**2. Research materials and methods**

In our study, we aimed to determine blood group antigens in patients with AT. The study material consisted of blood samples of 100 patients with AT which were further processed according to the appropriate methodology.

The research materials were collected at the Heart Disease Department of Batumi Referral Hospital (Republic of Georgia). The research was implemented of the two years of 2019–2020. This work received financial support from the Batumi Shota Rustaveli State University (Grant project №02–12/57. 15.02.2019). Also, the control group was studied. The Control group included individuals without cardiovascular disease during the period from 2019 to 2020. They were asymptomatic, healthy persons. All participants in the control group were from the Adjara Region (Republic of Georgia). The age range of male and female individuals in both groups (patients and controls) was 40–93. All donors and patients gave their written informed consent for participation in this study, which has been duly approved by the Ethics Committee from the "UnimedAdjara" Ltd. (Adjara, Georgia).(Ethic approval number is BAH-15-1026-4/1 26.10.2015).

The express forward and reversed method with universal monoclonal antibodies was applied in order to type blood groups and reveal the blood group system antigens. Anti-A, anti-B, anti-AB monoclonal antibodies were used for ABO blood group typing (**Figure 1**). Five monoclonal antibodies, anti-D; anti-C; anti-c; anti-E and anti-e were used to identify Rh system antigens. MN blood group phenotypes have been typed by two anti-M and anti-N antibodies and Kell system antigens were determined by anti-Kell and anti-k antibodies (Bio-Rad, cypress diagnostics) (**Figure 2**). Also, for research purposes, standard O(I), (II), (III) group erythrocytes and standard O(I), A(II), B(III), AB(IV) serums were used. For identifying erythrocyte blood group antigens ID cards such as ABO/D + Reverse Grouping (Bio-Rad) (**Figure 3**) were used. The Obtained material has been studied and processed statistically.

The results were processed with the bio statistical methods. The rates between donors and patients groups were compared by Chi-square analysis of proportions.

**91**

**Figure 3.**

*ABO/D + Reverse Grouping ID card.*

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian…*

*DOI: http://dx.doi.org/10.5772/intechopen.96121*

*ABO blood group typing by monoclonal antibodies.*

*ABO, Rh, MN, Kell blood group typing.*

**Figure 1.**

**Figure 2.**

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian… DOI: http://dx.doi.org/10.5772/intechopen.96121*

**Figure 1.** *ABO blood group typing by monoclonal antibodies.*

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

been revealed [22].

**2. Research materials and methods**

meaningful proportion of the population attributable fraction of VT, there may be a role for more widespread adoption of ABO(H) typing in testing strategies [19]. The study of the Iranian population has not shown a significant association between ABO blood groups and coronary artery diseases. Similar results have been shown in the cases of different blood groups as well [20]. Similar results were revealed according to the study of the cardiovascular risk factors and MI in the Turkish cohort. The study showed no difference according to the cardiovascular risk factors and different blood groups within patients with MI and healthy population [21]. No association of the blood group B with MI in the sample population in Malaysia has

Our research aimed to find a possible relationship between the blood groups, such as ABO, Rh, Kell, MN and AT, based on the cases of the Georgian population. The cardiovascular system diseases are also topical for Georgia, which is proved by "The European Heart Network" (EHN) (based in Brussels, EHN is an alliance of different countries together with non-governmental organizations and "Heart Foundations"). According to the research conducted by the alliance, in 2015, 29.007 deaths were reported in men and 33.509 in women ([European Cardiovascular

In our study, we aimed to determine blood group antigens in patients with AT. The study material consisted of blood samples of 100 patients with AT which were

The express forward and reversed method with universal monoclonal antibodies was applied in order to type blood groups and reveal the blood group system antigens. Anti-A, anti-B, anti-AB monoclonal antibodies were used for ABO blood group typing (**Figure 1**). Five monoclonal antibodies, anti-D; anti-C; anti-c; anti-E and anti-e were used to identify Rh system antigens. MN blood group phenotypes have been typed by two anti-M and anti-N antibodies and Kell system antigens were determined by anti-Kell and anti-k antibodies (Bio-Rad, cypress diagnostics) (**Figure 2**). Also, for research purposes, standard O(I), (II), (III) group erythrocytes and standard O(I), A(II), B(III), AB(IV) serums were used. For identifying erythrocyte blood group antigens ID cards such as ABO/D + Reverse Grouping (Bio-Rad) (**Figure 3**) were used. The Obtained material has been studied and

The results were processed with the bio statistical methods. The rates between donors and patients groups were compared by Chi-square analysis of proportions.

The research materials were collected at the Heart Disease Department of Batumi Referral Hospital (Republic of Georgia). The research was implemented of the two years of 2019–2020. This work received financial support from the Batumi Shota Rustaveli State University (Grant project №02–12/57. 15.02.2019). Also, the control group was studied. The Control group included individuals without cardiovascular disease during the period from 2019 to 2020. They were asymptomatic, healthy persons. All participants in the control group were from the Adjara Region (Republic of Georgia). The age range of male and female individuals in both groups (patients and controls) was 40–93. All donors and patients gave their written informed consent for participation in this study, which has been duly approved by the Ethics Committee from the "UnimedAdjara" Ltd. (Adjara, Georgia).(Ethic

Disease Statistics 2017 (http://www.ehnheart.org/cvd-statistics.html).

further processed according to the appropriate methodology.

approval number is BAH-15-1026-4/1 26.10.2015).

**90**

processed statistically.

**Figure 2.** *ABO, Rh, MN, Kell blood group typing.*

**Figure 3.** *ABO/D + Reverse Grouping ID card.*

The level of statistical significance was set at 0.05 in all analyses. The donors and patient cohort are the same profile. All participants have the same nationality. They are Georgians and age intervals are the same for both cohort.

We used the general formula for calculating chi-square (χ2).

$$\mathcal{X}\_{\varepsilon}^{2} = \Sigma \frac{\left(\mathbf{O}\_{\mathrm{i}} - \mathbf{E}\_{\mathrm{i}}\right)^{2}}{\mathbf{E}\_{\mathrm{i}}}$$

This test allows assessing the difference between observed (O) and expected (E) values. We used as on-variable chi-square as in some case two-variable chi-square criterion.

Gene distribution frequency of the ABO system in the studied cohorts was also analyzed. Their frequency was calculated using the formula used in the study of the three-allelic genetic system. Where O, A and B are the ration of people carrying 0 (I), A (II) and B(III) groups to the total number of research objects.

$$r = \sqrt{O}, \; p = \mathbf{1} - \sqrt{A + O}, \; q = \mathbf{1} - \sqrt{B + O}$$

Alleles of the Kell and Rh system were also analyzed in the studied cohorts. Different frequencies of p(K), p(D) and q (k), q (d) were found in the target groups.

$$\mathbf{q} \cdot \sqrt{\frac{n\_{av}}{N}} \quad \mathbf{p} = \mathbf{1} - \mathbf{q}$$

Where naa is a recessive homozygote according above mentioned locus (kk,dd), N is the total number of examined individuals.

The frequency of MN blood group alleles are calculated by bellow mentioned formula:

$$\mathbf{q} = \frac{n\_A + \frac{1}{2}n\_{AB}}{N} \mathbf{p} = \frac{n\_B + \frac{1}{2}n\_{AB}}{N}$$

where *nA* means the number of carriers of the phenotype M, *nAB* indicates MN phenotypes, *nB* means the number of carriers of the phenotype N.

### **3. Age, gender, smoking and arterial thrombosis**

#### **3.1 Age, gender and arterial thrombosis**

As already well known, aging is considered a physiological process with the gradually ongoing accumulation of numerous structural alterations in cells, tissues, and organs. Accordingly, most alterations are associated with functional changes and have significantly increased susceptibility toward some diseases. There is an exponential increase in the risk of both arterial and venous thrombotic events with aging. This fact has been proven by several studies that revealed the increased

**93**

**Table 1.**

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian…*

plasma concentration of V, VII, VIII, IX factors, fibrinogen as well as vWF levels which is increasing progressively along the age [12, 23]. In addition, it has revealed that high plasma levels of fibrinogen supposedly cause the high incidence of cardio-

The activity of proteolytic enzymes (PEs) in the blood serum changes with age [25]. Physiological aging also correlates with increased plasma levels of numerous blood coagulation proteins and fibrinolysis impairment. The studies confirm that there is an association between vascular and thromboembolic diseases and aging. Thus, the state of hypercoagulability tends to increase the risk for the development of thrombosis. Accordingly, hypercoagulability considers a significant marker associated with impairment in advanced age [26]. The blood coagulation capacity increases according to age in healthy individuals because of increases in the most procoagulant factors [27]. The blood coagulation potential a gradual increase during young adulthood and an almost 2-times elevated in old age [28]. The study confirmed that aged 53–64 years is associated with significantly higher fibrinogen levels than younger aged 20 [29]. The plasma concentration of several clotting factors increase with progressing age in healthy [30]. It suggests that thrombotic diseases are found more frequently in subjects with higher plasma levels of factor

In the human body PEs have a significant role. PEs is responsible for numerous processes in health and disease. Moreover, PEs is considered a significant factor in several physiological processes, including morphogenesis, cell differentiation, etc. [31]. According to the physiological coagulation mechanism, the calcium and platelet together contain a PEs like the trypsin, which transforms prothrombin to thrombin [32]. Paczek et al. aimed to investigate the associations between the aging process and PEs activity (including trypsin, elastase, plasmin, and active MMP-9). As they found, the active MMP-9 concentration and trypsin activity decreased according to age. Notably α 1-antitrypsin concentration and plasmin activity are elevated. This study suggests that individual PEs activity in the serum changes with

We have studied 100 patients with AT. The materials have been taken randomly. The 77% of patients were males and the rest of 23% were females. 100 volunteers were used as the controls group in the study. All volunteers were healthy. Age interval was the same for donor cohort as well. The gender ratio was not equal in patients and donors. 51% males and 49% females were studied in our controls group (**Table 1**). As our research has shown, the gender ratios between patients and controls are different. In this case, we used a two-variable chi-square criterion. Statistically revealed a high number of chi-square criteria for both variations. This data indicates the relationships between qualitative variables. In this particular case, the value χ2 effectively rejects the null hypothesis (E = 0). The value of χ2 in the case of males is 64.32, and in the case of females equals to 39.94. These numbers are much higher than the critical value (CV) of the criterion of the degree of freedom (d.f. = 1), which is equal to 3,841. The P-Value is < .00001. The result is significant

**Gender Patients Control** χ**2, (d.f.) = 1**

**Female** 23 ± 4,2 49 ± 4,99 **39,94**

**Male** 77 ± 4,2 51 ± 4,99 **64,32** CV = 3,841

*DOI: http://dx.doi.org/10.5772/intechopen.96121*

vascular events in age [24].

VII [26].

age [33].

at p < .05 (**Table 1**).

*Gender ratio in donors and in the control group.*

#### *The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian… DOI: http://dx.doi.org/10.5772/intechopen.96121*

plasma concentration of V, VII, VIII, IX factors, fibrinogen as well as vWF levels which is increasing progressively along the age [12, 23]. In addition, it has revealed that high plasma levels of fibrinogen supposedly cause the high incidence of cardiovascular events in age [24].

The activity of proteolytic enzymes (PEs) in the blood serum changes with age [25]. Physiological aging also correlates with increased plasma levels of numerous blood coagulation proteins and fibrinolysis impairment. The studies confirm that there is an association between vascular and thromboembolic diseases and aging. Thus, the state of hypercoagulability tends to increase the risk for the development of thrombosis. Accordingly, hypercoagulability considers a significant marker associated with impairment in advanced age [26]. The blood coagulation capacity increases according to age in healthy individuals because of increases in the most procoagulant factors [27]. The blood coagulation potential a gradual increase during young adulthood and an almost 2-times elevated in old age [28]. The study confirmed that aged 53–64 years is associated with significantly higher fibrinogen levels than younger aged 20 [29]. The plasma concentration of several clotting factors increase with progressing age in healthy [30]. It suggests that thrombotic diseases are found more frequently in subjects with higher plasma levels of factor VII [26].

In the human body PEs have a significant role. PEs is responsible for numerous processes in health and disease. Moreover, PEs is considered a significant factor in several physiological processes, including morphogenesis, cell differentiation, etc. [31]. According to the physiological coagulation mechanism, the calcium and platelet together contain a PEs like the trypsin, which transforms prothrombin to thrombin [32]. Paczek et al. aimed to investigate the associations between the aging process and PEs activity (including trypsin, elastase, plasmin, and active MMP-9). As they found, the active MMP-9 concentration and trypsin activity decreased according to age. Notably α 1-antitrypsin concentration and plasmin activity are elevated. This study suggests that individual PEs activity in the serum changes with age [33].

We have studied 100 patients with AT. The materials have been taken randomly. The 77% of patients were males and the rest of 23% were females. 100 volunteers were used as the controls group in the study. All volunteers were healthy. Age interval was the same for donor cohort as well. The gender ratio was not equal in patients and donors. 51% males and 49% females were studied in our controls group (**Table 1**).

As our research has shown, the gender ratios between patients and controls are different. In this case, we used a two-variable chi-square criterion. Statistically revealed a high number of chi-square criteria for both variations. This data indicates the relationships between qualitative variables. In this particular case, the value χ2 effectively rejects the null hypothesis (E = 0). The value of χ2 in the case of males is 64.32, and in the case of females equals to 39.94. These numbers are much higher than the critical value (CV) of the criterion of the degree of freedom (d.f. = 1), which is equal to 3,841. The P-Value is < .00001. The result is significant at p < .05 (**Table 1**).


#### **Table 1.**

*Gender ratio in donors and in the control group.*

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

are Georgians and age intervals are the same for both cohort. We used the general formula for calculating chi-square (χ2).

The level of statistical significance was set at 0.05 in all analyses. The donors and patient cohort are the same profile. All participants have the same nationality. They

( )

This test allows assessing the difference between observed (O) and expected (E) values. We used as on-variable chi-square as in some case two-variable chi-square

Gene distribution frequency of the ABO system in the studied cohorts was also analyzed. Their frequency was calculated using the formula used in the study of the three-allelic genetic system. Where O, A and B are the ration of people carrying 0

*r O p A Oq B O* = =− + =− + ; ; 1 **1**

Alleles of the Kell and Rh system were also analyzed in the studied cohorts. Different frequencies of p(K), p(D) and q (k), q (d) were found in the target groups.

> **<sup>q</sup>** p = **1 q** *naa <sup>N</sup>* <sup>=</sup> <sup>−</sup>

Where naa is a recessive homozygote according above mentioned locus (kk,dd),

The frequency of MN blood group alleles are calculated by bellow mentioned

2 2 q p *nn nn A AB B AB N N*

= =

phenotypes, *nB* means the number of carriers of the phenotype N.

**3. Age, gender, smoking and arterial thrombosis**

**3.1 Age, gender and arterial thrombosis**

1 1

+ +

where *nA* means the number of carriers of the phenotype M, *nAB* indicates MN

As already well known, aging is considered a physiological process with the gradually ongoing accumulation of numerous structural alterations in cells, tissues, and organs. Accordingly, most alterations are associated with functional changes and have significantly increased susceptibility toward some diseases. There is an exponential increase in the risk of both arterial and venous thrombotic events with aging. This fact has been proven by several studies that revealed the increased

i O E E

2 i i

<sup>−</sup> = ∑

c

χ

(I), A (II) and B(III) groups to the total number of research objects.

N is the total number of examined individuals.

2

**92**

criterion.

formula:


**Table 2.**

*Age category and gender in donors and control group.*

In our study we have been allocated six age categories (**Table 2**) as well in patients as in control cohort. As it has presented on the table in young categories, age range between 40–49, there are not female patients, however from the alternative gender, 11 patients belongs to current age group. The majority of patients (56 males and 20 females) are over the 60 years old.

As our data shows, AT is much more common in males than in females. Men have a higher risk of first and recurrent venous thrombosis than women. The pathophysiology of this phenomenon is yet unknown [34]. In the United States, cardiovascular diseases are the leading cause of death; for example, in 2013 death in both sexes were equal. However, several cardiovascular diseases occur early in ageonset within men than women, like MI and coronary heart disease (CHD), where women's prevalence is ~10-year delay in onset [35–37].

#### **3.2 Smoking and arterial thrombosis**

The harmful habits such as smoking, alcohol, drugs have a negative impact on human health. In particular, they have some implications to provoke diseases of the cardiovascular system. The cigarette smoke exposure (CSE) is known to increase the risk of AT. The almost 40% of smoking-related deaths are associated with cardiovascular disease. Both active and passive CSE predispose to cardiovascular events.

It is known that cigarette smoking increases inflammation, thrombosis, and oxidation of low-density lipoproteins (LDL). Several clinical and experimental data support that cigarette smoke increases the cell's oxidative stress that can promote cardiovascular dysfunction [38]. In some studies are reported that acute CSE leads to functional changes in both fibrinogen and platelets, affecting clot dynamics, which were correlated with an alteration in fibrin structure [39].

We have analyzed the possible association between cigarette smoking and AT. Accordingly, three target groups were allocated: active smokers, non-smokers, and ex-smokers. 35% of patients were non-smokers, 39% were ex-smokers and 26% were active smokers (**Table 3**).

As it is shown from the table (**Table 3**), 35% of patients with arterial thrombosis had no exposure to tobacco use, while in the controls group nonsmoker are 1.7 times more (60%). Statistically revealed a high number of chi-square criteria, which indicates the relationships between qualitative variables. In this particular case, the value χ2 is quite effective for rejecting the null hypothesis (E = 0). The value of χ2 in the case is 55, 05. These numbers are much higher than the critical value (CV) of

**95**

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian…*

**Patients** 26 ± 4,38 35 ± 4,76 39 ± 4,85 **55,05** 5,991

**Age Smokers (26) Non-smokers (35) Ex-smokers (39)**

**40-49** 8 0 0 0 3 0 **50-59** 2 0 3 2 4 1 **60-69** 12 0 2 4 11 0 **70-79** 3 0 6 10 15 1 **80-89** 1 0 3 4 3 0 **90-99** 0 0 0 1 1 0

**Male Female Male Female Male Female**

 **(d.f. = 2) CV**

**Category Smokers Non-smoking Ex-smokers** χ**<sup>2</sup>**

*Smokers, non-smokers and ex-smoker among patients with arterial thrombosis and control.*

**Control** 26 ± 4,38 60 ± 4,89 14 ± 3,46

the criterion of the degree of freedom (d.f. = 2), which is equal to 5,991. The result is

It is a wonder why the number of ex-smokers exceeds to the active smokers in our research? We do not have sufficient information when and with what a reason ex-smoker patient group gave up smoking. The high prevalence of ex-smokers in patients may be due to the fact that they had to quit smoking due to deterioration their health condition. On the other side the ex-smokers have a greater experience of smoking (at least 10 years), they have accumulated more toxic substances in the body, atherosclerotic plaques, have abnormal fibrin structure and etc., which may

Many studies have found that blood group phenotypes play as an important genetic risk factor in many diseases. For example, in hemostasis, the ABO blood group has a great influence since it is a crucial determinant of the vWF and therefore, to coagulation factor VIII (FVIII). Besides, an individual with O blood group has around 25% lower vWF and FVIII circulating levels comparing to other blood group individuals. Some experimental studies show that several inflammatory

also be one of the considerable reasons for high incidence [40, 41].

**4. Blood groups and arterial thrombosis**

**4.1 ABO blood group and arterial thrombosis**

and ex-smokers among patients with AT (**Table 4**). As it is shown on the table there are not female patients in the smokers' (n = 26) category. All 26 active smoker patients are males. As we can see, the highest rates of AT is revealed in the category of smokers, in which only men between 60–69 years of age were observed, (12 individuals in total) and further more in the category of ex-smokers males dominates as well with the age of 70–79 (15 males and 1 female). Among whole research group

*Smokers, non-smokers and ex-smokers among patients with arterial thrombosis (by age and sex).*

We have also been analyzed the age and gender groups of smokers, non-smokers

*DOI: http://dx.doi.org/10.5772/intechopen.96121*

significant at p < .05.

**Table 3.**

**Table 4.**

39% of patients were ex-smokers.

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian… DOI: http://dx.doi.org/10.5772/intechopen.96121*


**Table 3.**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

males and 20 females) are over the 60 years old.

*Age category and gender in donors and control group.*

women's prevalence is ~10-year delay in onset [35–37].

**3.2 Smoking and arterial thrombosis**

cardiovascular events.

**Table 2.**

were active smokers (**Table 3**).

In our study we have been allocated six age categories (**Table 2**) as well in patients as in control cohort. As it has presented on the table in young categories, age range between 40–49, there are not female patients, however from the alternative gender, 11 patients belongs to current age group. The majority of patients (56

**№ Age category Patients Control**

 40-49 11 0 6 3 50-59 10 3 6 7 60-69 25 4 16 20 70-79 23 11 17 11 80-89 7 4 6 6 90-99 1 1 0 2 **n** 77 23 51 49

**Male Female Male Female**

As our data shows, AT is much more common in males than in females. Men have a higher risk of first and recurrent venous thrombosis than women. The pathophysiology of this phenomenon is yet unknown [34]. In the United States, cardiovascular diseases are the leading cause of death; for example, in 2013 death in both sexes were equal. However, several cardiovascular diseases occur early in ageonset within men than women, like MI and coronary heart disease (CHD), where

The harmful habits such as smoking, alcohol, drugs have a negative impact on human health. In particular, they have some implications to provoke diseases of the cardiovascular system. The cigarette smoke exposure (CSE) is known to increase the risk of AT. The almost 40% of smoking-related deaths are associated

It is known that cigarette smoking increases inflammation, thrombosis, and oxidation of low-density lipoproteins (LDL). Several clinical and experimental data support that cigarette smoke increases the cell's oxidative stress that can promote cardiovascular dysfunction [38]. In some studies are reported that acute CSE leads to functional changes in both fibrinogen and platelets, affecting clot dynamics,

We have analyzed the possible association between cigarette smoking and AT. Accordingly, three target groups were allocated: active smokers, non-smokers, and ex-smokers. 35% of patients were non-smokers, 39% were ex-smokers and 26%

As it is shown from the table (**Table 3**), 35% of patients with arterial thrombosis had no exposure to tobacco use, while in the controls group nonsmoker are 1.7 times more (60%). Statistically revealed a high number of chi-square criteria, which indicates the relationships between qualitative variables. In this particular case, the value χ2 is quite effective for rejecting the null hypothesis (E = 0). The value of χ2 in the case is 55, 05. These numbers are much higher than the critical value (CV) of

with cardiovascular disease. Both active and passive CSE predispose to

which were correlated with an alteration in fibrin structure [39].

**94**

*Smokers, non-smokers and ex-smoker among patients with arterial thrombosis and control.*


**Table 4.**

*Smokers, non-smokers and ex-smokers among patients with arterial thrombosis (by age and sex).*

the criterion of the degree of freedom (d.f. = 2), which is equal to 5,991. The result is significant at p < .05.

We have also been analyzed the age and gender groups of smokers, non-smokers and ex-smokers among patients with AT (**Table 4**). As it is shown on the table there are not female patients in the smokers' (n = 26) category. All 26 active smoker patients are males. As we can see, the highest rates of AT is revealed in the category of smokers, in which only men between 60–69 years of age were observed, (12 individuals in total) and further more in the category of ex-smokers males dominates as well with the age of 70–79 (15 males and 1 female). Among whole research group 39% of patients were ex-smokers.

It is a wonder why the number of ex-smokers exceeds to the active smokers in our research? We do not have sufficient information when and with what a reason ex-smoker patient group gave up smoking. The high prevalence of ex-smokers in patients may be due to the fact that they had to quit smoking due to deterioration their health condition. On the other side the ex-smokers have a greater experience of smoking (at least 10 years), they have accumulated more toxic substances in the body, atherosclerotic plaques, have abnormal fibrin structure and etc., which may also be one of the considerable reasons for high incidence [40, 41].

### **4. Blood groups and arterial thrombosis**

#### **4.1 ABO blood group and arterial thrombosis**

Many studies have found that blood group phenotypes play as an important genetic risk factor in many diseases. For example, in hemostasis, the ABO blood group has a great influence since it is a crucial determinant of the vWF and therefore, to coagulation factor VIII (FVIII). Besides, an individual with O blood group has around 25% lower vWF and FVIII circulating levels comparing to other blood group individuals. Some experimental studies show that several inflammatory


**Table 5.**

*ABO blood group phenotypes distribution among patients with AT and control.*

cytokines and cholesterol levels may be the most likely mechanisms to explain an association between ABO blood group and cardiovascular diseases [42–45].

Numerous studies have established an association between venous thrombosis and the non-0 group. The mechanism of an association with the ABO blood group and cardiovascular disease has not been fully explored. However, there is a hypothesis that ABO glycosyltransferases modify glycoproteins and glycolipids on platelets' surface effects on the platelet function. The antigens of the ABO system are not presented only on the erythrocyte surface; There are also founded on the cells of epithelium platelet and vascular endothelium, as well [46]. Thus, they are extending potential pathophysiology into other areas of cardiovascular disease and postoperative outcomes.

According to the authors, it is suggested that venous and arterial thrombotic disorders have different pathophysiological backgrounds; for example, arterial thrombosis has extended by platelet activation, while venous thrombosis is based on the clotting system activation [3].

Our study aimed to find an association between blood groups and arterial thrombosis. 100 patients with arterial thrombosis and the same number (100) healthy control group's were investigated. In the control group, the distribution of ABO blood group phenotypes was as follows: group O – 48%; group A – 38%; group B –12%; and group AB – 2%. A similar distribution was revealed in the patients as well (**Table 5**). As the obtained data confirms, the correlation was not found between the ABO blood group and AT. The prevalence of ABO blood group phenotypes (O, A, B, AB) is the same in the patients and the controls. ABO system gene frequency is the same both cohort. The r allele frequency is 0,68, p – 0,08 and q – 0,23.

In this case, we have a Null hypothesis (E = 0). This means that there is no association between the two variables. The value of χ2 criteria is equal to 0,09, which is much less than the critical (CV) value of the degree of freedom (d.f. = 3), equal to 7,815. The P-Value is.029291. The result is significant at p < .05. Our result is the same as the study, including Iranian patients with coronary artery disease. The association with particular ABO blood groups was not found based on the comparison analysis between coronary artery disease general population [20]. The same data is in the study of risk factors and myocardial infarction (MI) in a Turkish cohort. The Turkish population study shows that ABO blood group antigens' frequency was the same in the patients and the control group [21]. There is no association of blood group B with MI in the sample population in Malaysia [22].

#### **4.2 Rh factor and arterial thrombosis**

Our aim was also to find a possible correlation between Rh blood group antigens and AT. Next to the ABO system, the Rh blood group system is the second significant human blood group system [47], which comprises 49 blood group antigens, among them the five D, C, c, E, and e antigens are the clinically most important

**97**

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian…*

**q p**

**Patients** 78 22 0,46 0,54 0,05 3,841

**(d.f. = 1)**

**CV**

**Category Rh + Rh− Rh system gene frequency** χ**<sup>2</sup>**

*Distribution of Rh positive and Rh-negative blood group in patients and control.*

antigens. Rh blood group is associated with the hemolytic disease of the newborn. The hemolytic condition occurs when there is an incompatibility between the

The majority of the available studies involved ABO blood group and risk of cardiovascular diseases, while there is a lack of data on the Rh factor's effect on MI's risk and prognosis. Serum cholesterol varied significantly in the Rh system, but not

The Rh family proteins include none erythroid Rh homologs found on the epithelial tissues of different organs, such as kidney, liver, brain, and skin [49]. Some data points to the coincidence of certain cardiovascular risk factors with the Rh blood group [50]. It has been described that positive Rh is an independent risk factor for low levels of high-density lipoprotein (HDL) cholesterol, which is a well-known risk factor for cardiovascular disorders. Except for that, several clinical studies reported that the Rh genotype is correlated with systolic blood pressure [51]. Rh factor was studied in both patients and control cohort. 78 studied patients were Rh-positive and the remaining 22 were Rh-negative. The prevalence of the Rh phenotypes was similar in the control group (79 – Rh-positive and 21 - Rh-negative) (**Table 6**). Rh system gene frequency is the same for both cohorts. The frequency of

In our study, the prevalence of the Rh blood group phenotypes (Rh-negative and Rh-positive) is the same in the patients and the controls. Based on our study, we could not found any correlation between arterial thrombosis and Rh blood group because the Rh phenotypes in the controls and patients groups are equally distributed. We are not able to reject the Null hypothesis (E = 0). There is no reliable difference between the two categories. The value of χ2 criteria, in this case, is equal to 0.05, which is much less than the Critical Value (CV) of the degree of freedom (d.f. = 1), which is equal to 3,841. The P-Value is.025347. The result is significant at

The MNS antigen system is a human blood group system based upon two genes (glycophorin A and glycophorin B) on the 4th chromosome 4q28.2-q13.1. There are currently 46 antigens in the system [52], but the five most important are called M, N, S, s, and U. We have studied 2 antigens from this system. Based on this, we have allocated 3 phenotypes (M group, N group, and MN group) in the control cohort and the patients. There is little information about MN blood group correlation with

No information could have been obtained through the scientific literature about

the correlation between MN blood groups and AT. But there are some information about association of MN blood group and hypertension. Males with the MN phenotype had significantly higher unadjusted systolic and diastolic blood pressures than those who were homozygous MM or NN [55]. The increased of

*DOI: http://dx.doi.org/10.5772/intechopen.96121*

mother and the fetus's Rh blood types.

**Control** 79 21

**Table 6.**

in the AB0 system [48].

q allele is 0,46, p allele – 0,54.

**4.3 MN blood group and arterial thrombosis**

some kind of infectious disease [53, 54].

p < .05 (**Table 6**).

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian… DOI: http://dx.doi.org/10.5772/intechopen.96121*


**Table 6.**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

*ABO blood group phenotypes distribution among patients with AT and control.*

**Control** 48 ± 4,99 3 8 ± 4,83 12 ± 3,24 2 ± 1,4

**Category O (I) A (II) B (III) AB (IV) ABO system Genes** 

postoperative outcomes.

on the clotting system activation [3].

cytokines and cholesterol levels may be the most likely mechanisms to explain an association between ABO blood group and cardiovascular diseases [42–45].

**Patients** 47 ± 4,99 38 ± 4,83 13 ± 3,36 2 ± 1,4 0,69 0,08 0,23 0,09 7,815

**frequency**

**r p q**

χ**2**

 **(d.f. = 3) CV**

Numerous studies have established an association between venous thrombosis and the non-0 group. The mechanism of an association with the ABO blood group and cardiovascular disease has not been fully explored. However, there is a hypothesis that ABO glycosyltransferases modify glycoproteins and glycolipids on platelets' surface effects on the platelet function. The antigens of the ABO system are not presented only on the erythrocyte surface; There are also founded on the cells of epithelium platelet and vascular endothelium, as well [46]. Thus, they are extending potential pathophysiology into other areas of cardiovascular disease and

According to the authors, it is suggested that venous and arterial thrombotic disorders have different pathophysiological backgrounds; for example, arterial thrombosis has extended by platelet activation, while venous thrombosis is based

Our study aimed to find an association between blood groups and arterial thrombosis. 100 patients with arterial thrombosis and the same number (100) healthy control group's were investigated. In the control group, the distribution of ABO blood group phenotypes was as follows: group O – 48%; group A – 38%; group B –12%; and group AB – 2%. A similar distribution was revealed in the patients as well (**Table 5**). As the obtained data confirms, the correlation was not found between the ABO blood group and AT. The prevalence of ABO blood group phenotypes (O, A, B, AB) is the same in the patients and the controls. ABO system gene frequency is the same both cohort. The r allele frequency is 0,68, p – 0,08 and

In this case, we have a Null hypothesis (E = 0). This means that there is no association between the two variables. The value of χ2 criteria is equal to 0,09, which is much less than the critical (CV) value of the degree of freedom (d.f. = 3), equal to 7,815. The P-Value is.029291. The result is significant at p < .05. Our result is the same as the study, including Iranian patients with coronary artery disease. The association with particular ABO blood groups was not found based on the comparison analysis between coronary artery disease general population [20]. The same data is in the study of risk factors and myocardial infarction (MI) in a Turkish cohort. The Turkish population study shows that ABO blood group antigens' frequency was the same in the patients and the control group [21]. There is no association of blood

Our aim was also to find a possible correlation between Rh blood group antigens and AT. Next to the ABO system, the Rh blood group system is the second significant human blood group system [47], which comprises 49 blood group antigens, among them the five D, C, c, E, and e antigens are the clinically most important

group B with MI in the sample population in Malaysia [22].

**4.2 Rh factor and arterial thrombosis**

**96**

q – 0,23.

**Table 5.**

*Distribution of Rh positive and Rh-negative blood group in patients and control.*

antigens. Rh blood group is associated with the hemolytic disease of the newborn. The hemolytic condition occurs when there is an incompatibility between the mother and the fetus's Rh blood types.

The majority of the available studies involved ABO blood group and risk of cardiovascular diseases, while there is a lack of data on the Rh factor's effect on MI's risk and prognosis. Serum cholesterol varied significantly in the Rh system, but not in the AB0 system [48].

The Rh family proteins include none erythroid Rh homologs found on the epithelial tissues of different organs, such as kidney, liver, brain, and skin [49]. Some data points to the coincidence of certain cardiovascular risk factors with the Rh blood group [50]. It has been described that positive Rh is an independent risk factor for low levels of high-density lipoprotein (HDL) cholesterol, which is a well-known risk factor for cardiovascular disorders. Except for that, several clinical studies reported that the Rh genotype is correlated with systolic blood pressure [51].

Rh factor was studied in both patients and control cohort. 78 studied patients were Rh-positive and the remaining 22 were Rh-negative. The prevalence of the Rh phenotypes was similar in the control group (79 – Rh-positive and 21 - Rh-negative) (**Table 6**). Rh system gene frequency is the same for both cohorts. The frequency of q allele is 0,46, p allele – 0,54.

In our study, the prevalence of the Rh blood group phenotypes (Rh-negative and Rh-positive) is the same in the patients and the controls. Based on our study, we could not found any correlation between arterial thrombosis and Rh blood group because the Rh phenotypes in the controls and patients groups are equally distributed. We are not able to reject the Null hypothesis (E = 0). There is no reliable difference between the two categories. The value of χ2 criteria, in this case, is equal to 0.05, which is much less than the Critical Value (CV) of the degree of freedom (d.f. = 1), which is equal to 3,841. The P-Value is.025347. The result is significant at p < .05 (**Table 6**).

#### **4.3 MN blood group and arterial thrombosis**

The MNS antigen system is a human blood group system based upon two genes (glycophorin A and glycophorin B) on the 4th chromosome 4q28.2-q13.1. There are currently 46 antigens in the system [52], but the five most important are called M, N, S, s, and U. We have studied 2 antigens from this system. Based on this, we have allocated 3 phenotypes (M group, N group, and MN group) in the control cohort and the patients. There is little information about MN blood group correlation with some kind of infectious disease [53, 54].

No information could have been obtained through the scientific literature about the correlation between MN blood groups and AT. But there are some information about association of MN blood group and hypertension. Males with the MN phenotype had significantly higher unadjusted systolic and diastolic blood pressures than those who were homozygous MM or NN [55]. The increased of


**Table 7.**

*MN group system phenotype frequency in patients and control group.*

erythrocyte sodium-lithium counter-transport activity associated with hypertension. Erythrocyte sodium-lithium counter-transport genetically is heritable, but it is not monogenic inheritance [56]. It is complex genetically characteristics. Some authors studied the association of blood pressure, sodium-lithium countertransport and two genetic markers – MN Blood group system and haptoglobin in Michigan population example. They find genetic linkage between the MN locus and red blood cell sodium-lithium counter-transport activity. The authors suggested that the relation between MN phenotype and systolic blood pressure is different in men and women. The men with the MN phenotype and having high systolic blood pressure had significantly elevated red blood cell sodium-lithium counter-transport activity [57].

Delanghe et al., study suggested that detection age of hypertension was lower for patients with MN phenotype. The increasing age of detection the distribution of MN phenotype gradually decreases. The hypertensive with MM blood group had a lower prevalence of cerebrovascular accidents [58].

Sobha R. et al., found that the distribution of MN blood groups differs significantly in asthma patients and controls (chi-square - 15.1160, d.f. 2, Pp > .2) suggesting that blood group MM is resistant to asthma. The comparison of NN blood type reveals non-significant differences between asthma patients and controls [59].

In our studies the prevalence of MN blood group phenotypes was not equal in controls and patients groups (**Table 7**). The frequency of M+ N+ (MN) phenotype was relatively high in the patient groups compared to the controls. 43/100 patients and 30/100 donors carry the M+ N+ (MN) phenotype. In contrast, the major part of the control group (n = 55) had M+ N- (MM) phenotype, which is 1,14 times higher than in the patients' group (n = 48). The study also has revealed the differences according to the prevalence of the M-N+ (NN) phenotype control and patients' groups. The nine patients had the M-N+ phenotype characteristics, which is 1,8 times less than in the donors (n = 15). The study of MN system alleles frequency do not show any significant differences between to cohorts (**Table 7**).

The χ2 criteria is 8,95, which is only 3 units higher then CV (d.f. = 3). In this case, the chi-square criterion indicates the existence of relationship between the two qualitative variables and the rejection of the null hypothesis (E = 0). The P-Value is < .00001. The result is significant at p < .05 (**Table 7**).

#### **4.4 Kell blood group system and arterial thrombosis**

The Kell blood group system is a complex and contains many highly immunogenic antigens. The locus of Kell blood group system is highly polymorphic and gives rise to many Kell antigens in human being. The Kell system gene locus is found on chromosome 7, at 7q33 and contains 19 exons. This locus encoding the 25 antigens and majority of Kell blood group system antigens are glycoproteins. Some of them are highly immunogenic. The Kell antigen is expresses only by erythroid progenitor cells and mature erythroid cells. There are two codominant allelic genes that produce two main antigens: Kell (K) and Cellano (k). These antigens differ to

**99**

these groups.

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian…*

**q p**

**, (d.f. = 1) CV**

**Category k Kell Kell system alleles frequency** χ**<sup>2</sup>**

**Control** 87 13 0.93 0,07

each other by a single amino acid. The prevalence of these antigens is not same. The k antigen is more common in most world population. K–K+ phenotype is found in 98% of Blacks and 91% of Caucasians. These antigens are the third most potent antigens after ABO and Rh blood groups, triggering an immune reaction [60]. Kell blood group system is important not only transfusion but also clinically is associated with the Hemolytic Disease of the Newborn (HDN). HDN caused by anti-Kell1 antibody is the second most common after Rh disease. Anti-Kell is becoming relatively more important as the prevention of Rh disease is also becom-

**Patients** 79 21 0,88 0,12 51,73 3,841

Unlikely to Rhesus and ABO immunosensibilization, HDN attributable to Kell sensitization is causes by anti-K antibody suppressing the fetal red blood cell (RBCs) production. The main reason of this is well known. The Kell antigens generally expressed on the surface of RBCs precursors, and anti-K antibodies initiated immune destruction of K positive elytroid precursor cells by macrophages in the fetal liver. The RBCs progenitor cells do not contain hemoglobin. There is releasing less amount of bilirubin and newborn very rarely presents a jaundice phenotype of

We could not have obtained any information about the correlation between the Kell blood group antigens and cardiovascular disease. The study according to the Kell system 2 phenotypical groups (K+ and K- phenotypes), showed that the frequency of K+ phenotype (KK or Kk) is a high in the patients compared to the controls. 87 of the studied control have the K-negative (kk) phenotype, and as for the patients with arterial thrombosis, the prevalence of the above-mentioned phenotype is 79. The Kell antigen prevalence was relatively high in the studied patients (n = 21) and 13 healthy control were carriers of Kell antigen. K+ phenotype characteristics are highly prevalent in patients than in donors. Kell system alleles frequency are not same between to cohort (**Table 8**). In this case, quite a high number of χ2 criteria was observed, which indicates the relationship between qualitative variables. χ2 value is equal to 51,73. Which is much more than CV (3,841) of df (d.f. = 1). The P-Value is < .00001. The result is significant at < .05 (**Table 8**).

Thus, our study has shown maleness as a higher risk factor for AT. The study also revealed that smokers have a more predicted chance for AT compared to nonsmokers. K+ phenotype and M+ N+ characteristics are of the high prevalence in the patients than in the donors. The correlations of the Kell and MN blood groups with AT have practical significance. There is no correlation between the ABO and Rh blood groups with AT based on our research; however, we think that finding the possible association needs to be identified with multicenter, prospective, and large-scale studies. Based on our research, it is possible to single out high-risk groups of disease and implement preventive arrangements on the individuals of

*DOI: http://dx.doi.org/10.5772/intechopen.96121*

*Kell and k phenotype frequency in patients and control group.*

ing more effective.

**Table 8.**

anemia [61].

**5. Conclusion**

*The Study of Some Possible Risk Factors for Arterial Thrombosis in the Example of Georgian… DOI: http://dx.doi.org/10.5772/intechopen.96121*


**Table 8.**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

**Category M+N+ M+N- M-N+ MN allele frequency** χ**<sup>2</sup>**

**Control** 30 55 15 0,7 0,3

*MN group system phenotype frequency in patients and control group.*

lower prevalence of cerebrovascular accidents [58].

not show any significant differences between to cohorts (**Table 7**).

P-Value is < .00001. The result is significant at p < .05 (**Table 7**).

**4.4 Kell blood group system and arterial thrombosis**

The χ2 criteria is 8,95, which is only 3 units higher then CV (d.f. = 3). In this case, the chi-square criterion indicates the existence of relationship between the two qualitative variables and the rejection of the null hypothesis (E = 0). The

The Kell blood group system is a complex and contains many highly immunogenic antigens. The locus of Kell blood group system is highly polymorphic and gives rise to many Kell antigens in human being. The Kell system gene locus is found on chromosome 7, at 7q33 and contains 19 exons. This locus encoding the 25 antigens and majority of Kell blood group system antigens are glycoproteins. Some of them are highly immunogenic. The Kell antigen is expresses only by erythroid progenitor cells and mature erythroid cells. There are two codominant allelic genes that produce two main antigens: Kell (K) and Cellano (k). These antigens differ to

erythrocyte sodium-lithium counter-transport activity associated with hypertension. Erythrocyte sodium-lithium counter-transport genetically is heritable, but it is not monogenic inheritance [56]. It is complex genetically characteristics. Some authors studied the association of blood pressure, sodium-lithium countertransport and two genetic markers – MN Blood group system and haptoglobin in Michigan population example. They find genetic linkage between the MN locus and red blood cell sodium-lithium counter-transport activity. The authors suggested that the relation between MN phenotype and systolic blood pressure is different in men and women. The men with the MN phenotype and having high systolic blood pressure had significantly elevated red blood cell sodium-lithium counter-transport

**Patients** 43 48 9 0,695 0,305 8,95 5,991

**q P**

**, (d.f. = 2) CV**

Delanghe et al., study suggested that detection age of hypertension was lower for patients with MN phenotype. The increasing age of detection the distribution of MN phenotype gradually decreases. The hypertensive with MM blood group had a

Sobha R. et al., found that the distribution of MN blood groups differs significantly in asthma patients and controls (chi-square - 15.1160, d.f. 2, Pp > .2) suggesting that blood group MM is resistant to asthma. The comparison of NN blood type reveals non-significant differences between asthma patients and controls [59]. In our studies the prevalence of MN blood group phenotypes was not equal in controls and patients groups (**Table 7**). The frequency of M+ N+ (MN) phenotype was relatively high in the patient groups compared to the controls. 43/100 patients and 30/100 donors carry the M+ N+ (MN) phenotype. In contrast, the major part of the control group (n = 55) had M+ N- (MM) phenotype, which is 1,14 times higher than in the patients' group (n = 48). The study also has revealed the differences according to the prevalence of the M-N+ (NN) phenotype control and patients' groups. The nine patients had the M-N+ phenotype characteristics, which is 1,8 times less than in the donors (n = 15). The study of MN system alleles frequency do

**98**

activity [57].

**Table 7.**

*Kell and k phenotype frequency in patients and control group.*

each other by a single amino acid. The prevalence of these antigens is not same. The k antigen is more common in most world population. K–K+ phenotype is found in 98% of Blacks and 91% of Caucasians. These antigens are the third most potent antigens after ABO and Rh blood groups, triggering an immune reaction [60].

Kell blood group system is important not only transfusion but also clinically is associated with the Hemolytic Disease of the Newborn (HDN). HDN caused by anti-Kell1 antibody is the second most common after Rh disease. Anti-Kell is becoming relatively more important as the prevention of Rh disease is also becoming more effective.

Unlikely to Rhesus and ABO immunosensibilization, HDN attributable to Kell sensitization is causes by anti-K antibody suppressing the fetal red blood cell (RBCs) production. The main reason of this is well known. The Kell antigens generally expressed on the surface of RBCs precursors, and anti-K antibodies initiated immune destruction of K positive elytroid precursor cells by macrophages in the fetal liver. The RBCs progenitor cells do not contain hemoglobin. There is releasing less amount of bilirubin and newborn very rarely presents a jaundice phenotype of anemia [61].

We could not have obtained any information about the correlation between the Kell blood group antigens and cardiovascular disease. The study according to the Kell system 2 phenotypical groups (K+ and K- phenotypes), showed that the frequency of K+ phenotype (KK or Kk) is a high in the patients compared to the controls. 87 of the studied control have the K-negative (kk) phenotype, and as for the patients with arterial thrombosis, the prevalence of the above-mentioned phenotype is 79. The Kell antigen prevalence was relatively high in the studied patients (n = 21) and 13 healthy control were carriers of Kell antigen. K+ phenotype characteristics are highly prevalent in patients than in donors. Kell system alleles frequency are not same between to cohort (**Table 8**). In this case, quite a high number of χ2 criteria was observed, which indicates the relationship between qualitative variables. χ2 value is equal to 51,73. Which is much more than CV (3,841) of df (d.f. = 1). The P-Value is < .00001. The result is significant at < .05 (**Table 8**).

### **5. Conclusion**

Thus, our study has shown maleness as a higher risk factor for AT. The study also revealed that smokers have a more predicted chance for AT compared to nonsmokers. K+ phenotype and M+ N+ characteristics are of the high prevalence in the patients than in the donors. The correlations of the Kell and MN blood groups with AT have practical significance. There is no correlation between the ABO and Rh blood groups with AT based on our research; however, we think that finding the possible association needs to be identified with multicenter, prospective, and large-scale studies. Based on our research, it is possible to single out high-risk groups of disease and implement preventive arrangements on the individuals of these groups.
