Experimental estimation of the effects of exogenous carbon monoxide on blood cells

The aim of the study was to investigate the effect of the carbon monoxide (CO) donor on the Ca 2+ -activated potassium permeability of the erythrocyte membrane and platelet aggregation ability. Materials and methods. Healthy volunteers ( n = 27) and patients with chronic coronary heart disease (CHD) ( n = 32) of both sexes were examined. The material of the study was packed red blood cells and platelet-rich plasma obtained from patient’s venous blood. The change of Ca 2+ -dependent potassium conductivity of the erythrocyte membrane was evaluated by potentiometric method, and the platelet aggregation was studied by turbidimetric method. Carbon monoxide releasing molecule-2 (CORM-2) was used as a CO donor. The amplitude of A23187- and redox-induced hyperpolarization response (HR) of erythrocytes, and the rate and degree of platelet aggregation were estimated. Results. It was shown that the addition of CORM-2 (10 and 100 μM) in the erythrocyte suspension caused a dose-dependent decrease in the amplitude of A23187- and redox-dependent HR in healthy donors, as well as in patients with chronic CHD. The maximum decrease was observed in the presence of 100 μM CORM-2. The effect of CORM-2 at concentrations of 10 and 100 μM on collagen-induced platelet aggregation led to a decrease in the degree and rate of aggregation in healthy donors. The maximum effect was shown at 100 μM of CO donor. However, such an unambiguous effect of CORM-2 on the aggregation parameters in patients with CHD was not observed. Conclusion. The results suggest that CO has a significant effect on the ion transport function of the erythrocyte membrane and platelet aggregation activity of both healthy donors and patients with CHD.


INTRODUCTION
Carbon monoxide (CO), nitric oxide (NO) and hydrogen sulfide (H2S) are a new class of gas regulatory molecules [1,2]. The formation of CO occurs during the degradation of the hemoprotein in the heme molecule (hemoglobin, myoglobin, catalase, etc.), which is catalyzed by the heme oxygenase (HO) enzyme, that has inducible (HO-1) and constitutive (HO-2) isoforms [3].
Currently, CO is considered as an important mediator in the cardiovascular system that regulates vascular tone and has anti-inflammatory, anti-apoptotic, and antiproliferative effects [4]. There is evidence that CO modulates surface architectonics and red blood cell energy metabolism [5]. At the same time, changes in the structural and functional status of red blood cells can be an indicator of the degree of damage to membranes during various pathological processes in the body.
Abnormalities in the rheological properties of blood are of great importance among the factors determining hemodynamic disturbances in patients with coronary heart disease (CHD) [6,7]. It has been shown that in patients with CHD, there is a change in the phospholipid composition of the erythrocyte membrane due to the increased incorporation of cholesterol, as well as possible phosphatidylserine externalization [8].
Structural disorganization and changes in the biomechanical properties of the erythrocyte membrane cause disruption in its ion transport function [9,10], in which Gardos channels -Ca 2+ -activated potassium channels (K Сa -channels)are significant. An increase in the activity of these channels causes eryptosis and also reduces the erythrocyte deformability. [2,11]. A special place in the pathogenesis of CHD is given to the increased platelet aggregation and related to it relevant antiplatelet therapy. The literature data indicate that CO donors can produce an antiplatelet effect [12].
Thus, the aim of this work was to study the effect of CO on the ionic permeability of the erythrocyte membrane and platelet aggregation.

MATERIAL AND METHODS
The study included 32 patients (20 men and 12 women) aged 40 to 65 years, with a clinically verified chronic form of CHD, functional class II-III. Of these, 21 (65.6%) patients had myo-cardial infarction in past medical history. The patients had 5 (2; 8) years' experience of CHD. Arterial hypertension (AH) was diagnosed in 14 (43.7%) of the examined individuals. Clinical and laboratory studies were conducted before the start of conservative treatment for chronic CHD. The comparison group consisted of 27 healthy volunteers (16 men and 11 women) aged 38 to 62 years who did not have cardiovascular, endocrine or genetic diseases in the medical history. The characteristics of patient groups are given in Table 1. The work complied with the ethical standards developed in accordance with the Declaration of Helsinki (as amended in 2000) and the Guideline for good clinical practice. Each individual in this study signed a written informed consent.
Venous blood taken from elbow vein of fasted patients in the morning in test tubes of the BD Vacutainer® type with an anticoagulant served as material for the study. Hematological (XN-1000 analyzer, Sysmex, Japan), biochemical (Konelab 60i analyzer, Thermo Scientific, USA) and hemostasiological tests (coagulation analyzer ACL TOP 700, Instrumentation Laboratory Company, USA) were performed.
The erythrocyte suspension was obtained by centrifugation (5 min, 1,000 g, 4 °C) of whole blood (heparin, 17 IU / ml). Plasma and white blood cells were removed, and then the red blood cells were washed twice with 150 mM NaCl containing PBS (5 mM, pH 7,4) under the same centrifugation conditions. The erythrocyte sediment was washed with iso-osmotic medium (320 mOsm/L) containing 150 mM NaCl, 10 mM glucose, 1 mM KCl, and 1 mM MgCl 2 . The red blood cells were transferred to ice and stored for no more than 12 hours. For the study, packed red blood cells were diluted in 1:5 ratio in their incubation medium. To obtain platelet-rich plasma, blood with sodium citrate (blood: citrate ratio -9:1) was centrifuged for 7 min at 800 g.
The study of Ca 2+ -activated potassium permeability of the erythrocyte membrane was conducted by the potentiometric method. The erythrocyte hyperpolarization response (HR) was estimated in response to the addition of 0.5 μM Ca 2+ -ionophore A23187 or the ascorbate (10 mM)phenazine methosulfate (PMS, 0,1 mM) system. The quazi stationary pH i level was determined during cell hemolysis in the presence of detergent Triton X-100 (0.2%).
The aggregation ability of platelets was studied by the turbidimetric method with the use of a laser analyzer (220 LA "NPF Biola", Russia). Platelet aggregation was caused by collagen at a final concentration of 2 mg/ml. The degree and rate of aggregation were estimated from the curve of the average aggregate size.
Statistical analysis of the data was performed in SPSS Statistics 17.0 using Mann -Whitney U test and Chi-square test. Data are presented as median (Me), interquartile range (Q 1 ; Q 3 ), and n (%). p < 0.05 was considered statistically significant.
T a b l e 1

RESULTS
An increase in the cytosolic Ca 2+ concentration in red blood cells, induced by the Ca 2+ -ionophore A23187, as well as the effect of the ascorbate-PMS redox system lead to the development of a hyperpolarization response (HR). Change in the HR amplitude characterizes the conductivity of the K Ca -channels of the erythrocyte membrane [11,13].
To study the role of CO in the regulation mechanisms of erythrocyte Gardos channels, its donor, tricarbonyldichlororuthenium(II) dimer (CORM-2) -ruthenium carbonyl, was used. Despite the release of CO, which binds to hemoglobin of erythrocytes with the formation of carboxyhemoglobin (COHb), it is noted that the COHb content is less than 5%, and the effective concentration of CO is in the range of those observed in vivo [1].
It was found that under the effect of 10 and 100 μM CORM-2, the amplitudes of A23187and redox-dependent HR of healthy donors and patients with chronic CHD decreased dose-dependently. The maximum decrease in HR was observed in the presence of 100 μM CO donor.
Under the action of 10 μM CORM-2, the amplitude of Ca 2+ -ionophore induced HR in patients' erythrocytes decreased more than that of healthy donors. For the ascorbate-PMS-caused HR this dependence was detected only in the presence of 100 μM CO donor (Table 2).
Platelet aggregation was caused by collagen, which interacts with glycoprotein VI (GPVI) and integrin α 2 β 1 of platelets, and, thus, triggers a complex cascade of processes, including activation of phospholipase C and phospholipase A 2 , protein kinases C (PKC), MAP kinases (MAPKs), an increase in cytosolic Ca 2+ level, thromboxane A 2 synthesis and platelet granule secretion [14,15].
The effect of CORM-2 in concentrations of 10 and 100 μM on the collagen-caused processes in platelets led to a dose-dependent decrease in the degree and rate of aggregation in healthy donors. The maximum decrease in platelet aggregation was observed with the effect of 100 μM CO donor. However, there was no unambiguous effect of CORM-2 on the aggregation parameters in patients with coronary artery disease, although the initial values did not differ between CHD patients and healthy volunteers (Table 3).

DISCUSSION
During the past three decades, electrophysiological studies revealed that human red blood cell membrane includes a large variety of ion transporting systems that are involved in the homeostasis of cationic and, to a lesser extent, anionic cell conductivity [10]. It is known that activation of Gardos channels contributes to the release of potassium ions to the outside, causes a shift in the membrane potential towards hyperpolarization and creates a driving force for the displacement of chlorine from red blood cells. The release of cations and anions is accompanied by a loss of water, which leads to dehydration and cell shrinkage [2].
A decrease in the HR amplitude in response to the action of various concentrations of the CO donor indicates a decrease in the Ca2+-activated potassium conductivity of the membrane and leakage of potassium ions from the cell. This, probably, happens due to the interaction of CO with the channel proteins or its regulatory protein kinases [16]. At the same time, the more significant effect of CO in patients with chronic CHD, unlike healthy donors, can be associated not only with structural membrane restructuring and increased lipid peroxidation [9], but also with its antioxidant properties and increased levels of reduced glutathione (GSH) in blood cells [17]. It was found that the electron-donor system, ascorbate-PMS, promotes the formation of redox agents, which influence the Gardos channels of the erythrocyte membrane through oxidation or reduction of SH groups [18].  N o t e . The level of statistical significance of differences in comparison with control for given parameter (р 1 ), CORM-2 (10 μM) for given parameter (р 2 ); the same parameter for healthy donors (р 3 ).
Platelets are small anucleate cell fragments that circulate in blood playing a crucial role in managing vascular integrity and regulating hemostasis. Nevertheless, the functional reaction of platelets can be changed either by increasing pro-aggregation stimuli or by reducing the number of antiaggregation substances. These factors contribute to increased platelet aggregation and often occur in cardiovascular diseases. It is known that CHD is associated with a systemic imbalance in hemostasis caused by the presence of a hypercoagulable state and a decrease in fibrinolysis [19]. The proportion of large platelets which are metabolically and enzymatically more active increases in patients with chronic CHD [20]. The number of platelets incapable of expressing P-selectin and having a significantly greater tendency to form microaggregates in a citrate anticoagulant increases at the same time. [21]. Larger platelets contain more prothrombotic material, with increased thromboxane A 2 and B 2 per unit volume and glycoprotein IIb-IIIa receptor expression.
In our study, it was shown that a CO donor reduced the degree and rate of collagen-induced aggregation in healthy donors and patients with CHD. A higher concentration of CO was required for the latter. Our data are consistent with the results of Chlopicki S. and colleagues [12], confirming the anti-aggregation effect of exogenous CO donors.
Also, in the presence of a guanylate cyclase inhibitor (ODQ), the decrease in collagen-induced aggregation caused by CORM-3 was not blocked, but increased. This fact indicates additional effector targets of CO in platelets. It is noted that CO, not being a powerful inhibitor of platelet activation, acquires this property when there is a lack of other antiplatelet agents (NO and prostacyclin) [12]. It is known that antiplatelet therapy, usually with aspirin, may not be effective, because there are other important ways of platelet activation that are not affected by cyclooxygenase block [22]. In this regard, CO donors appear to have high potential as antiplatelet agents.

CONCLUSION
We have found that CO has a significant effect on the ion transport function of the erythrocyte membrane and platelet aggregation in both healthy donors and patients with CHD. The CO-dependent decrease in the amplitude of Ca 2+ -and redox-induced HR can have a positive effect in the mechanisms of regulation of erythrocyte deformability. The CO influenced decrease in platelet aggregation creates the basis for the development of methods for optimizing antiplatelet therapy in patients with CHD, in which this gasomediator participates.