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Cardiovascular Research 2006 71(2):393-401; doi:10.1016/j.cardiores.2006.03.011
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Copyright © 2006, European Society of Cardiology

Carbon monoxide released by CORM-3 inhibits human platelets by a mechanism independent of soluble guanylate cyclase

Stefan Chlopickia,*, Rafal Olszaneckia, Ewa Marcinkiewicza, Magdalena Lomnickaa and Roberto Motterlinib

aDepartment of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University Medical College, 31-531 Krakow 16 Grzegorzecka, Poland
bVascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, United Kingdom

* Corresponding author. Tel.: +48 12 421 11 68; fax: +48 12 421 72 17. Email address: s.chlopicki{at}cyfronet.krakow.pl

Received 25 October 2005; revised 4 March 2006; accepted 6 March 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Objective Carbon monoxide (CO) modulates several physiological functions through activation of a cGMP-dependent pathway similar to that of nitric oxide (NO). Here we investigated the possible involvement of soluble guanylate cyclase in the anti-aggregatory effect of micromolar concentrations of CO released by a novel, water-soluble, CO releasing molecule (CORM) in human platelets.

Methods Human platelet aggregation was induced by collagen or thrombin, and the effects of CO releasing molecule (CORM-3) and an NO donor on platelet aggregation were compared.

Results CORM-3 liberated CO in a time- and concentration-dependent manner as evidenced by the formation of carbon monoxy myoglobin (MbCO) using a spectrophotometric assay. When added to washed platelets, CORM-3 (10–300 µM) inhibited collagen- and thrombin-induced aggregation in a concentration-dependent manner. The anti-aggregatory effect of CORM-3 was reversed by deoxy-Mb (50 µM). Interestingly, in the presence of an inhibitor of guanylate cyclase (ODQ, 5 µM), inhibition of collagen-induced aggregation by CORM-3 was not blocked but potentiated. Under the same experimental conditions, inhibition of platelet aggregation by an NO donor (SNAP, 1 µM) was prevented by ODQ. In collagen-induced or thrombin-induced platelet aggregation, a stimulator of guanylate cyclase (YC-1, 0.3 µM) did not alter the effect of CORM-3, whereas it markedly potentiated the inhibition of platelet aggregation mediated by SNAP. Notably, CORM-3-induced inhibition of platelet aggregation was of similar degree when platelets were activated by a low (20 mU/ml) or by high concentration of thrombin (100–200 mU/ml), whereas NO donors (SNP and SNAP)- or carbaprostacylin (cPGI2)-induced effects were considerably attenuated when platelets were activated by high concentrations of thrombin.

Conclusions Inhibition of platelet aggregation by CO released by a novel, water-soluble CORM is not mediated by activation of soluble guanylate cyclase. In contrast to NO and PGI2, CO effectively inhibits platelets even when cells are activated excessively. We suggest that despite the fact that CO is not a potent inhibitor of platelet activation, it may gain importance when NO and PGI2 alone are insufficient to overcome excessive platelet activation.

KEYWORDS Platelets; Carbon monoxide; Nitric oxide; Guanylate cyclase; Signal transduction; sGC; CORM-3; Vasoactive agents


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Carbon monoxide (CO) is the end product of heme degradation by a family of heme oxygenases (HO), consisting of at least 3 isoforms: an inducible stress enzyme (HO-1) and two constitutive proteins (HO-2 and HO-3). HO decomposes heme into CO, ferrous iron and biliverdin, the latter being rapidly converted to bilirubin by biliverdin reductase [1,2]. A growing number of studies point to a possible role for endogenous CO as a signalling molecule in the regulation of vascular function within the cardiovascular system. Originally it has been demonstrated that endogenous CO is a vasodilator that acts primarily through a stimulation of soluble guanylate cyclase (sGC) and cGMP production [3,4]. At molecular level, this is achieved by a direct binding of CO to the iron present in the heme moiety of the guanylate cyclase protein [5]. Recently, other potential targets for the vasodilatory action of gaseous CO have been proposed, including calcium-dependent potassium channels [6]. In an elegant study, heme has been identified to bind covalently to potassium channels, which could form a sensitive molecular switch for the activation by CO [7,8]. Additional evidence suggests that inhibition of cytochrome P450-mediated production of endothelin-1 could also contribute to the vasoactive properties of CO [9,10].

The anti-inflammatory and anti-proliferative actions of CO were shown to be mediated via multiple pathways, involving for example activation of both guanylate cyclase and p38 MAPK [11,12]. In addition, in macrophages activated with endotoxin, CO seems to inhibit both TNF-{alpha} and nitric oxide (NO) generation the latter by binding to the heme moiety of iNOS [13,14].

In contrast to the numerous reports on the vasoactive or anti-inflammatory effects mediated by CO, there is only a limited number of studies on the mechanisms by which CO inhibits platelet aggregation. Interestingly, all of them use gaseous CO and reveal a similarity with NO in the ability of both gases to inhibit platelet function via a common target (i.e. soluble guanylate cyclase) [15,16]. It is important to note that in platelets the biological anti-aggregatory activity of CO has been considered relatively low compared to the effect elicited by other endogenous agents released from the endothelium, such as NO or PGI2 [16]. Indeed, only high concentrations of gaseous CO (100%) appear to inhibit aggregation of human platelets via the activation of guanylate cyclase [16]. Furthermore, YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole)], a compound that sensitizes guanylate cyclase to the action of NO, was also shown to amplify the action of gaseous CO on guanylate cyclase activity in platelets [17]. The mechanism of this effect was suggested to be similar to that found for YC-1-NO co-operation, involving the inhibition of deactivation of NO-sensitive guanylate cyclase [18,19].

Recently, a novel class of compounds, termed carbon monoxide-releasing molecules (CORMs), has been discovered and their chemical and biochemical features characterized [20,21]. These compounds have been demonstrated to liberate CO in biological systems providing a useful research tool for exploring the mechanism by which CO exerts its pharmacological activities [22]. One of these compounds, CORM-3 ((tricarbonylchloro(glycinato)ruthenium(II)) has a unique feature of being fully water-soluble and has been shown to simulate the bioactivities of gaseous CO including vessel relaxation [23,24], protection against ischemia–reperfusion injury [23,25,26], prevention of organ rejection following transplantation [23] and inhibition of the inflammatory response [14]. Therefore, the present study was undertaken to characterize a possible action of CORM-3 against platelet aggregation and analyze the involvement of sGC in this effect using ODQ, a selective and potent inhibitor of sGC, and the sGC sensitizer, YC-1.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgment
 References
 
2.1 Isolation of human platelets
The investigation conforms with the principles outlined in the Declaration of Helsinki. Venous blood was obtained from human volunteers at the University Hospital Blood Bank Centre. Volunteer donors had not taken any medicines for the preceding 2 weeks. Blood was collected into vials containing sodium citrate (3.2%, 1:9 v/v) as anti-coagulant agent. To obtain platelet-rich plasma (PRP), blood was centrifuged at 250 x g for 20 min. The PPP fraction was obtained by centrifugation of the remaining blood for 5 min at 2000 x g. Washed platelets (WP) were obtained from PRP which were washed twice in PGI2-containing PBS according to the method of Radomski et al. [27] and finally suspended (2 x 108 platelet/ml) in Ca++-free PBS containing 0.1% albumin. Contamination of neutrophils in WP was less then 1/108 [28].

2.2 Platelet aggregation assay
Aggregation of blood platelets was assessed in WP with a dual channel Chronolog aggregometer using a method previously described by Born [29]. The baseline value on the aggregometer was set using WP whereas distilled water was used to set the full transmittance. WP (500 µl) were equilibrated for 3 min at 37 °C with a continuous stirring at 1100 rev/min and then stimulated with collagen or thrombin to cause aggregation. At the beginning of each experiment, concentrations of collagen and thrombin that induced sub-maximum aggregation response were determined. These were in the range of 1.2–2 µg/ml and 10–20 mU/ml for collagen and thrombin, respectively.

CORM-3 (tricarbonylchloro(glycinato)ruthenium(II)), of which chemical structure is represented in Fig. 1A, and other agents such as carbaprostacyclin (cPGI2), S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP), were added 2 min before stimulation of platelets with collagen (2 µg/ml) or thrombin (20–100 mU/ml). ODQ (5 µM), YC-1 (300 nM), OxyHb (10 µM) or deoxy-Mb (10 or 50 µM) were added 1 min before CORM-3 or SNAP treatment. Transmittance was read 3 min after simulation of platelets with an agonist.


Figure 1
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  		Fig. 1 Time- and concentration-dependence of CO liberated from CORM-3. A. Chemical structure of CORM-3 (tricarbonylchloro(glycinato)ruthenium(II)). B. Kinetics of formation of MbCO by addition of CORM-3. The graph shows spectra read every 20 s after addition of CORM-3 (30 µM) to a deoxy-Mb solution (5 µM). B. Original tracing from experiment showing concentration- and time-dependent formation of MbCO after addition of CORM-3 (final concentrations: 10, 30, and 100 µM) to deoxy-Mb (5 µM). Change in absorbance at 541 nm (background wavelength 558 nm) was measured every 6 s for 4 min.

 
2.3 In vitro detection of CO release
The release of CO from CORM-3 was assessed spectrophotometrically by measuring the kinetics of conversion of deoxymyoglobin (deoxy-Mb) or oxymyoglobin (oxy-Mb) to carbonmonoxy myoglobin (MbCO) [21]. Myoglobin solutions (5 µmol/l final concentration) were prepared freshly by dissolving the protein in 0.1 mol/l phosphate buffer (pH 7.4). Sodium dithionite (0.1%) was added to convert myoglobin to deoxy-Mb prior to each experiment. In order to obtain oxy-Mb, deoxy-Mb solution was vortexed in the open air for 3 min. CO released from CORM-3 (final concentrations: 10, 30 and 100 µM) was quantified by adding aliquots of stock solutions (10 µl) of the carbonyl complex (in pure distilled water) directly to the deoxy-Mb or oxy-Mb solutions. The kinetics of MbCO formation was quantified by measuring the change in absorbance at 541 nm (background wavelength 558 nm) for deoxy-Mb and 565 nm (background wavelength 584 nm) for oxy-Mb at 23 °C using a Beckman DU 640 B spectrophotometer equipped with kinetic software package. The rates of MbCO formation were calculated within the initial 10 s after addition of CORM-3 and expressed as change in absorbance per 1 min (dA/min).

2.4 Reagents and drugs
Collagen was obtained from Chronolog (USA) and thrombin was from Polfa–(Krakow, Poland). Aspirin was obtained from Bayer (Germany) and CORM-3 was synthesized as described previously [23]. YC-1 and SNAP were from Sigma-Aldrich Chemicals International, whereas PGI2 and cPGI2 were from Biomol Research Lab, Inc., (USA).

2.5 Data analysis
Results were expressed as means±SEM. Differences between means were evaluated by one-way ANOVA or two-way ANOVA followed by Scheffe test. In Figs. 2A and 4GoB–D where data were not normally distributed Kruskall–Wallis test followed by Wilcoxon two-sample comparisons was performed. In Fig. 2B significance of difference from control response (100%) as well as comparison between effects of RuCl3 and CORM-3 was evaluated by Student t-test. P value of less than 0.05 was considered statistically significant.


Figure 2
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  		Fig. 2 Inhibition of human platelet aggregation by CORM-3 (10–500 µM). A. Concentration-dependent inhibition of platelets aggregation by CORM-3 in washed human platelets. Platelets aggregation was induced by sub-maximal concentration of collagen (2.0 µg/ml), or thrombin (20 mU/ml). Data represent the mean±SEM of 5–34 experiments. * indicates p<0.05 between CORM-3 effects on collagen and thrombin-induced response. B. Comparison between effects of CORM-3 (500 µM) and RuCl3 (500 µM) on platelet aggregation induced by thrombin (100 mU/ml). Data represent mean±SEM of 3 parallel experiments. *, ** indicate p<0.05 and p<0.001, respectively vs control response. ## indicates p<0.001, between effect of RuCl3 and CORM-3.

 

Figure 4
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  		Fig. 4 Differential effects of ODQ (5 µM) on the anti-aggregatory effect of CORM-3 (10–300 µM) (A, C) and SNAP (0.1–1 µM) (B, D). Platelets aggregation was induced by collagen (2.0 µg/ml) (A, B) or thrombin 20 mU/ml (C, D). Data represent mean±SEM of 5–9 experiments. *, ** indicate p<0.05 and p<0.001, respectively between CORM or SNAP response with and without ODQ.

 

   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgment
 References
 
3.1 Biochemical assessment of CO release from CORM-3
As shown in Fig. 1B, addition of CORM-3 (30 µM) to a deoxy-Mb (5 µM) solution promoted the formation of MbCO as evidenced by the typical spectrum changes. In contrast, RuCl3 (300 µM), a compound that is lacking of CO groups but contains ruthenium as CORM-3, did not change the spectrum of deoxy-Mb (data not shown). Addition of CORM-3 (10–100 µM) to the deoxy-Mb solution resulted in formation of MbCO in a time- and concentration-dependent manner (Fig. 1B, C). Importantly, when oxy-Mb was used instead of deoxy-Mb, the rate of MbCO formation after addition of CORM-3 was significantly slower: for CORM-3 at concentration of 10 µM it was 0.012±0.002 and 0.0033±0.0004 dA/min for deoxy-Mb and oxy-Mb, respectively, and for CORM-3 at concentration of 100 µM it was 0.16±0.008 and 0.047±0.0033 dA/min for deoxy-Mb and oxy-Mb, respectively (p<0.01 between values for oxy-Mb and deoxy-Mb).

3.2 Inhibition of platelet aggregation by CORM-3
When added to washed human platelets, CORM-3 (10–300 µM) inhibited platelet aggregation induced by collagen or thrombin in a concentration-dependent manner (Fig. 2A). The effect of CORM-3 on collagen and thrombin-induced aggregation was similar. In contrast to the results obtained with CORM-3, addition of RuCl3 (500 µM) did cause marginal inhibition of platelet aggregation induced by either collagen (data no shown) or thrombin (Fig. 2B).

When oxy-Mb (10 µM) was added to the solution containing platelets, the inhibitory effect of CORM-3 on aggregation was unaffected. In contrast, oxy-Mb fully reversed the inhibitory effect on platelet aggregation induced by the NO donor, SNAP (data not shown). Similarly, deoxy-Mb (10 µM) fully reversed the anti-aggregatory effect of SNAP (30 µM) (Fig. 3A), whereas the effect of CORM-3 (300 µM) was only slightly affected by 10 µM deoxy-Mb (data not shown). However, in the presence of a higher concentration of deoxy-Mb (50 µM) the anti-aggregatory effect of CORM-3 was nearly completely prevented (Fig. 3B, C).


Figure 3
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  		Fig. 3 Reversal of the anti-aggregatory effect of SNAP and CORM-3 by deoxy-Mb at concentration of 10 µM (A) and 50 µM (B, C), respectively. Platelets aggregation was induced by low thrombin (20 mU/ml) or high thrombin (100 mU/ml) concentration, (A) and (B, C), respectively. Data represent mean±SEM of 5 experiments. ** indicates p<0.001 vs SNAP or CORM-3 response.

 
3.3 Effect of soluble guanylate cyclase inhibition or sensitization on the anti-aggregatory effect of CORM-3
In the presence of ODQ (5 µM), an inhibitor of soluble guanylate cyclase activity, the inhibitory effect of CORM-3 on collagen-stimulated platelets aggregation was potentiated (Fig. 4A), whereas SNAP-mediated inhibition of platelet aggregation was reversed (Fig. 4B). On the other hand, the guanylate cyclase sensitizer, YC-1 (0.3 µM), significantly potentiated the inhibitory effect of SNAP on collagen-induced platelets aggregation, while YC-1 had a negligible effect on the anti-aggregatory action of CORM-3 (Fig. 5A and B). When platelets were stimulated with thrombin (20 mU/ml) instead of collagen, the inhibitory effect of SNAP (30 µM) on thrombin-induced platelet aggregation was reversed by 5 µM ODQ whereas the effect elicited by 300 µM CORM-3 was not reversed by ODQ (Fig. 4 C, D). Interestingly, in presence of L-NAME (300 µM) CORM-3 effect tended to be potentiated (53.8±5.9% vs 27.4±4.1%, n=7, p=0.09 for CORM-3 (100 µM) without and with L-NAME, respectively).


Figure 5
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  		Fig. 5 Differential effects of YC-1 (300 nM) on the anti-aggregatory effects of CORM-3 (A) and SNAP (B). Platelets aggregation was induced by collagen (2.0 µg/ml). Data are presented as mean±SEM from 4 to 8 experiments. *, ** indicate p<0.05 and p<0.001, respectively between SNAP or CORM-3 response with and without YC-1.

 
3.4 Comparison between the anti-aggregatory activity of CORM-3, sodium nitroprusside and carbaprostacyclin in platelets stimulated with high concentration of thrombin
As shown in Fig. 6, when platelets were activated by a low concentration of thrombin (20 mU/ml), the inhibitory effect of 300 µM CORM-3 was approximately equivalent to that induced by 20 µM SNP, 30 µM SNAP, or 20 nM cPGI2 (carbaprostacylin). However, when platelets were activated by a higher concentration of thrombin (100–200 mU/ml), the inhibitory effect of 20 µM SNP, 30 µM SNAP or 20 nM cPGI2, but not that of CORM-3, was nearly completely lost. Approximately 10 times higher concentration of cPGI2 (200 nM) was necessary to inhibit 200 mU thrombin-induced platelet aggregation to a similar degree (8±3.8% of control response, n=4) as that obtained by 20 nM cPGI2 in 20 mU/ml thrombin-induced platelet aggregation (4.4±2.3% of control response, n=5). Furthermore, 10 times higher concentration of SNAP or even 100 times higher concentration of SNP was not enough to inhibit 200 mU/ml thrombin-induced platelet aggregation to a similar degree as that observed in the case of 20 mU/ml thrombin-induced platelet aggregation. In the presence of SNP (20 µM) or SNAP (30 µM) platelet aggregation induced by 20 mU/ml thrombin was inhibited by 96.2±0.8% (n=5) and 83±3.147% (n=17), respectively. However, the aggregation of platelets induced by 200 mU/ml thrombin was inhibited only by 16±1.8% (n=4) with 1 mM SNP and by 14.5±2.3% (n=4) with 200 µM SNAP. In contrast to cPGI2 and NO donors, CORM-3 (300 µM) inhibited platelet aggregation to an approximately similar degree when 20 or 200 mU/ml of thrombin were used (Fig. 6).


Figure 6
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  		Fig. 6 Comparison between the anti-aggregatory actions of CORM-3, SNP, SNAP and cPGI2 in washed human platelets activated by high thrombin concentrations. Data represent the mean±SEM of 3–41 experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgment
 References
 
The major finding originating from this study reveals that a novel water-soluble metal carbonyl compound (CORM-3) releasing CO in a time and concentration dependent manner [19,21,22] displays anti-aggregatory effects in human platelets. Importantly, in contrast to the effect of a typical NO donor such as SNAP (or SNP), the effect of CORM-3 on platelet aggregation was not mediated by activation of soluble guanylate cyclase (sGC). We present here evidence that the bioactivity of CORM-3 in platelets is mediated by CO. Firstly, the inactive analogue of CORM-3, which is devoid of CO groups (RuCl3), did not inhibit platelet aggregation. Secondly, prolonged incubation of CORM-3 in buffer (>48 h) did not produce significant amount of MbCO with the myoglobin assay and did not inhibit platelet aggregation. Finally, the effect of CORM-3 was reversed by deoxymyoglobin, a known scavenger of CO. Moreover, using spectrophotometric measurements we have confirmed that CO released by CORM-3 reacts more avidly with deoxy-Mb than Oxy-Mb.

It has been consistently shown that soluble guanylate cyclase (sGC) is strongly activated by NO and to a lesser extent by CO [17,30,31]. Indeed, CO like NO has also the ability to bind the iron of the heme moiety associated with soluble guanylate cyclase and thereby to activate the enzyme. Here we show that in contrast to NO donors, the anti-aggregatory activity of the CO releasing agent is not mediated by sGC. In our experiments, ODQ, a potent and selective inhibitor of sGC [32], completely reversed the antiplatelet effect of SNAP, while it was ineffective against CORM-3. Furthermore, YC-1, a benzylindazole derivative that directly activates sGC and sensitizes guanylate cyclase toward NO as well as CO [17] has failed to potentiate the effect of CO liberated by CORM-3 in human platelets. At the same time, YC-1 amplified the anti-aggregatory effect of NO released by SNAP. YC-1 has been described as both vasodilator [33] and anti-thrombotic agent due to its properties of increasing cGMP [34]. In vascular tissue, YC-1, at concentrations that are by themselves ineffective, substantially augmented NO- or CO-induced sGC-dependent vasodilation [19,21,22,30]. In platelets, high concentration of gaseous CO (100%) also inhibited platelets via the activation of sGC [16] and YC-1 amplified this activity [17].

Using a similar pharmacological approach, we provide evidence that in contrast to NO, inhibition of platelet aggregation by physiologically relevant concentrations of CO liberated by CORM-3 appears to be mediated by sGC-independent mechanisms. The discrepancies between previous data and the results presented in this study could be due to the fact that we used micromolar concentrations of CO released by the CO-carrier while previous results were obtained with high (100%) concentrations of gaseous CO bubbled for a short period of time in a suspension of platelets. Administering gaseous CO to study biological response to CO poses some constraints, let alone the difficulty of storing and delivering CO in a controlled manner. Furthermore, the anti-aggregatory effect of gaseous CO analysed by Brune and Ullrich [15] was difficult to quantitate as it was an "all or none" response. Also Friebe et al. reporting on the synergistic activation of sGC by CO and YC-1 [17] did not show a concentration-dependent inhibition of platelet aggregation by CO. In contrast, here we show that CO liberated from CORM-3 displayed a concentration-dependent inhibition of platelet aggregation. Importantly, the amount of CO delivered by CORM-3 at micromolar concentration is lower than that delivered by 100% gaseous CO which is in the order of millimolar concentrations.

It seems unlikely that gaseous CO and CO released by CORM-3 are chemically distinct entities so the most possible explanation for the apparent discrepancy is that CO released by CORM-3 or applied as a gas reaches different targets. This discrepancy might originate from the various concentrations of CO applied. Indeed, CO has poor sCG-stimulating properties [35] and CORM-3 activated purified guanylate cyclase activity only at concentrations higher than 300 µM in the presence of YC-1 [25], that is above the range of concentrations of CORM-3 used in our experiments. In contrast to platelets, in vascular preparation gaseous CO or CORM-3 relaxed blood vessel via cGMP dependent mechanisms, albeit some reports underscore the involvement of endogenous NO in this response [36]. Interestingly, repeated addition of CORM-3 to rat aorta induced vasodilatation that was no longer dependent on sGC [22]. Accordingly, in platelets as well as in vascular tissue under certain conditions sGC appears not to be a primary target for the bioactivity of micromolar concentration of CO. A surprising finding from this study was that in collagen-stimulated platelets, inhibition of guanylate cyclase by ODQ potentiated the anti-aggregatory effect of CO released by CORM-3 suggesting that the action of CO may be accentuated by the removal of sGC function. Similar effects were found with L-NAME suggesting that elimination of platelet NO–cGC pathway amplifies the inhibition of platelet aggregation elicited by CO. The mechanism and physiological meaning of these findings remain to be elucidated.

In other experimental systems it has been found that inhibition of NO synthase blocked CO-induced cerebral vasodilation [37,38] and a permissive role of NO in CO-mediated vasodilation has been suggested [38,39]. On the other hand, inhibition of NOS accentuated the renal vascular response to heme oxygenase inhibition [38,40] and CO attenuated NO-induced activation of guanylate cyclase [38,41]. As the vasoregulatory functions of the HO and NOS systems are interrelated, it is tempting to speculate that interactions between NO and CO may also be crucial in regulating activity of platelets. Circulating platelets remain under constant influence of endothelial NO and lack of endothelial NO may well accentuate the inhibitory effect of CO on platelets.

Apart from sGC [3,42,43], there is a number of other possible targets for CO such as calcium-activated potassium channels [6], cytochrome P450 [44], mitochondrial respiratory chain [45] or p38MAPK [46,47]. Their role in mediating the anti-aggregatory effects of CO remains to be studied. Platelets contain calcium-activated potassium channels [48] but still the role of membrane potential in the regulation of platelet activation is controversial [49]. It seems unlikely, that CO-induces effects by potassium channels in platelet membrane as CORM-3-induced inhibition of platelets was not affected by high extracellular KCl concentration (data not shown). Since CO was almost equally effective as an inhibitor of platelet aggregation induced by both low and high thrombin concentration, also p38MAPK seems to be an unlikely target for CO action in platelets [50]. It is also unlikely that CORM-3 mediates its bioactive effect in platelets by inhibition of the mitochondrial respiratory chain since the contribution of mitochondrial respiration in aggregation of platelets is minimal. Whether other non-mitochondrial heme or non-heme iron represent the targets for CO released by CORM-3 remains to be determined.

Despite the fact that the target for antiplatelet action of a low concentration of CO remains unknown, our data point out that in in vivo setting CO represents another mediator released from vascular wall that along with PGI2 and NO buffers platelet activation. It is important to note that in our experiments, in contrast to PGI2 and NO, CO effectively inhibited platelet aggregation even when platelets were excessively activated. These results suggest that CO may play a role of a retaliatory mediator that comes into play when PGI2 and NO are insufficient to overcome excessive platelet activation.

In conclusion, CORM-3 effectively liberates CO into human platelets to exert antiplatelet action that, in contrast to NO-mediated effect, does not involve activation of soluble guanylate cyclase. Interestingly, even though NO- or CO-mediated bioactivity in platelets utilizes distinct mechanisms, they may be interlinked, as the inhibition of sGC accentuates inhibitory effect of CO. The physiological significance as well as mechanisms of this phenomenon remains to be determined. Also, an intracellular target for antiplatelet activity of CO released by CORM-3 has to be established.


   Acknowledgment
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
References
 
We would like to thank Jolanta Reyman for her excellent technical assistance. This work was supported by Polish Ministry of Science and Information Society Technologies (MNiI) (grants No P05A 003 25, PBZ-KBN-101/T09/2003/6). Professor Stefan Chlopicki is the recipient of a Professorial grant from the Foundation for Polish Science (SP/04/04). We thank Prof. Brian E. Mann and Dr. Tony Johnson for synthesizing and providing CORM-3.


   Notes
 
Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 

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E. Masini, A. Vannacci, P. Failli, R. Mastroianni, L. Giannini, M. C. Vinci, C. Uliva, R. Motterlini, and P. F. Mannaioni
A carbon monoxide-releasing molecule (CORM-3) abrogates polymorphonuclear granulocyte-induced activation of endothelial cells and mast cells
FASEB J, September 1, 2008; 22(9): 3380 - 3388.
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