ROLE PLASMA LEVEL OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE -1 (PAI-) AND GENETIC POLYMORPHISM OF PAI-1 IN PATIENTS WITH ISCHEMIC HEART DISEASE IN UZBEK NATIONALITY

Полный текст

Introduction

State committee statistics of the republic of Uzbekistan published a report on the demographic situation in 2022. According to the statistics agency, the number of deaths in January-December 2022 amounted to 172.1 thousand people. Of the number of registered deaths in January-December 2022, 55.5% were diseases of the circulatory system (State committee statistics of the republic of Uzbekistan). Young survivors of an arterial thrombotic event have an increased mortality and morbidity compared with the general population, mainly related to the high risk of cardiovascular recurrences [1, 2, 3].  This information is cause of finding a new methods of diagnostics and treatment of cardiovascular disease. This is particularly important at a young age, because the impact on quality of life and on socioeconomic costs, considering life expectancy, is the highest [4, 5].

Common knows that hypercoagulability is a stronger risk factor for ischaemia in myocard. Hypercoagulability increases the risk of arterial thrombosis; however, this effect may differ between various manifestations of arterial disease [6]. Platelets play a pivotal role in the formation and propagation of the thrombus, and therefore are the primary targets of antithrombotic therapy in arterial disease [7]. However, arterial thrombus formation is also determined by the activation of the coagulation cascade [8, 9]. Fibrinolysis is the resulting of the interactions among multiple plasminogen activators and inhibitors constituing the enzymatic cascade, and ultimately leading to the degradation of fibrin. The plasminogen activator system plays a key role in a wide range of physiological and pathological processes. Plasminogen activator inhibitor-1 (PAI-1) is a member of the superfamily of serine-protease inhibitors (or serpins), and the principal inhibitor of both the tissue-type and the urinary-type plasminogen activator, the two plasminogen activators able to activate plasminogen.[10]. Plasminogen is primarily present in the plasma, and the liver represents its primary site of synthesis. The activation of plasminogen into plasmin is mediated by two types of activators, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). The activity of both is regulated by specific plasminogen activator inhibitors (PAIs). The principal PAIs are PAI type 1 (PAI-1), initially called the endothelial cell PAI[11]. The increased expression of PAI-1 in vivo suppresses fibrinolysis, consequently leading to the pathological fibrin deposition and tissue damage[12, 13]. PAI-1 level is influenced by several factors such as patients’ age, renal insufficiency, systolic blood pressure, insulin resistance, obesity, and triglyceride levels; however, it is not associated with cholesterol levels and smoking[14]. There is increasing evidence indicating the importance of the internal fibrinolysis system and particularly PAI-1 level in the progression of atherosclerosis and that inhibiting glycoproteins can inhibit the development of atherosclerosis or even treat it. Thus, there are wide ongoing activities for the invention of compounds that inhibit the function of PAI-1. [15]. PAI-1 and TPA antigen levels are predictive of cardiovascular disease (CVD) events after accounting for established risk factors. A serial increase in PAI-1 is associated with a further increase in risk. These findings support the importance of fibrinolytic potential in CVD[16].

Additionally, there is evidence that the PAI-1 plasma concentration depends on genetic variability. Many genetic polymorphisms have been described at the PAI-1 gene locus on chromosome 7 [ 17,18.]. One of them, guanine insertion/deletion polymorphism known as −675 4G/5G located within the promoter region, is reported in many studies to be associated with the plasma concentration of PAI-1 [19.]. Among the known polymorphic sites within the region of PAI-1 gene, only the insertion/deletion 4G/5G polymorphism at position −675 is suspected of functional relevance [20, 21]. This polymorphism is considered to be an independent risk factor for ischemic heart disease and/or acute MI. A large cohort study (n = 1179) showed that the 4G4G genotype is observed more frequently in first-degree relatives of patients with coronary heart disease than in individuals without a family history of heart disease [22].

The association's strength may vary significantly between populations, with some ethnic groups showing a weak or non-existent link. This variability could be due to the complex interplay of genetic and environmental factors characteristic of each population.

The association between the PAI-1 4G/5G polymorphism and traditional risk factors of ischemic heart disease in patients with stable coronary artery disease is a topic of ongoing research. Al-Wakeel [23] found no significant association between the PAI-1 4G/5G polymorphism and the risk of coronary artery disease in an Egyptian population. However, Lima (2011) reported a significant association between the 4G/4G genotype and high plasma PAI-1 levels with severe coronary stenosis in Brazilian individuals [32]. Saely [24] found that the PAI-1 -675 4G/5G polymorphism was significantly associated with coronary stenoses in non-diabetic subjects, but not in those with type 2 diabetes. A meta-analysis by Zhang (2014, PMCID: PMC4238518.) also suggested that the PAI-1 4G/5G polymorphism is a risk factor for coronary artery disease. These studies collectively indicate a potential role of the PAI-1 4G/5G polymorphism in the development of coronary artery disease, but further research is needed to confirm these findings.

This study was undertaken to analyze the association between polymorphism gene of PAI-1 and plasma level of PAI-1 with the traditional risk factors of ischemic heart disease in patients with stable coronary artery disease.  

Material and methods. In our scientific research included 63 patients with the diagnosis ischemic heart disease (IHD) especially with stable angina (48 men and the 15 woman) passing on hospitalization in I-cardiology department of Multidisciplinary clinic of Tashkent Medical Academy. Average age of patients was 56,8±6,40 years (42 up to 66 years). Average age of men were 56,4±6,60 years and women age were 58,0±5,52 years.

The diagnosis of IHD (stable angina) was made in compliance with classification of IHD accepted at the IV congress of cardiologists (2000). Functional class of stable angina (SA) was established on the basis of Canadian society of cardiologists classification of stenocardia and exciting test veloergometry.

Criteria of an exception of a research patients with unstable stenocardias, an acute and chronic heart, renal, liver failure, patients with an arrhythmia, acute disorders of a cerebral circulation, the diabetes mellitus transferred MI; malignant neoplasms.

For solution of tasks all patients were devided into 2 groups depending on functional class (FC) of SA. 24 patients (38,1%) with SA II FC entered in the first group. The second group consist of 39 patients (61,9%) with SA III FC.

The state of hypercoagulability was assessed by measures of polymorphism gene of PAI-1 and plasma level of PAI-1. The material for the detection of polymorphic  genes in this study served as the venous blood from the cubital vein of 3 ml. DNA analysis for the gene PAI-I (4G / 5G)  carried by the multiplex standard PCR thermal cycler and CG - 1-96 «Corbett Research » (Australia) and 2720 «Applied Biosystem » ( USA), using kits of " Geno Technology ", according to the manufacturer's instructions. Plasma level of PAI-1 was investigated with ELISA-Kit method. Besides in our research work we assessed the main risk factor of IHD such as, obesity, smoking, anxiety-depressive syndrome (ADS). Obesity was calculated with Quetelet's formula. Smoking was estimated by Fagerstrem’s test.  ADS was assesses by using Hospital Anxiety and Depression Scale (HADS).

Results

We for the first time studied the frequency of occurrence of different options of genotypes of a gene of PAI-1, clarification of its role in association with the main RF of IHD. Selection criteria for determination of additional prognostic criterion were patients with SA of Uzbek nationality. For clarification of pathogenetic value of a polymorphism of a gene of PAI-1 at patients with SA of various functional classes in the studied group was carried out the PCR analysis of a condition of a gene of PAI-1.

At a stage of carrying out of genotyping from 63 IHD patients (SA). 2 (3,3%) patients were excluded  from the research in connection with errors in storage of a blood.

The following data on the frequency of occurrence of alleles and genotypes of 4G/5G polymorphism of a gene of PAI in groups with SA specified were obtained in tables 2-3.

Table 2

Frequency of distribution of alleles and genotypes of G5/G4 polymorphism of PAI gene in group of patients with IHD and healthy individuals

№	Groups	n	Frequency alleles				Frequency of genotype distribution
			5G		4G		5G/5G		G5/G4		4G/4G
			n	%	N	%	N	%	N	%	N	%
1	Main Group (n=61)	61	67	54,9	55	45,1	19	31,1	29	47,5	13	21,3

 

 

Population distribution of alleles of PAI-1 gene is investigated at 61 patients with IHD (122 chromosomes). Frequency of 4G allele in this group made 55(45,1%). 13 homozygous carriers and  29 heterozygous carriers of this allele are revealed. Frequency 5G allele at patients of the main group made 67(54,9%). This allele in a homozygous state was revealed at 19 people.

Options of distribution of genotypes of alleles of a gene PAI-1 of patients of IHD it is visualized on an elektroforegramma of the PCR of products and it is presented on Fig.1

Fig. 1 Electrofphoregramme of PCR products of G5/G4  polymorphism of PAI gene.

Table  3

Distribution of frequencies of genotypes under Hardy-Weinberg's law. The expected and observed frequencies of distribution of genotypes on DHW in the main and control groups:

№	Groups	Frequencies of distribution of genotypes			Total	χ2	P
		5G/5G	G5/G4	4G/4G
1	Main Group (n=61)	19	29	13	n=61	7,00	0.03
	Expected frequency(n=61)	25.66	26.14	9.2
2	Control Group(n=65)	34	25	6	n=65
	Expected frequency(n=65)	27.34	27.86	9.8
	Total	53	54	19	n=126

 

Notes:  χ2 =AMOUNT (observed - expected)2/ expected =

((19-25,66)2/25,66)+((29-26,14)2/26,14)+((13-9,2)2/9,2)+((34-27,34)2/27,34)+((25-27,86)2/27,86)+((6-9,8)2/9,8)=7,00

Degreeoffreedom (df)=( number of columns-1)*( Number of lines-1)=(3-1)*(2-1)=2

Ourindicatorisinarea р<0.05, calculated with the help of Microsoft Excel p=0.03

 

For this polymorphism at patients with SA and conditionally healthy donors, observed distribution of genotypes of this polymorphism correspond to theoretical and has rather high h_obs and h_exp level (the observed and expected heterozygosis) at Hardy-Weinberg's equilibrium (р<0.05) on the basis of the Chi-square indicator (χ2=7,00) it is established statistically significant differences on distribution of genotypes 5G/5G, G5/G4, 4G/4G between patients of the main group and probands of control group (р<0.05).

The analysis of genotyping of the studied persons of the Uzbek nationality showed that, at IHD patients mutagen of 4G allele of gene PAI-1 in a homozygous and heterozygous state what makes 21,3% and 47,5%, respectively, meets more than in group of healthy faces at which given distributions of genotypes made 9,2% and 38,5%. Points these data probability of influence of existence of 4G allele of PAI-1 gene, especially in a heterozygous state, to development of IHD.

5G/5G the group of healthy faces in number of 34 people in 52.3% of cases, than at 19 (31,1%) IHD patients caused a stir in the greatest occurrence of a favorable genotype. Thus, these differences have the high statistical importance and wear, non random character (р<0.05).

Distribution of frequencies of genotypes of a polymorphic marker 4G (-675)5G of PAI-1 gene in groups of IHD patients depending on a functional class of stenocardia are presented in table 4.

Table 4.

Distribution of frequencies of genotypes of a polymorphic marker 4G(-675)5G of PAI-1 gene in the main group.

Group	Gene PAI-1, detected genotypes, n (%)			Total
	5G/5G	4G/5G	4G/4G
Patients with SA FC II	6 (25%)	14 (58,3%)	4 (16,7%)	24 (39,4%)
Patients with SA FC III	13 (35,1%)	15 (40,5%)	9 (24,3%)	37 (60,6%)

 

During the research between subgroups of IHD patients depending on FC on polymorphic markers 4G(-675)5G of PAI-1 gene, difference on the frequency of distribution of alleles and genotypes of rs1799768 became perceptible. At patients with SA FC II of 24 investigated 4 (16.7%) patients - 4G/4G, at 14 (58.3%) - 4G/5G and at 6 (25,0%) - 5G/5G, whereas in group of patients with SA FC III of 37 at 9 (24,3%) patients with 4G/4G, 15 (40,5%) with 4G/5G and 13 (35,1%) with 5G/5G genotypes. 

          Table 5.

Distribution of frequencies of genotypes of a polymorphic marker 4G(-75)5G of PAI-1 gene in subgroups of IHD patients.

Groups	Gene PAI-1,Detected genotypes, N			Total	χ2	p
	5G/5G	4G/5G	4G/4G
Patients with SA FC II	6	14	4	n=24	1,85	0.39
Expected	7.48	11.41	5.11
Patients with SA FC III	13	15	9	n=37
Expected	11.52	17.59	7.89
Total	19	29	13	n=61

Notes:χ² =AMOUNT (observed - expected)²/observed = 1,85

Degree of freedom (df)=( Number of columns-1)*( Number of lines-1)=(3-1)*(2-1)=2

On the р<0.05 significance level, with 2^nd degree of freedom, the number in the table must be equal to 5,99.  But we have 1,85.

Our indicatorрis in the areaр>0.05, calculated in calculations with the help of Microsoft Excel  p=0.39

 

Assessment of occurrence of various genotypes of a polymorphic marker 4G(-675)5G of PAI-1 gene, established that differences between distribution 5G/5G,4G/5G,4G/4G of genotypes depending on FC of CVD weren't reliable as CHI – a square on persen turned out χ2=1,85 that corresponds (р>0.05). On the basis of these results it is possible to assume that, existence of heterozygous and homozygous mutagen genotypes of a gene of PAI-1 doesn't influence disease severity in a particular on degree of FC of SA.

When genotyping of the studied groups in relation to the frequency of occurrence of a favorable homozygous genotype 5G/5G of PAI gene, in group of patients of CVD, the low frequency of this genotype in 31,1% cases, in comparison to statistically significant frequent occurrence of a genotype 5G/5G in 52,3% cases among healthy faces is taped. Occurrence at patients of SSN of a heterozygous polymorphic genotype 4G/5G of PAI gene, made statistically significantly 47,5% of cases, in comparison with control group in 38,5% cases (р<0.05) that demonstrates probability of high prevalence of this genotype at persons of the Uzbek nationality.

Interconnection of IHD RF with polymorphism of gene PAI-1.

We for the first time studied the frequency of occurrence of mutagen genotypes of PAI-1 gene and clarification of its role in association with the main IHD RF in development of CVD.

There were no clear indications of synergistic interaction effects involving the PAI-1 4G/5G polymorphism and the environmental exposures considered (cigarette smoking, physical inactivity, overweight, diabetes mellitus, hypercholesterolemia, hypertension, high C-reactive protein and hypertriglyceridaemia) [26].

Taking into account polyfactorial characteristics of IHD pathogenesis, i.e. existence of a set of RF leading to development and advance of a disease the analysis on existence of associations of a polymorphism 4g (-675) 5g PAI-1 gene with clinical-anamnestic data, such as smoking, the accompanying ADS, obesity, hypercholesterolemia (HCHE and a hypodynamia in the studied groups was carried out. The analysis of genotyping on favorable (5G/5G), polymorphic (4G/5G) and mutagen (4G/4G) genotypes of PAI-1 gene depending on existence of these or those IHD RF is presented in Table 6.

Table 6

The characteristic of RF at patients with different genotypes of a polymorphic marker 4g (-675) 5g of gene PAI-1

Indicators	Patients with IHD (n=61)
	Genotype 4G/4G n=13 (21.3)	Genotype 4G/5G n=29 (47.5)	Genotype 5G/5G n=19 (31.1)
Age, years	59.4	56.7	57.6
Heredity	8 (61.5)	19 (65.5)	9 (47,4)
Degrees of obesity (kg/m2): Normal BM Overweight 1   degree 2  degree 3 degree	1 (7.6) 6 (46.2) 4 (31) 1 (7.6) 1 (7.6)	3 (10.3) 15 (51.7) 5 (17.2) 6 (20.7) 0	5 (26.3) 9 (47.4) 3 (15.8) 2 (10.5) 0
Smoking, n(%)	8 (61.5)*	9 (31.0)	5 (26.3)
ADS, n(%) Subclin.evid. ADS, n(%)  clin.evid.	3 (23.1) 9 (69.3)	7 (24.1) 21 (72.4)	2 (10.5) 12 (63.1)
HCHE	11(84.6)*	18 (62.1)	9 (47.4)

P < 0.001

 

The analysis of interrelation of not modified and modified risk factors with 5G/5G, 4G/5G and 4G/4G genotypes of PAI-1 gene established that genotyping between groups considerably did not differ depending on age whereas heriditary burdeness on IHD met at a heterozygous polymorphic genotype (61,5%) more often, and also a homozygous mutagen genotype (65,5%) of patients with IHD. Among patients of the main group with genotypes 4G/4G an obesity of various degree is taped in 46,2% of cases, at a genotype of 4G/5G in 37,9% and at 5G/5G 26,3%, therefore is rather more often at patients with SA with an obesity including the third degree of PAI-1 gene mutation was genotyped.

Connection of mutagen genotypes with smoking appeared to be one of the most significant as among patients with a heterozygous genotype smokers in 61,5% of cases, in comparison with patients with a polymorphic genotype and a homozygous genotype of wild allele 5G made 31% and 26,3% of smokers, respectively prevailed. Existence and degree of expression of the accompanying ADS at patients of the main group, prevailed at persons with mutagen 4G/4G in 92,4% and a polymorphic 4G/5G genotype in 96,5% of cases, and at patients with a genotype 5G/5G ADS in rather smaller degree is revealed at 73,6% of cases

 Besides we evaluated plasma level of PAI-1 and got following results (Table 7).

 

 

Table 7

Plasma level of PAI-1 depending of genotypes of PAI-1 gene in patients with ischemic heart disease

Groups	Gene PAI-1,Detected genotypes,
	5G/5G	4G/5G	4G/4G
PAI-1 level in patients  with SA (ng/ml)	33,3±2,07*	54,8±3,47	72,0±7,6*

*P < 0.001

Average level of PAI-1 in patients with 5G/5G genotype was 33,3±2,07 and with in 4G/4G  genotype was 72,0±7,6 and it was statistically significant (P < 0.001). The plasma level of PAI-1 was higher in patients with SA FC III than patients with SA FC II and this was statistically significant. All patients had no differences in age, lipid profile, and coagulogram level. Moreover, patients with 4G/4G  genotype and with  SA FC III are at a higher risk of high PAI-1 levels.

Discussion

Cardiovascular disease with disturbances in the haemostatic system, might lead to thrombotic complications with clinical manifestations like acute myocardial infarction (AMI) and stroke. [27] Certain individuals may have an abnormal propensity to develop venous or arterial thrombosis and either experience thromboembolic events relatively early in life or suffer recurring events [28].
Based on the above data, we set a goal to study the violation of the hemostasis system at gene levels in patients with stable coronary disease. Сlearly defined associations have been described for hypercoagulable states and thrombosis within the venous system, the establishment of causative or contributing roles for these same thrombophilic conditions and the occurrence of arterial thrombosis has been considerably more elusive [29].

 Our findings suggest that an increased coagulation tendency is associated, in a high plasma level PAI-1, with a polymorphic genotypes of gene PAI-1 especially genotype 4G/4G increased risk of cardiovascular events in patients with stable ischemic heart disease. The applied coagulation evaluation of manifestations has fibrinolytic markers of hypercoagulability and is weighted by the risk of an acute myocardial ischemia event, since these markers were found to be the main marker in the state of stable coronary heart disease, in which hypercoagulability plays the greatest role. In some scientific researchs PAI-1 has been an informative marker to study the degree of hypercoagulability in patients with acute coronary syndrome. In 1995 work about association allele 4G of PAI-1 gene with higher risk MI at 100 young men (35-45 years) from the Swedish population was for the first time published [30].People, homozygous on 4G allele, have higher PAI-1 level in plasma, and homozygous on 5G allele— lower. The mechanism which is the cornerstone of allelic differences in the level of synthesis PAI–1 was installed after ability of both alleles to be bound to the gene transcription activator whereas the allele 5G also has the website of linking with a transcriptional repressor [30.]. According to the data obtained by different researchers, the PAI-1 level is about 25% higher at carriers of a genotype 4G/4G, in comparison with carriers of a genotype 5G/5G. It is shown that, in comparison with 5G allele, at 4G allele carriers concentration of PAI-1 and risk of clottages is higher. At 4G allele carriers both in hetero - and in a homozygous state also higher PAI-1 level of plasma [31] and larger risk of development of acute coronary syndromes becomes perceptible [32].

Several studies have shown the relationship between levels of plasminogen activator inhibitor-1, and either stable or unstable coronary artery disease [33, 34] However, in patients with MI, there is a clear association between PAI-1 levels and patient response to treatment with fibrinolytic agents; this has been demonstrated in several studies. [35]. There are however, limited data on the association between stable ischemic heart disease and PAI-1level and his polymorphism. In our research no difference was found between groups regarding age, gender. Plasma PAI-1 levels and 4G/4G genotype frequency were significantly higher, especially in patients with SA FC III compared to the SA FC II. Furthermore, patients with 4G/4G genotype had significantly higher plasma PAI-1 levels than those with 5G/5G genotype. [36] The results of  meta-analysis from 2022 indicated that PAI-1 4G > 5G SNP was associated with decreased risk of CAD in the overall population as well as in the Asians, Caucasians, and Arab populations. However, the PAI-1 gene - 844 G > A polymorphism had no significant association with susceptibility to CAD [37].

Our genetic analysis revealed that individuals with the 4G4G genotype had higher plasma concentration of the PAI-1. This finding is consistent with the data from in vitro studies, which have shown that the 4G allele is associated with higher transcriptional activity of the PAI-1 promoter compared to the 5G allele, and is related to the creation of an additional repressor-binding site in the 5G carriers [36].

This finding is of clinical importance because fibrinolysis plays an important role in the development of acute MI as well as stable coronary artery disease.

 

Conclusion

Our investigation results show that PAI-1 4G/5G polymorphism was significant associated with CAD risk in uzbek nationality. In the subgroup analysis according to FC of SA, the results suggested that PAI-1 4G/5G polymorphism was associated with CAD risk and high level of PAI-1 in plasma.

 

Об авторах

Наргиза Кадирова

ташкентский медицинский университет

Email: xasanova_nargiza@bk.ru
ORCID iD: 0000-0003-0156-1304

кандидат медицинских наук

Узбекистан

Наргиза Мухтархановна Нуриллаева

Ташкентский медицинский Университет

Автор, ответственный за переписку.
Email: nargizanur@yandex.ru
ORCID iD: 0000-0001-5520-675X

Professor Head of the Department of Internal Diseases in Family Medicine №1 and Fundamentals of Preventive Medicine,Tashkent Medical University (Tashkent,Uzbekistan)

Узбекистан, 100109,Фараби 2, Ташкент,Узбекистан

Виктория Petrova

North-Western State Medical University  named after I.I. Mechnikov, St. Petersburg, Russian Federation 

Email: nikki007@mail.ru
ORCID iD: 0000-0002-1672-4922

Associate Professor, Department of Geriatrics, Propaedeutics and Management of  Nursing Activities, North-Western State Medical University  named after I.I. Mechnikov, St. Petersburg, Russian Federation 

Россия

Ekaterina Lapteva

North-Western State Medical University named after I.I. Mechnikov, St. Petersburg, Russian Federation 

Email: ekaterina.lapteva@szgmu.ru
ORCID iD: 0000-0002-4897-6606

Head of the departmentб Department of Geriatrics, Propaedeutics and Management of  Nursing Activities North-Western State Medical University named after I.I. Mechnikov, St. Petersburg, Russian Federation 

Россия

V. Shumkov

North-Western State Medical University named after I.I. Mechnikov, St. Petersburg, Russian Federation

Email: shumkovVA@mail.ru
ORCID iD: 0000-0001-9055-432X

Assistant, Department of Geriatrics, Propaedeutics and Management of  Nursing Activities North-Western State Medical University named after I.I. Mechnikov, St. Petersburg, Russian Federation

Россия

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