Introduction

Over the last decade, diabetes mellitus (DM) has acquired an epidemic prevalence in the population: according to WHO experts, by 2025 the number of patients with this pathology worldwide is expected to reach 380 million, of which more than 90% are cases of type 2 DM. The primary cause of mortality in type 2 DM is vascular complications, including cerebrovascular complications [1].

It is known that cerebral atherosclerosis is one of the leading factors in the development of ischemic cerebrovascular accidents, and DM is an independent risk factor for their development. Several authors have suggested a commonality in the pathogenetic mechanisms of atherosclerosis, cardiovascular diseases (CVD), and type 2 DM [1, 2].

Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia, which results from defects in insulin secretion, insulin action, or a combination of these factors. Chronic hyperglycemia in diabetes is associated with damage, dysfunction, and eventual failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels [3, 4].

Vascular complications, requiring prolonged and costly treatment, are among the most common causes of disability and death in patients with DM. At the same time, early diagnosis and effective therapy of this formidable disease can delay or prevent the occurrence of complications through a thorough investigation of changes in the hemostasis gene system and genetic thrombophilia. Among causes of blindness in the age group under 50, DR ranks first. Twenty years after the onset of the disease, DR develops in 90-100% of patients with type 1 DM (T1DM) and 60% of patients with type 2 DM (T2DM), leading to low vision in 10% and blindness in 2% of patients. The causes of diabetic complications remain not fully understood. Specific microvascular lesions contribute significantly to the pathogenesis of DM complications, leading to vision impairment. Traditionally, T2DM is considered a consequence of prolonged decompensation of carbohydrate metabolism, and angiopathy is often diagnosed only in the late stages of T2DM. Against the background of DM, virtually all components of the hemostasis system are affected in patients, leading to the development of thrombophilia. At the same time, clinical experience and analysis of literature sources indicate the presence of specific changes in the retina at the time of diagnosis [3, 4, 5].

In the early stages of the disease, dysfunction of the coagulation and fibrinolysis systems leads to the appearance of retinopathy and nephropathy [6].

It is a well-established fact that the blood clotting process is determined by the state of three components of the hemostasis system: platelets, coagulation factors, and the integrity of the vascular wall. If at least one component is disturbed, the process leading to thrombus formation is activated. The basis of pathological changes in the body of patients with DM is generalized damage to the vascular system – macro- and microangiopathy. The duration and quality of life of DM patients are currently determined by the development of late vascular complications of this disease, one of which is diabetic retinopathy (DR) [7, 8].

In domestic and foreign literature, the aspects of hereditary thrombophilia in the pathogenesis of vascular complications in patients with T2DM are poorly covered. A number of authors state that multifactorial hereditary thrombophilia is a risk factor for retinal arterial occlusions (RAO) and retinal vein occlusions (RVO) in patients with DR. Subsequent scientific research has led to a significant expansion of understanding regarding the possible causes of an increased tendency to thrombosis [9].

Associated genes, as well as endothelial dysfunction (ED) genes, include genes of the renin-angiotensin system (RAS) and endothelial nitric oxide synthase (eNOS), which are involved in the pathogenesis of CVD. Mutations in the angiotensin-converting enzyme (ACE), angiotensinogen (AGT), angiotensinogen receptor type 1 (AGTR1), and endothelial nitric oxide synthase (eNOS) genes may serve as markers of CVD risk and blood clotting disorders. Common gene polymorphisms may also strongly interact both with each other and with less common mutations, leading to potentially life-threatening conditions [8, 9].

The well-known domestic researcher in this field, Prof. Z.S. Barkagan, proposed that thrombophilia be understood to include not only the aforementioned causes but also acquired (secondary, symptomatic) hemostasis disorders that predispose to the early onset and recurrence of thromboses, thromboembolisms, organ ischemia and infarctions. In the classification proposed by the author, particular importance was attached to conditions associated with metabolic diseases – diabetes, obesity, autoimmune and infectious-immune diseases – immune thrombovasculitis, sepsis, etc., as well as drug effects, the so-called iatrogenic forms of thrombophilia.

Researchers – scientists and clinicians from the Research Institute for Complex Issues of Cardiovascular Diseases, Siberian Branch of the Russian Academy of Medical Sciences, Kemerovo; and the Altai Branch of the Hematological Research Center, Russian Academy of Medical Sciences, Barnaul – studied the prevalence of intergenic associations in patients with severe angina, type 2 DM, and healthy individuals. It was found that the frequency of mutations, Factor V Leiden, and MTHFR was significantly higher compared to the occurrence of PAI-I and prothrombin gene abnormalities in practically healthy individuals. It can also be observed that allelic polymorphisms of PAI-I and MTHFR were most frequently identified in patients [10]. There is a lack of research in the scientific literature regarding mutations and polymorphisms of hemostasis genes in patients with DR and DME.

Objective

To investigate and analyze changes in the hemostasis system, mutations, and polymorphisms of hemostasis genes in patients with clinical forms of DR and DME associated with T2DM.

Materials and Methods

An analysis was performed on 50 outpatient records of patients with DR (100 eyes), including clinical and laboratory changes in the hemostasis system and genetic thrombophilia in these patients.

Patients were divided into 3 groups. The following clinical forms of diabetic retinopathy were identified: non-proliferative diabetic retinopathy 2 (NPDR-2) – 20 patients; non-proliferative diabetic retinopathy 3 (NPDR-3) – 15 patients; pre-proliferative diabetic retinopathy (PPDR) – 15 patients. Patient age was 45–55 years: 30 were female, 20 were male.

The control group, consisting of 50 individuals for the study of genetic thrombophilia markers, included persons with no history of thrombotic events and with diabetes excluded. The control group was composed based on literature data for patients aged 45–55 years.

Concomitant diseases associated with DM: hypertension and coronary heart disease in 18 patients; hypercholesterolemia in 10 patients (5.8-6.4 μmol/L) and hyperhomocysteinemia (HHcy) in 20 patients (homocysteine (Hcy) 15-17 μmol/L); lower extremity macroangiopathy in 8 patients; diabetic nephropathy in 4 patients. Under treatment, glucose levels were 8.7 ± 2.4 mmol/L. Follow-up duration ranged from 2 weeks to 5 years.

Ophthalmological examinations

Standard methods: visometry, tonometry, perimetry, direct ophthalmoscopy. Specialized methods: fundus examination with a Goldmann lens, fundus photography, fundus fluorescein angiography (FFA), optical coherence tomography (OCT) of the retina, computer perimetry.

Hemostasis system studies were conducted using screening and specialized automated coagulometry methods: determination of von Willebrand factor (vWF) activity, antithrombin III, activated protein C (APC), factor VIII (FVIII), quantitative content of factor V (FV) in plasma; determination of factor V resistance to activated protein C, APC resistance or APC resistance index; quantitative determination of soluble fibrin monomer complexes (SFMC); quantitative content of fibrinogen (Clauss method). Detection of lupus anticoagulant (LA) was performed using venom-based tests, as well as confirmatory tests with donor plasma and phospholipid correction; plasma testing for LA was repeated after 12 weeks. Reference values for LA were up to 0.8 U – 1.2 U. LA is heterogeneous in its functional properties and, depending on its ability to prolong phospholipid-dependent and phospholipid-independent coagulation tests, can be divided into 4 types. While LA types 1 and 2 do not possess significant thrombogenicity, LA types 3 and 4 are highly thrombogenic. LA levels of 1.25-1.35 U were detected in 16% of patients with DR and DME, classified as moderately thrombogenic.

Plasma homocysteine (Hcy) levels were studied using ELISA. The reference range for Hcy in the blood of healthy adults is 5-15 μmol/L, but in individuals with risk factors, a borderline level is considered not 15 μmol/L, but 10 μmol/L (Russian Clinical Recommendations).

Molecular genetic testing using real-time polymerase chain reaction (PCR): F2 gene mutation: 20210G>A; F5 gene mutation: 1691 G>A; FGB gene mutation: -455 G>A; F13A polymorphism; platelet integrin receptor gene polymorphism: ITGA2: 807 C>T; ITGB3: 1565T>C; plasminogen activator inhibitor type 1 (PAI-1): -675 5G>4G; folate gene polymorphisms: MTHFR C677T, MTHFR 1268, MTRR; renin-angiotensin system gene polymorphisms: ACE, ATGR1, AGT; NOS3 gene polymorphism.

Statistical processing of results was performed on a personal computer using the Statistica 6.0 (Statsoft) for Windows application package. Differences were considered statistically significant at a significance level of p < 0.05.

Results and Discussion

High levels of vWF, FVIII, and protein C correlated with macular retinal thickness in patients with NPDR and PPDR (R1 – 0.93 correlation coefficient for vWF with macular retinal thickness in patients with clinical forms of DR; R2 – 0.93 correlation coefficient for factor VIII activity level with macular retinal thickness in patients with clinical forms of DR; R3 – 0.84 correlation coefficient for protein C activity with macular retinal thickness in patients with clinical forms of DR; R4 – 0.96 correlation coefficient for SFMC with macular retinal thickness in patients with clinical forms of DR) (Table 1).

Table 1. Changes in the Hemostasis System in Patients with Diabetic Retinopathy

Note:

* – significant difference from the norm p ≤ 0.05;

** – significant difference between NPDR-2 and NPDR-3 patient groups p ≤ 0.05;

*** – significant difference between NPDR-3 and PPDR patient groups p ≤ 0.05.

vWF – von Willebrand factor activity, %, elevated level indicates endothelial dysfunction (ED); FVIII – factor VIII activity, %, elevated level indicates ED and ischemia. Protein C – protein C activity, %; SFMC – mg%, elevated level indicates thrombinemia, tendency to thrombosis; Macular OCT – retinal thickness, µm.

Compared with the control group, patients with DR showed activation of hemostatic components responsible for endothelial dysfunction. Factor VIII was most activated: by 25% in NPDR-2 patients, by 39% in NPDR-3 patients, and by 44% in PPDR patients. The increase in factor VIII activity was accompanied by an increase in vWF activity in NPDR-2 patients by 18% compared to healthy individuals, by 28% in NPDR-3 patients, and by 37% in PPDR patients. Protein C activity also increased significantly in NPDR-2 patients by 13%, in NPDR-3 patients by 22%, and in PPDR patients by 39%, which is associated with the progression of clinical manifestations of DR.

The increase in markers of thrombogenic activity (vWF, factor VIII, APC, SFMC) across DR stages indicates its progression, characterizes retinal capillary occlusion leading to extensive zones of impaired blood supply, and increased permeability of retinal microvessels.

The increase in endothelial dysfunction markers (vWF, factor VIII, APC, SFMC) correlates with an increasing incidence of diabetic macular edema in patients with NPDR (NPDR-2, NPDR-3) and PPDR (R1 – 0.93 correlation coefficient for vWF with macular retinal thickness in patients with clinical forms of DR; R2 – 0.93 correlation coefficient for factor VIII activity level with macular retinal thickness in patients with clinical forms of DR; R3 – 0.84 correlation coefficient for protein C activity with macular retinal thickness in patients with clinical forms of DR; R4 – 0.96 correlation coefficient for SFMC with macular retinal thickness in patients with clinical forms of DR).

Leakage from retinal capillaries leads to retinal edema: diffuse due to plasma leakage and focal resulting from the formation of microaneurysms. The former results in "soft exudates" and cystoid degeneration, while the latter results in "hard exudates".

In patients with non-proliferative DR associated with T2DM, focal macular edema was observed in 17% of cases. In patients with pre-proliferative DR, moderate focal macular edema was observed in 4% of cases, flat diffuse macular edema in 18%, and high diffuse macular edema in 8%.

Fig. 1. Fundus photograph and macular OCT of patient P., 52 years old. Diagnosis: PPDR, diffuse cystoid macular edema. MHT: FGB gene: -455 G>A, PAI-1 gene: -675 5G>4G with ACE gene, with NOS3, HPA-2 genes.

The study of HPA-2 (A1/A2) polymorphism of the platelet glycoprotein 1b (GP1b) gene has prognostic value, allowing the assessment of the risk of ischemic stroke. This polymorphism determines the antigenic status of platelets, corresponding to either HPA group 1 or 2 (variants A1 and A2). Several studies have shown that carriers of the A2 variant have an increased risk of coronary thrombosis, ischemic stroke, and a younger age of onset [10,11,12].

Multifactorial hereditary thrombophilia (MHT) was found in 100% of patients with retinal vein occlusion, including 16% in combination with LA. In groups 1, 2, and 3, the FGB gene mutation (-455 G>A) combined with endothelial dysfunction genes (ACE, ATGR1, AGT, NOS3) predominated – 40%; the F2 gene mutation (20210G>A) combined with the F5 gene mutation (1691 G>A), with ACE, ATGR1, AGT genes, combined with the platelet gene P2Y12 ins801A (AP), with folate cycle genes and endothelial genes = 8%; the PAI-1 gene (-675 5G>4G) with endothelial and folate genes = 14%. Polymorphism in the F13A gene combined with the platelet receptor gene polymorphism ITGA2: 807 C>T = 12%.

High cystoid diabetic macular edema was identified in patients with NPDR (NPDR-2, NPDR-3) and PPDR in 47% of cases. There is a prevalence of intergenic associations in patients with DR and DME in combination with cardiovascular pathology in DR patients.

It is important to carefully select patients with DME associated with DR and T2DM for conservative medication, surgical, and laser treatment, paying special attention to the study of hemostasis, the presence of LA, and hemostasis gene mutations and polymorphisms. The effectiveness of DR treatment significantly decreases as it progresses; therefore, diagnostic and therapeutic/preventive measures should ideally begin at the earliest stages of its development, alongside the correction of hyperglycemia with modern hypoglycemic agents, and timely prescription of anticoagulants and antiplatelet agents, considering the polymorphism of platelet integrin receptor genes (ITGA2: 807 C>T; ITGB3: 1565T>C) and rare platelet genes such as P2Y12 ins801A (AP) and HPA-2. In patients with T2DM and genetic thrombophilia involving platelet integrin receptor gene polymorphisms ITGA2: 807 C>T, aspirin resistance occurs. In patients with T2DM and genetic thrombophilia involving platelet integrin receptor gene polymorphisms ITGB3: 1565T>C, resistance to aspirin and clopidogrel occurs. The polymorphism in the platelet receptor gene for the ADP inducer P2Y12 ins801A (AP) MN increases platelet aggregation capacity and causes insensitivity to clopidogrel, which increases the risk of post-traumatic and postoperative bleeding [11,12].

Fig. 2. Fundus photograph and OCT of patient K., 50 years old, with PPDR and diffuse cystoid DME. Polymorphism in the F13A gene combined with the platelet receptor gene polymorphism: ITGA2: 807 C>T, with polymorphism in the PAI-1 gene: -675 5G>4G with folate genes: MTHFR C677T; MTHFR 1298; MTRR (66 A>G).

Endothelial plasminogen activator inhibitor suppresses the fibrinolysis process; the heterozygous mutation PAI-1: -675 5G>4G indicates an elevated level of this enzyme in the blood, which reduces its fibrinolytic activity; this condition is associated with hypercoagulation.

MTRR 66GG is a risk factor for deep vein thrombosis, and the risk increases in the presence of MTHFR 677CT/1298 polymorphisms. The MTRR gene encodes the enzyme methionine synthase reductase (MSR), which participates in methionine synthesis by restoring methionine synthase to its functional state. In Kuzbass, individuals with the MTRR 66GG polymorphism tended to have higher homocysteine levels than 66AA homozygotes (wild type) – 27.9%; homocysteine levels positively correlated with an increased frequency of DNA damage.

HHcy contributes to vascular damage by inhibiting endothelial cell growth and endothelial repair, and also leads to the proliferation of vascular smooth muscle cells, differentiation of inflammatory monocytes, oxidation of low-density lipoproteins (LDL), and a prothrombotic state; a number of clinical observations demonstrate the significance of hyperhomocysteinemia as a risk factor for retinal vascular thrombosis [10,11,12].

Genetic polymorphisms of MTHFR occur in 46.3% of patients with progression of the atherosclerotic process despite achieving target lipid profile values and with persistent hypercoagulation despite receiving standard doses of antiplatelet agents and anticoagulants. In the structure of polymorphisms, heterozygous carriage predominates over homozygous. The presence of folate cycle gene polymorphisms in an individual contributes to the development of HHcy. The most common hereditary cause of HHcy is point mutations in the gene for the enzyme methylenetetrahydrofolate reductase (MTHFR), located on chromosome 1 [9,10].

In all DR patients with LA, there is a significant increase in the imbalance of the hemostasis system and endothelial dysfunction, as well as the activation not only of the cytokine cascade of inflammatory reactions but also increasing ischemia in the microvasculature of the retina and choroid. This is accompanied by a pronounced retinal thrombohaemorrhagic syndrome manifested as recurrent RVO in 12% with high diffuse persistent DME with serous retinal detachment in 8% of cases. RAO occurred in DR patients with LA in 5.3%. LA levels were 1.25-1.35 U. LA disrupts the physiological regulation of the hemostasis system – reduces endothelial thromboresistance, activates platelet hemostasis, reduces the activity of natural anticoagulants, leading to the development of hypercoagulation and exacerbating ED. Some scientific publications describe the hereditary nature of thrombophilia associated with LA [3].

Fig. 3. Fundus photograph, OCT angiography of patient P., 55 years old, with T2DM with partial central retinal vein occlusion (CRVO) associated with FV Leiden mutation and LA, with pronounced retinal thrombohaemorrhagic syndrome, zones of ischemia and leakage, high diffuse cystoid macular edema (852 µm). MHT: FGB gene mutation: -455 G>A, F2 gene mutation: 20210G>A combined with F5 gene mutation: 1691 G>A, PAI-1 gene: -675 5G>4G combined with MTHFR C677T; MTHFR 1298. ACE gene polymorphism combined with NOS3 gene polymorphism + P2Y12 ins801A (AP).

The polymorphism in the platelet receptor gene for the ADP inducer P2Y12 ins801A (AP) MN increases platelet aggregation capacity and causes insensitivity to clopidogrel, which increases the risk of post-traumatic and postoperative bleeding [10,11,12]. In patients with diabetic macular edema, multifactorial hereditary thrombophilia was identified in 47% of cases: mutations in PAI-I, prothrombin, Factor V Leiden genes combined with LA = 16%, mutation in the MTHFR gene – 21% of cases. Defective factor V, such as Factor V Leiden, is inactivated much more slowly than normal factor V, leading to stabilization of the prothrombinase complex and an increased rate of thrombin generation, which under certain conditions leads to thrombosis at any age [9,10,11,12].

Conclusions

1. MHT involving high-thrombogenic-risk hemostasis genes (FGB gene mutation: -455 G>A, F2 gene mutation: 20210G>A combined with F5 gene mutation: 1691 G>A, PAI-1 gene: -675 5G>4G combined with MTHFR C677T; MTHFR 1298) underlies thrombohaemorrhagic syndrome in the retinal microvasculature with DR, DME, with zones of ischemia in the center and periphery of the retina.

2. Pathological changes in the hemostasis system, mutations and polymorphisms of hemostasis genes, intergenic associations, and the circulation of LA in the blood are associated with the risk of high cystic macular edema in DR patients, as well as with vascular catastrophes: RVO, RAO in patients with cardiovascular pathology associated with T2DM.

3. The obtained results can be used in the laboratory-clinical monitoring of hemostasis in patients with DR and DME for timely diagnosis, adequate conservative pathogenetic therapy, and proper selection of patients for surgical treatment, including retinal laser coagulation.