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THROMBOSIS IN DIFFERENT LOCATIONS: CANDIDATE GENES AND THE FUNCTIONAL STATE OF THE HAEMOSTATIC SYSTEM

Tsvetovskaja G.A. Lifshits G.I. Chikova E.D. Belevantseva A.V. Voronina E.N. Novikova Y.V. Danilkina S.T.

Thrombotic diseases, such as ischemic disturbances of cerebral and coronary circulation and thrombotic diseases of the venous system, have spread widely in recent years. Many see the reasons of this pathology in disturbed hemostasis, hemorheology, as well as in the interaction of environmental factors and genetic predisposition [3, 4, 5, 8, 9].

Prevention, diagnostics and treatment of thrombotic conditions are the topical questions facing the modern medicine. At the same time, it has been proved, that the revealed markers of thrombinemia do not let identify the reason of predisposition to intravascular coagulation. Consequently, this method is not sufficient for selecting a pathogenetic therapy. In this regard, clinical practice focuses nowadays on tracking the causes of predisposition to recurrent thrombosis - finding «risk genes», responsible for the predisposition to multifactorial diseases, including the thrombotic ones [1, 6].

Aims and objectives

The aim of this study was to analyze the candidate genes in patients of the West Siberian region with a verified diagnose of thrombosis in different locations, and try to prove, that genetic tests give more diagnostic opportunities to cardiologists, phlebologists and other doctors in their everyday practice. We have examined the venous blood, estimated the functional state of the haemostatic system and thrombophilia predisposition genes.

Materials and methods

We observed 64 patients from the West Siberian region (Novosibirsk and the Novosibirsk region) with different forms of thrombophilia, aged between 6 and 71 years old. Our test persons were divided into two groups: 22 patients with arterial thrombosis - ischemic stroke and cardiosclerosis after a heart attack, and 42 patients with deep venous thrombosis (DVT). We studied 19 candidate genes (25 allelic variants), whose products are involved in coagulation cascade, fibrinolysis system, sustaining of vascular tone and methionine metabolism (folat cycle genes).

Results

Analysis of changes in haemostatic values - platelet, coagulation and fibrinolysis - in patients with ischemic stroke and deep venous thrombosis showed, that AT III activity, content of protein С, fibrinogen and D-dimer had no statistically considerable differences from the normal values. At the same time, in 73% of all patients from the both groups, a higher level of soluble fibrin complex (SFC) could be seen, which points to hemostasis activation, i.e. intensification of thrombin and fibrin formation. In both groups of patients, a higher functional platelet activity was observed, but the frequency of induced platelet activity among the patients with ischemic stroke was higher, than in the patients with DVT. It should be mentioned, that the higher platelet functional activity in patients with the deep venous thrombosis was induced by collagen, in patients with the arterial thromboses - by ADP and adrenalin.

The main candidate genes, which take part in thrombophilia development in the examined patients are listed in the following table.


Frequency rate of genetic polymorphisms in patients with thrombosis in different locations

Gene

Gene name, (polymorphic substitution)

Frequency

(% )

Homozygous
variant (%)

PAI1

Plasminogen activator inhibitor (675 5G -> 4G )

80,6

38,4

α2 РLI

Plasminogen inhibitor

50,0

53,8

PLAT

Tissue plasminogen activator (7351 С -> Т)

34,4

20,0

MTHFD

Methylenetetrahydrofolate Dehydrogenase 1 (1958 А- >G)

76,6

47,8

MTHFR

Methylenetetrahydrofolate Reductase (С677Т)

58,0

5,5

MTHFR

Methylenetetrahydrofolate Reductase (А1298С)

58,2

33,3

MTRR

Methionine synthase reductase (66A > G)

70,0

40,9

MTR

Methionine synthase ( 2756 A- >G )

10,3

0

NSO3

Endothelial NO-synthase (VNTR)

18,5

60,0

NSO3

Endothelial NO-synthase C->T (Glu298Asp)

42,8

16,6

Gp Ia

Integrin-alpha-2, glycoprotein 1а of platelets (807С- > Т)

50,0

0

GpIIIa

Platelet glycoprotein IIIA, integrin beta 3, (1565 Т- С) (Leu33Pro)

45,2

21,4

Gp Iba

Platelet glycoprotein Iba (VNTR)

0

 

FGB

Fibrinogen-beta-peptide (455 G - >A)

37,5

8,3

FV

Leiden factor (1691 G->A (R506Q)

14,0

0

FII

II coagulation factor (20210 G - >A)

6,0

 

Analyzing gene Gp-Ia integrin-аlpha-2, polymorphic variant Т Gp-Ia was revealed in 50% of all cases. As for polymorphic variant Т of this gene, we observed, that platelets adhere to vessel walls faster, which can increase the risk of thrombophilia. Our data let regard variant Т as a marker for high risk of thrombus formation, especially, in combination with gene defects - markers of endothelial dysfunction and folat cycle enzyme genes. Comparative analysis of distribution of alleles and genotypes of this polymorphic marker in patients with arterial and venous thromboses revealed no reliable differences.

Platelet glycoprotein GPIIIa (integrin β3) encodes amino acid sequence of platelet receptor subunits for fibrinogen and Willebrand factor. A leicine-to-proline substitution, determined by the substitution of Т by С in exon 2 of gene GPIIIa at position 1565, is accompanied by a higher platelet predisposition to aggregation, which increases the risk of cardio-vascular diseases. Polymorphic substitution 1565 Т- > С was found in 45,2% of the examined patients of the both groups. None of the examined patients had polymorphic variant Gp1ba (VNTR) - gene that encodes amino acid sequence 1beta - subunits of specialized platelet receptors, which organize interaction between platelets and the wall of a damaged vessel or damaged surface of a atherosclerotic plaque.

Polymorphic substitution 675 5G -> 4G of the plasminogen activator inhibitor gene (PAI-1) points at the predisposition to endothelial dysfunction. The gene encodes protein - plasminogen activator inhibitor, which is one of the main components of the blood anticoagulation system. Polymorphic variant 4G, which is accompanied with increased gene expression and causes higher PAI-1 level in blood, was found in 80,6 % of the patients, both with arterial and venous thromboses. It is well known, that in endothelial dysfunction, fibrinolytic activity is mainly inhibited by stronger endothelial synthesis and PAI-1 secretion [2]. 45% of patients had a combination of PAI-1 gene polymorphism with gene α 2 PLI -plasminogen inhibitor, 34,8% had a polymorphic variant of tissue plasminogen activator (PLAT), polymorphic substitution 7351 С->Т, which is a sign of falling expression of tissue plasminogen activator, leading to ineffective fibrinolysis.

We also estimated the predisposition for endothelial dysfunction analyzing mutation of genes, which regulate the vessel wall condition - NOS(е) endothelial NO-synthase, VNTR-polymorphism and polymorphic alternative С- Т (Glu298Asp), as well as endothelin and hANP. Polymorphic variant of endothelial NО-synthase gene - NOS(e), VNTR-polymorphism and polymorphic substitution С -> Т (Glu298 Asp) were registered in 42,8 and 18,5% of cases correspondingly, which could be a reason for reduced NО synthesis, and, as a result, growing vasoconstriction, lower vasodilation and higher predisposition to thrombus formation [9,10].

We studied the system of folat cycle genes, as they play an important pathogenetic role in thrombophilia development. Six folat cycle candidate genes (7 allelic variants) have been examined. Polymorphisms of genes MTHFD, MTRR and MTHFR were revealed frequently. The majority of homozygous variants were found in genes MTHFD and MTRR. It should be mentioned, that in most cases, we found combinations of several folat cycle gene polymorphisms. Three-genetic-polymorphism combination was observed in у 45,2% of patients, homozygous variants of one or two polymorphisms were revealed in 81,2% of patients. Combination of four polymorphisms was found at 22,6% of the examined patients, and two polymorphisms - at 19,3%. Obviously, the polymorphic variant combination is a serious risk factor of functional disturbances in folat cycle enzymes, which leads to excessive homocysteine accumulation in blood and raises its thrombogenicity dramatically (1, 6).

Our research revealed a relatively low percent of mutations in factor Leiden and prothrombin gene (II coagulation factor (20210 G - >A) - 14% and 8% correspondingly.

Conclusion. To sum up, among the population of Novosibirsk and the Novosibirsk region, platelet glycoprotein and endothelial NO-synthase genes, as well as genes that encode endothelial proteins of fibrinolysis and folat cycle system, are the mostly frequent revealed thrombophilia-related polymorphic sites. The obtained data prove, that one of the key elements of thrombosis pathogenesis in different locations is the inhibition of fibrinolytic blood activity. Mutations in tissue plasminogen activator gene (PLAT) combined with mutations of the most important fibrinolysis inhibitors PAI 1 и α 2 PLI, create serious conditions for a lower plasmin level in blood and cleaning the clogged blood vessels. Higher risk of thrombus formation and pulmonary embolism have PAI1-allele carriers, especially in combination with additional genetic defects - mutations in folat cycle genes ( MTHFD, МТРР, MTHFR), platelet glycoprotein genes (GP III а, Gp-Ia) and genes, responsible for the vessel wall regulation.

Endothelial dysfunction, which determines the ineffective fibrinolysis, combined with the above mentioned risk genes of thrombophilia, can turn the predisposition into the pathology. Despite the fact, that arterial and venous thromboses develop differently, we found some syntrope genes involved in thrombophilia development, i.e. polymorphic genes responsible for both arterial and venous thromboses. We do not rule out the possibility, that the polymorphic gene frequency rate in patients with different forms of thrombophilia could change, if a larger number of people would be examined. As for the population of the West Siberian region - patients with pulmonary embolism, post-thrombotic disease, recurrent thrombosis, patients who need cava filter, testing of the above mentioned genes is getting more and more important, as it lets determine a pathogenetically reasonable therapy and reduce the number of thrombosis recurrences. Besides, diagnostics of predisposition for thrombophilia lets considerably reduce the number of the post-operative thromboembolic venous complications, which are challenging for modern medicine and life threatening for patients after surgeries.

References

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8. Takada A/, Urano T., Takada Y. Stress and hemostasis. Recent progress in blood coagulation and fibrinolysis. International congress series 1129. Exsepta Medika 1997; 213 - 221.

9. Вeckman J.S., Crapo J.D. The role of nitric oxide in limiting gene transfer: parallels to viralhost defenses // Am.J. Respir. Cell. Mol. Biol. 1977. V.16. №.5. P. 495-496.

10. Cooke J.P. NO and atherogenesis // PNAS. 2003 b.v. 100. 3. p.768 - 770.

The work is submitted to Scientific Conference "Basic and applied research in medicine", France (Paris), October 13-20, 2009. Came to the Editor´s Office on 31.08.2009.


Библиографическая ссылка

Tsvetovskaja G.A., Lifshits G.I., Chikova E.D., Belevantseva A.V., Voronina E.N., Novikova Y.V., Danilkina S.T. THROMBOSIS IN DIFFERENT LOCATIONS: CANDIDATE GENES AND THE FUNCTIONAL STATE OF THE HAEMOSTATIC SYSTEM // Международный журнал прикладных и фундаментальных исследований. – 2009. – № 2. – С. 22-24;
URL: http://applied-research.ru/ru/article/view?id=358 (дата обращения: 02.06.2020).

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