). CORM-2 (at higher concentrations: 10, 50 and 100 μM) after long incubation times (30 and 60 minutes) significantly also reduced lipid peroxidation induced by H₂O₂/Fe (Fig 3).

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Fig 1. Chemical structure and characteristics of CORM-2 [14; modified].

https://doi.org/10.1371/journal.pone.0184787.g001

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Fig 2. The effect of CORM-2 (0.1–100 μM, incubation time: 5–60 min) on plasma lipid peroxidation induced by H₂O₂.

Data represents means ± SD of 6 experiments (from different donors). * p<0.05 vs. control (+ H₂O₂), n.s. p>0.05 vs. control (+ H₂O₂); # p<0.05 vs. control (- H₂O₂), ## p<0.02 vs. control (- H₂O₂).

https://doi.org/10.1371/journal.pone.0184787.g002

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Fig 3. The effect of CORM-2 (0.1–100 μM, incubation time: 5–60 min) on plasma lipid peroxidation induced by H₂O₂/Fe.

Data represents means ± SD of 6 experiments (from different donors). * p<0.05 vs. control (+ H₂O₂/Fe), n.s. p>0.05 vs. control (+ H₂O₂/Fe).

https://doi.org/10.1371/journal.pone.0184787.g003

Although CORM-2 did not change the level of carbonyl groups in plasma proteins when administered without the addition of H₂O₂ or H₂O₂/Fe (p>0.05) (data are not presented), a significant decrease was observed in the level of carbonyl groups in plasma proteins compared with control—H₂O₂/Fe, when human plasma was incubated with the highest tested concentrations of CORM-2–50 and 100 μM and with H₂O₂/Fe (Fig 4). In the presence of highest concentration of CORM-2 (100 μM) protein carbonylation was reduced by about 30% (for time incubation– 30 min) and by about 15% (for time incubation– 60 min) (Fig 4).

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Fig 4. The effect of CORM-2 (0.1–100 μM, incubation time– 30 and 60 min) on carbonyl group formation (plasma protein oxidation) induced by H₂O₂/Fe.

Data represents means ± SD of 6 experiments (from different donors). * p<0.05 vs. control (+ H₂O₂/Fe), n.s. p>0.05 vs. control (+ H₂O₂/Fe).

https://doi.org/10.1371/journal.pone.0184787.g004

Another set of experiments focused on the determination of oxidation of plasma protein thiols. We observed that CORM-2 (at all tested concentrations) had strong protector effect on the changes of the level of thiol groups in plasma proteins induced by H₂O₂/Fe (Fig 5). The CORM-2 activity was concentration—dependent (Fig 5).

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Fig 5. The effect of CORM-2 (0.1–100 μM, incubation time– 30 and 60 min) on the oxidation of protein thiols induced by H₂O₂/Fe.

Data represents means ± SD of 6 experiments (from different donors). * p<0.05 vs. control (+ H₂O₂/Fe), ** p<0.01 vs. control (+ H₂O₂/Fe), *** p<0.0002 vs. control (+ H₂O₂/Fe), # p<0.0005 vs. control (+ H₂O₂/Fe), ## p<0.00002 vs. control (+ H₂O₂/Fe), n.s. p>0.05 vs. control (+ H₂O₂/Fe).

https://doi.org/10.1371/journal.pone.0184787.g005

Incubation (30 minutes) with CORM-2 resulted in changes of coagulation activity of human plasma (Fig 6). We demonstrated that incubation of plasma with CORM-2 (at high tested concentrations: 50 and 100 μM) significantly prolonged the prothrombin time. The studied also showed that the exposure of plasma to CORM-2 (0.1–100 μM) did not change in the TT and APTT (Fig 6).

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Fig 6. The effect of CORM-2 (0.1–100 μM, incubation time– 30 min) on the hemostatic parameters of human plasma (APPT, PT and TT).

Data represents means ± SD of 10 experiments (from different donors). * p<0.05 vs. control, n.s. p>0.05 vs. control.

https://doi.org/10.1371/journal.pone.0184787.g006

iCORM-2 had no effect on biomarkers of oxidative stress, which we studied in vitro (in compare to control—plasma treated with H₂O₂/Fe) (Fig 7). However, 100 μM CORM-2 significantly reduced lipid peroxidation, protein carbonylation and oxidation of protein thiols in plasma treated with H₂O₂/Fe (in compare not only to plasma treated only with H₂O₂/Fe, but also to plasma treated with iCORM-2 and H₂O₂/Fe). The same process was observed in presence of albumin and CORM-2 (Fig 7). In addition, iCORM (100 μM) had no effect on hemostatic parameters of plasma. It did not change the PT (Fig 8) and the TT and APTT (data not shown). Moreover, CORM-2 (100 μM) in presence of albumin did not change in the level of tested hemostatic parameters of plasma (Fig 8).

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Fig 7. The effect of iCORM-2 (100 μM, incubation time—30 min) or CORM-2 (100 μM, incubation time—30 min) and albumin (10%, incubation time—30 min) on biomarkers of oxidative stress (lipid peroxidation (A), carbonyl group formation (B), oxidation of protein thiols (C)) in plasma treated with H₂O₂/Fe.

Data represents means ± SD of 5–11 experiments (from different donors).

https://doi.org/10.1371/journal.pone.0184787.g007

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Fig 8. The effect of iCORM-2 (100 μM, incubation time—30 min) or CORM-2 (100 μM, incubation time—30 min) and albumin (10%, incubation time—30 min) on the PT of plasma.

In these experiments, the PT in control samples was 45.6 ± 10.2 s. Data represents means ± SD of 5–15 experiments (from different donors).

https://doi.org/10.1371/journal.pone.0184787.g008

In control experiments, we observed that DMSO (the solvent of CORM-2) added to plasma at a final concentration <0.05% did not change the level of biomarkers of oxidative stress and tested hemostatic parameters (data not shown).

Discussion

CORMs are releasing CO in a concentration-dependent manner, modulated by temperature, pH and environment (solvents) [22]. The effects of various donors of CO on different components of hemostasis, i.e. blood platelets, coagulation and fibrinolysis process may be manifold due to their pleiotropic character. However, there are only a few experiments addressing the anti-caoagulatory, pro-coagulatory and anti-platelet effects of CORMs [10, 12, 23, 24]. Liu et al. [10] have observed that CORM-2 inhibits the abnormal activation of blood platelets stimulated by endotoxin. The results of Nielsen and Pretorius [24] have demonstrated that CO derived from CORM-2 interacts with coagulation and fibrinolytic process, resulting in thrombi that begin to form more quickly, grow faster, become stronger, and are more resistant to lysis. In these experiments, the effects of CORMs on coagulation were determined by thrombelastography using either tissue factor or celite activation. The same author have noted the effects of six Agkistrodon species’ venoms on plasmatic coagulation, with iron and CORM-2 addition attenuating the degradation of coagulation in a species—specific manner [25]. In addition, CO and iron have modulated plasmatic coagulation in Alzheimer’s disease [7]. Results of Motterlini et al. [26] have indicated that CORMs, including CORM-2 cause vasodilation in precontracted rat aortic rings, attenuate coronary vasoconstriction in hearts ex vivo, and reduce acute hypertension in vivo.

The present study provides more information on the biological property of CORM-2 in human plasma. First, the results of our studies showed that exogenous CO (as CORM-2) causes changes in hemostasis (determined coagulametrically); at concentrations of 50 and 100 μM prolonges the prothrombin time, which is a measure of the efficiency of extrinsic coagulation system. Prolongation of prothrombin time by CORM-2 can attest to its effect on prothrombin activity or clotting factors V, VII, and X. This obtained results indicate the anti-coagulant activities of CORM-2 in model system in vitro. We also demonstrated changes in lipid peroxidation, formation of carbonyl groups and oxidation of thiol groups in human plasma treated with CORM-2. Our present experiments have demonstrated that in human plasma, the properties of CORM-2 depend on very often its concentration and incubation time. Our work was designed to estimate the antioxidative or prooxidative effects of CORM-2 on human plasma lipid and protein changes stimulated by two biological oxidants: H₂O₂ and H₂O₂/Fe (in vitro). In our present experiments, we used two oxidative conditions, namely (I) H₂O₂ and (II) H₂O₂/Fe, because the concentration of Fe is low in isolated human plasma, and the level of markers of oxidative stress (i.e. TBARS) is also low. However, when we added Fe to plasma, we observed higher level of these biomarkers of oxidative stress. In addition, some authors believe that some complexes of iron ions (i.e. EDTA) may also react with H₂O₂ to form OH [27,28]. The results from our experiments show that CORM-2 (at the highest tested concentrations) had an inhibitory action on H₂O₂ or H₂O₂/Fe—induced oxidation in plasma. These results are consistent with other studies on the role of CO donors in protecting against oxidative stress, i.e. studies of Tsai et al. [8] demonstrated that CORM-2 inhibits angiotensin II-induced human aortic smooth muscle cells migration through inactivation of NADPH oxidase/reactive oxygen species generation. Babu et al. [14] also noted that CORM-2 shows antioxidant property in murine intestinal epithelial MODE-K cells under oxidative stress (induced by H₂0₂). Moreover, it is very important that our used concentrations of CORM-2 in our model in vitro are consistent with other studies [8, 11, 14, 25, 29], i.e. Tsai et al. [8] observed that 50 μM CORM-2 has antioxidant activity. Results of Srisook et al. [29] have also demonstrated that 50 μM CORM-2 inhibits the production of superoxide anion (O₂^-·) and nitric oxide (NO^·) in macrophages stimulated by bacterial lipopolysaccharide (LPS) in vitro. The amount of CORM-2 used in vitro in the present experiment and by other authors is significantly less than has been administrated in vivo [30, 31]. In addition, our present results show that CORM-2 especially at high tested concentrations (10, 50 and 100 μM) could be used as an antioxidant (when the oxidative stress is stimulated not only by H₂O₂, but also by a hydroxyl radical donor—H₂O₂/Fe).

It is very important that in cardiovascular system, CO does not work often in isolation, but it may interact with reactive oxygens species or reactive nitrogen species, i.e. nitric oxide, which may be secondary messengers in various biological systems. The obtained results indicate not only the antioxidant activities, but also anticoagulant properties of CORM-2 in vitro. Therefore, we suppose that CORM-2 may be promising compound to prevent thrombosis in pathological processes where plasma procoagulant activity and oxidative stress are observed. However, the mechanism(s) of CORM-2 action on hemostasis has not yet well know. Recognition of CO donors, including CORM-2 as both an antioxidant and a prooxidant may enhance the treatment of various diseases associated with oxidative stress. Moreover, further experiments are need to gain a more complete understanding of the dual nature of the procoagulant and anticoagulant of CORMs. It may be especially very important in cardiovascular diseases, in which oxidative stress may modulate various elements of hemostasis. The further experiments should be done to establish the effects of CORM-2 (as an antioxidant) on activity of different elements of hemostasis, including blood platelets.

Supporting information

S1 Table. The effect of CORM-2 (0.1–100 μM, incubation time: 5–60 min) on plasma lipid peroxidation induced by H₂O₂.

https://doi.org/10.1371/journal.pone.0184787.s001

(TIF)

S2 Table. The effect of CORM-2 (0.1–100 μM, incubation time: 5–60 min) on plasma lipid peroxidation induced by H₂O₂/Fe.

https://doi.org/10.1371/journal.pone.0184787.s002

(TIF)

S3 Table. The effect of CORM-2 (0.1–100 μM, incubation time—30 and 60 min) on carbonyl group formation (plasma protein oxidation) induced by H₂O₂/Fe.

https://doi.org/10.1371/journal.pone.0184787.s003

(TIF)

S4 Table. The effect of CORM-2 (0.1–100 μM, incubation time—30 and 60 min) on the oxidation of protein thiols induced by H₂O₂/Fe.

https://doi.org/10.1371/journal.pone.0184787.s004

(TIF)

S5 Table. The effect of CORM-2 (0.1–100 μM, incubation time—30 min) on the hemostatic parameters of human plasma (APPT, PT and TT).

https://doi.org/10.1371/journal.pone.0184787.s005

(TIF)

S6 Table. The effect of iCORM-2 (100 μM, incubation time—30 min) or CORM-2 (100 μM, incubation time—30 min) and albumin (10%, incubation time—30 min) on biomarkers of oxidative stress (lipid peroxidation, carbonyl group formation, oxidation of protein thiols) in plasma treated with H₂O₂/Fe.

https://doi.org/10.1371/journal.pone.0184787.s006

(TIF)

S7 Table. The effect of iCORM-2 (100 μM, incubation time—30 min) or CORM-2 (100 μM, incubation time—30 min) and albumin (10%, incubation time—30 min) on the PT of plasma.

https://doi.org/10.1371/journal.pone.0184787.s007

(TIF)

References

1. Foresti R, Bani-Hani MG, Motterlini R. Use of carbon monoxide as a therapeutic agent: promises and challenges. Inten Care Med J. 2008;34: 649–658.
- View Article
- Google Scholar
2. Durante W, Johnson FK, Johnson RA. Role of carbon monoxide in cardiovascular function. J Cell Mol Med. 2006;10: 672–686. pmid:16989727
- View Article
- PubMed/NCBI
- Google Scholar
3. Olas B. Carbon monoxide is not always a poison gas from human organism: physiological and pharmacological features of CO. Chem-Biol Inter. 2014;222: 37–43.
- View Article
- Google Scholar
4. Motterlini R, Forsti R. Biological signaling by carbon monoxide and carbon monodxide-reeasing molecules. Am J Physiol Cell Physiol. 2017;312: C302–C313. pmid:28077358
- View Article
- PubMed/NCBI
- Google Scholar
5. Vreman HJ, Wong RJ, Stevenson DK. Carbon monoxide in breath, blood, and other tissues. [in] Carbon Monoxide Toxicity, Penney D.G., Ed., CRC Press Boca Raton. FL., 2000, pp. 19–59.
6. Fredenburgh LE, Merz AA, Cheng S. Heme oxygenase signalling pathway: implications for cardiovascular disease. Eur Heart J. 2015;36: 1512–1518. pmid:25827602
- View Article
- PubMed/NCBI
- Google Scholar
7. Nielsen VG, Pretorius E, Bester J, Jacobsen WK, Boyle PK, Reinhard JP. Carbon monoxide and iron modulate plasmatic coagulation in Alzheimer's disease. Curr Neurovasc Res. 2015;12: 31–39. pmid:25557378
- View Article
- PubMed/NCBI
- Google Scholar
8. Tsai MH, Lee CW, Hsu LF, Li SY, Chiang YC, Lee MH, et al. CO-releasing molecules CORM2 attenuates angiotensin II-induced human aortic smooth muscle cell migration through inhibition of ROS/IL-6 generation and matrix metalloproteinases-9 expression. Redox Biol. 2017;12: 377–388. pmid:28292711
- View Article
- PubMed/NCBI
- Google Scholar
9. Truss N, Warner TD. Gasotransmiters and platelets. Pharm Therap. 2011;132: 196–203.
- View Article
- Google Scholar
10. Liu D, Zhuang M, Zhang J, Chen J, Sun B. Study of exogenous carbon monoxide-releasing molecules 2 on endotoxin/lipopolysaccharide-induced abnormal activation of platelets of healthy human donors. Zhonghua Shao Shang Za Zhi 2015;31: 354–360. pmid:26714404
- View Article
- PubMed/NCBI
- Google Scholar
11. Liu D, Wang X, Qin W, Chen J, Wang Y, Zhuang M, et al. Suppressive effect of exogenous carbon monoxide on endotoxin-stimulated platelet over-activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway. Sci Rep. 2016, 1–14.
- View Article
- Google Scholar
12. Nielsen HG, Pretorius E. Iron and carbon monoxide enhance coagulation and attenuate fibrinolysis by different mechanisms. Blood Coagul Fibrinol. 2014;25: 695–702.
- View Article
- Google Scholar
13. Sun B, Sun Z, Jin Q, Chen X. CO-releasing molecules (CORM-2)-liberated CO attenuates leukocytes infiltration in the renal tissue of thermally injured mice. Int J Biol Sci. 2008;4: 176–183. pmid:18566696
- View Article
- PubMed/NCBI
- Google Scholar
14. Babu D, Leclercq G, Motterlini R, Lefebvre RA. Differential effects of CORM-2 and CORM-401 in murine intestinal epithelial MODE-K cells under oxidative stress. Front. Pharm. 2017;8: 1–17.
- View Article
- Google Scholar
15. Whitaker JR, Granum PE. An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Anal Biochem. 1980;109: 156–159. pmid:7469012
- View Article
- PubMed/NCBI
- Google Scholar
16. Wachowicz B. Adenine nucleotides in thrombocytes of birds. Cell Bioch Fun. 1984;2: 167–170.
- View Article
- Google Scholar
17. Bartosz B. Druga twarz tlenu. PWN, Warszawa 2008;1.7: 99–120.
18. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. Determination of carbonyl content in oxidatively modified proteins. Meth Enzymol. 1990;186: 464–478. pmid:1978225
- View Article
- PubMed/NCBI
- Google Scholar
19. Ando Y, Steiner M. Sulphydryl and disulphide groups of platelet membranes: determination of disulphide groups. Biochim Biophys Acta 1973;311: 26–37. pmid:4718241
- View Article
- PubMed/NCBI
- Google Scholar
20. Ando Y, Steiner M. Sulphydryl and disulphide groups of platelet membranes: determination of sulphydryl groups. Biochim Biophys Acta 1973;311: 38–44. pmid:4718242
- View Article
- PubMed/NCBI
- Google Scholar
21. Malinowska J, Kołodziejczyk-Czepas J, Moniuszko-Szajwaj B, Kowalska I, Oleszek W, Stochmal A, et al. Phenolic fractions from Trifolium pallidum and Trifolium scabrum aerial parts in human plasma protect against changes induced by hyperhomocysteinemia. Food Chem Toxicol. 2012;50: 4023–4027. pmid:22898612
- View Article
- PubMed/NCBI
- Google Scholar
22. Nielsen VG, Kirklin JK, George JF. Carbon monoxide releasing molecule-2 increases the velocity of thrombus growth and strength in human plasma. Wolters Kluwer Health | Lippincott Williams & Wilkins 2009
23. Nielsen VG, Garza JI. Comparison of the effects of CORM-2, CORM-3 and CORM-A1 on coagulation in human plasma. Blood Coagul Fibrinol. 2014;25: 801–805.
- View Article
- Google Scholar
24. Nielsen V, Pretorius E. Carbon monoxide: antioagulant or procoagulant? Thromb Res. 2014;133: 315–321. pmid:24360115
- View Article
- PubMed/NCBI
- Google Scholar
25. Nielsen VG, Redford DT, Boyle PK. Effect of iron and carbon monoxide on fibrinogenase-like degradation of plasmatic coagulation by venoms of six Agkistrodon species. BCPT 2016;118: 390–395.
- View Article
- Google Scholar
26. Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, Green CJ. Carbon monoxide-releasing molecules. Characterization of biochemical and vascular activities. Circ Res. 2002;90: e17–e24. pmid:11834719
- View Article
- PubMed/NCBI
- Google Scholar
27. Luzzatto E, Cohen H, Stockheim C, Wieghardt K, Meyerstein D. Reactions of low valent transition metal complexes with hydrogen peroxide. Are they “Fenton-like” or not? The case of Fe(II)L, L = edta, hedta and tcma. Free Radic Res. 1995;23: 453–463. pmid:7581828
- View Article
- PubMed/NCBI
- Google Scholar
28. Sandstrom BE, Svoboda P, Granstrom M, Harms-Ringdahl M, Candeias LP. H₂O₂-driven reduction of the Fe³⁺—quin2 chelate and the subsequent formation of oxidizing species. Free Radic Biol Med. 1997;23: 744–753. pmid:9296451
- View Article
- PubMed/NCBI
- Google Scholar
29. Srisook K, Han SS, Choi HS, Li MH, Ueda H, Kim C, et al. CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages. Biochem Pharmacol. 2006;71: 307–318. pmid:16329999
- View Article
- PubMed/NCBI
- Google Scholar
30. Nielsen VG, Arkebauer MR, Wasko KA, Malayaman SN, Vosseller K. Carbon monoxide releasing molecule-2 decreases fibrinolysis in vitro and in vivo in the rabbit. Blood Coagul Fibrin. 2012;23: 104–107.
- View Article
- Google Scholar
31. Kshirsagar AV, Freburger JK, Ellis AR, Wang L, Winkelmayer WC, Brookhart AM. Intravenous iron supplementation practices and short-term risk of cardiovascular events in hemodialysis patients. PLOS ONE 2013;8: e78930. pmid:24223866
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Foresti R, Bani-Hani MG, Motterlini R. Use of carbon monoxide as a therapeutic agent: promises and challenges. Inten Care Med J. 2008;34: 649–658.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Durante W, Johnson FK, Johnson RA. Role of carbon monoxide in cardiovascular function. J Cell Mol Med. 2006;10: 672–686. pmid:16989727
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Olas B. Carbon monoxide is not always a poison gas from human organism: physiological and pharmacological features of CO. Chem-Biol Inter. 2014;222: 37–43.
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref4] 4. Motterlini R, Forsti R. Biological signaling by carbon monoxide and carbon monodxide-reeasing molecules. Am J Physiol Cell Physiol. 2017;312: C302–C313. pmid:28077358
View Article
PubMed/NCBI
Google Scholar

[12] View Article

[13] PubMed/NCBI

[14] Google Scholar

[ref5] 5. Vreman HJ, Wong RJ, Stevenson DK. Carbon monoxide in breath, blood, and other tissues. [in] Carbon Monoxide Toxicity, Penney D.G., Ed., CRC Press Boca Raton. FL., 2000, pp. 19–59.

[ref6] 6. Fredenburgh LE, Merz AA, Cheng S. Heme oxygenase signalling pathway: implications for cardiovascular disease. Eur Heart J. 2015;36: 1512–1518. pmid:25827602
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref7] 7. Nielsen VG, Pretorius E, Bester J, Jacobsen WK, Boyle PK, Reinhard JP. Carbon monoxide and iron modulate plasmatic coagulation in Alzheimer's disease. Curr Neurovasc Res. 2015;12: 31–39. pmid:25557378
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref8] 8. Tsai MH, Lee CW, Hsu LF, Li SY, Chiang YC, Lee MH, et al. CO-releasing molecules CORM2 attenuates angiotensin II-induced human aortic smooth muscle cell migration through inhibition of ROS/IL-6 generation and matrix metalloproteinases-9 expression. Redox Biol. 2017;12: 377–388. pmid:28292711
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref9] 9. Truss N, Warner TD. Gasotransmiters and platelets. Pharm Therap. 2011;132: 196–203.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref10] 10. Liu D, Zhuang M, Zhang J, Chen J, Sun B. Study of exogenous carbon monoxide-releasing molecules 2 on endotoxin/lipopolysaccharide-induced abnormal activation of platelets of healthy human donors. Zhonghua Shao Shang Za Zhi 2015;31: 354–360. pmid:26714404
View Article
PubMed/NCBI
Google Scholar

[32] View Article

[33] PubMed/NCBI

[34] Google Scholar

[ref11] 11. Liu D, Wang X, Qin W, Chen J, Wang Y, Zhuang M, et al. Suppressive effect of exogenous carbon monoxide on endotoxin-stimulated platelet over-activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway. Sci Rep. 2016, 1–14.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref12] 12. Nielsen HG, Pretorius E. Iron and carbon monoxide enhance coagulation and attenuate fibrinolysis by different mechanisms. Blood Coagul Fibrinol. 2014;25: 695–702.
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref13] 13. Sun B, Sun Z, Jin Q, Chen X. CO-releasing molecules (CORM-2)-liberated CO attenuates leukocytes infiltration in the renal tissue of thermally injured mice. Int J Biol Sci. 2008;4: 176–183. pmid:18566696
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref14] 14. Babu D, Leclercq G, Motterlini R, Lefebvre RA. Differential effects of CORM-2 and CORM-401 in murine intestinal epithelial MODE-K cells under oxidative stress. Front. Pharm. 2017;8: 1–17.
View Article
Google Scholar

[46] View Article

[47] Google Scholar

[ref15] 15. Whitaker JR, Granum PE. An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Anal Biochem. 1980;109: 156–159. pmid:7469012
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref16] 16. Wachowicz B. Adenine nucleotides in thrombocytes of birds. Cell Bioch Fun. 1984;2: 167–170.
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref17] 17. Bartosz B. Druga twarz tlenu. PWN, Warszawa 2008;1.7: 99–120.

[ref18] 18. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. Determination of carbonyl content in oxidatively modified proteins. Meth Enzymol. 1990;186: 464–478. pmid:1978225
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref19] 19. Ando Y, Steiner M. Sulphydryl and disulphide groups of platelet membranes: determination of disulphide groups. Biochim Biophys Acta 1973;311: 26–37. pmid:4718241
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref20] 20. Ando Y, Steiner M. Sulphydryl and disulphide groups of platelet membranes: determination of sulphydryl groups. Biochim Biophys Acta 1973;311: 38–44. pmid:4718242
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref21] 21. Malinowska J, Kołodziejczyk-Czepas J, Moniuszko-Szajwaj B, Kowalska I, Oleszek W, Stochmal A, et al. Phenolic fractions from Trifolium pallidum and Trifolium scabrum aerial parts in human plasma protect against changes induced by hyperhomocysteinemia. Food Chem Toxicol. 2012;50: 4023–4027. pmid:22898612
View Article
PubMed/NCBI
Google Scholar

[69] View Article

[70] PubMed/NCBI

[71] Google Scholar

[ref22] 22. Nielsen VG, Kirklin JK, George JF. Carbon monoxide releasing molecule-2 increases the velocity of thrombus growth and strength in human plasma. Wolters Kluwer Health | Lippincott Williams & Wilkins 2009

[ref23] 23. Nielsen VG, Garza JI. Comparison of the effects of CORM-2, CORM-3 and CORM-A1 on coagulation in human plasma. Blood Coagul Fibrinol. 2014;25: 801–805.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref24] 24. Nielsen V, Pretorius E. Carbon monoxide: antioagulant or procoagulant? Thromb Res. 2014;133: 315–321. pmid:24360115
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref25] 25. Nielsen VG, Redford DT, Boyle PK. Effect of iron and carbon monoxide on fibrinogenase-like degradation of plasmatic coagulation by venoms of six Agkistrodon species. BCPT 2016;118: 390–395.
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref26] 26. Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, Green CJ. Carbon monoxide-releasing molecules. Characterization of biochemical and vascular activities. Circ Res. 2002;90: e17–e24. pmid:11834719
View Article
PubMed/NCBI
Google Scholar

[84] View Article

[85] PubMed/NCBI

[86] Google Scholar

[ref27] 27. Luzzatto E, Cohen H, Stockheim C, Wieghardt K, Meyerstein D. Reactions of low valent transition metal complexes with hydrogen peroxide. Are they “Fenton-like” or not? The case of Fe(II)L, L = edta, hedta and tcma. Free Radic Res. 1995;23: 453–463. pmid:7581828
View Article
PubMed/NCBI
Google Scholar

[88] View Article

[89] PubMed/NCBI

[90] Google Scholar

[ref28] 28. Sandstrom BE, Svoboda P, Granstrom M, Harms-Ringdahl M, Candeias LP. H₂O₂-driven reduction of the Fe³⁺—quin2 chelate and the subsequent formation of oxidizing species. Free Radic Biol Med. 1997;23: 744–753. pmid:9296451
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

[ref29] 29. Srisook K, Han SS, Choi HS, Li MH, Ueda H, Kim C, et al. CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages. Biochem Pharmacol. 2006;71: 307–318. pmid:16329999
View Article
PubMed/NCBI
Google Scholar

[96] View Article

[97] PubMed/NCBI

[98] Google Scholar

[ref30] 30. Nielsen VG, Arkebauer MR, Wasko KA, Malayaman SN, Vosseller K. Carbon monoxide releasing molecule-2 decreases fibrinolysis in vitro and in vivo in the rabbit. Blood Coagul Fibrin. 2012;23: 104–107.
View Article
Google Scholar

[100] View Article

[101] Google Scholar

[ref31] 31. Kshirsagar AV, Freburger JK, Ellis AR, Wang L, Winkelmayer WC, Brookhart AM. Intravenous iron supplementation practices and short-term risk of cardiovascular events in hemodialysis patients. PLOS ONE 2013;8: e78930. pmid:24223866
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

Figures

Abstract

Purpose

Methods

Results

Conclusion

Introduction

Materials and methods

Exposure of human plasma to CORM-2

Lipid peroxidation measurement

Carbonyl group measurement

Thiol group measurement

The measurement of PT

The measurement of TT

The measurement of APTT

Data analysis

Results

Discussion

Supporting information

S1 Table. The effect of CORM-2 (0.1–100 μM, incubation time: 5–60 min) on plasma lipid peroxidation induced by H2O2.

S2 Table. The effect of CORM-2 (0.1–100 μM, incubation time: 5–60 min) on plasma lipid peroxidation induced by H2O2/Fe.

S3 Table. The effect of CORM-2 (0.1–100 μM, incubation time—30 and 60 min) on carbonyl group formation (plasma protein oxidation) induced by H2O2/Fe.

S4 Table. The effect of CORM-2 (0.1–100 μM, incubation time—30 and 60 min) on the oxidation of protein thiols induced by H2O2/Fe.

S5 Table. The effect of CORM-2 (0.1–100 μM, incubation time—30 min) on the hemostatic parameters of human plasma (APPT, PT and TT).

S6 Table. The effect of iCORM-2 (100 μM, incubation time—30 min) or CORM-2 (100 μM, incubation time—30 min) and albumin (10%, incubation time—30 min) on biomarkers of oxidative stress (lipid peroxidation, carbonyl group formation, oxidation of protein thiols) in plasma treated with H2O2/Fe.

S7 Table. The effect of iCORM-2 (100 μM, incubation time—30 min) or CORM-2 (100 μM, incubation time—30 min) and albumin (10%, incubation time—30 min) on the PT of plasma.

References

S1 Table. The effect of CORM-2 (0.1–100 μM, incubation time: 5–60 min) on plasma lipid peroxidation induced by H₂O₂.

S2 Table. The effect of CORM-2 (0.1–100 μM, incubation time: 5–60 min) on plasma lipid peroxidation induced by H₂O₂/Fe.

S3 Table. The effect of CORM-2 (0.1–100 μM, incubation time—30 and 60 min) on carbonyl group formation (plasma protein oxidation) induced by H₂O₂/Fe.

S4 Table. The effect of CORM-2 (0.1–100 μM, incubation time—30 and 60 min) on the oxidation of protein thiols induced by H₂O₂/Fe.