Retinal vein occlusion (RVO) accounts for up to 60 % of acute vascular disorders of the eye; it ranks second to diabetic retinopathy in terms of severity of damage to the retina and carries a poor prognosis [1, 2]. Retinal vein thrombosis often precedes the onset of such life threatening conditions as acute myocardial infarction and stroke. RVO is traditionally seen as imbalance between thrombogenic and antithrombogenic factors. In the recent years, the number of patients with retinal vein occlusion has been increasing, especially among young and working-age individuals with genetic predisposition to thrombosis [3].

Thrombophilia is susceptibility to thrombosis associated with inherited or acquired defects of procoagulant and anticoagulant pathways. Among the risk factors for venous thrombosis are inherited resistance to activated protein C (activated protein C resistance, APCR) and a mutant variant of factor V (factor V Leiden, FV Leiden). Activated protein C breaks down the mutant factor V more slowly than it occurs in healthy individuals, which increases the rate of thrombin production and under certain conditions may lead to thrombosis at any age. The nature of APCR can be genetic associated with FV Leiden or acquired associated with the presence of antiphospholipid antibodies and the effects of oral contraceptives [4, 5, 6].

Antiphospholipid antibodies (APA) alter the homeostatic regulation of blood coagulation. The exact mechanism of thrombosis involving APA (in particular, the lupus anticoagulant, LA) has not been identified yet. The prothrombotic mechanism of APA action is putatively based on the inhibition of endogenous anticoagulant pathways: reduced antithrombotic potential of the vessel wall and impaired activity of natural coagulants cause hypercoagulation [7, 8, 9, 10, 11].

Hemostatic defects and their clinical manifestation in patients with RVO who also have FV Leiden and LA are understudied. The aim of this work was to study some aspects of clinical laboratory testing for these disorders in patients with RVO who have FV Leiden alone or a combination of FV Leiden and LA.

METHODS

The study included 63 male and 87 female patients (a total of 150 individuals or 150 eyes) with retinal vein occlusion. Mean age was 42 ± 10 years. Branch retinal vein occlusion was detected in 78 patients (52 %), central retinal vein thrombosis was detected in 72 patients (48 %); 56 patients had hypertensive disease; 30 patients were diagnosed with coronary artery disease; 15 had varicose veins of the lower extremities. The length of the observation period varied from 2 weeks to 2 years.

The patients were divided into 3 groups. Group 1 included 12 patients with both FV Leiden and LA; group 2 included 11 individuals with FV Leiden; group 3 included 107 patients with RVO and without activated protein C resistance. In group 3, we analyzed data from 30 patients to make sample sizes comparable. The control group included 50 individuals without RVO who had hypertensive disease, no signs of systemic or autoimmune damage to the connective tissue, no coronary artery disease, cancer, or severe chronic infections. Male to female ratio in the control group was 20 : 30.

The clinical diagnosis was made using standard ophthalmic techniques: a visual acuity test, ocular tonometry, perimetry, direct ophthalmoscopy, and a number of specific techniques, such as the examination of the ocular fundus using a Goldmann lens, fundus fluorescein angiography, optical coherence tomography of the retina, computed perimetry.

To study the hemostatic system, automated coagulation screening was performed. A number of measurements were taken, including determination of von Willebrand factor (vWF), antithrombin III, activated protein C, and coagulation factor VIII activities; determination of factor V levels in blood plasma, levels of soluble fibrin monomer complexes (SFMC) and fibrinogen (the Clauss assay); resistance of factor V to activated protein C (APCR index).

The presence of the lupus antigen was detected by venom tests and confirmed by donor plasma and phospholipid tests using reagent kits by Technology-Standard (Russia) and Instrumentation Laboratory (Italy). Six weeks later, the tests were repeated.

Factor V Leiden and other polymorphisms responsible for susceptibility to thrombophilia were detected using real time PCR.

For this study we used the CL4 homeostasis analyzer (Behnk Elektronik, Germany) and a platelet aggregometer (Research and Production Company Biola, Russia). All tests were carried out at the facilities of the Laboratories for Hemostasis and Genetics (Kemerovo Regional Clinical Hospital).

Statistical analysis was performed using Statistica 6.0 software by StatSoft, USA. To describe the groups, we used the mean and the standard error of the mean. The groups were compared using Student’s T-test. Differences were considered statistically significant at p < 0.05.

RESULTS

Of 150 patients with RVO, the lupus anticoagulant was found in 32 (12 %) individuals; 20 of them did not have FV Leiden, while 12 did. There were 11 (7.3 %) patients with RVO who had only FV Leiden. The hemostatic analysis (see the table) demonstrated that SFMC levels in group 2 (mutant FV) and group 1 (FV Leiden + LA) were increased by 20 and 15 %, respectively, compared to group 3 (no FV Leiden or LA). Fibrinogen levels in groups 1 and 2 were by 37.5 and 20.0 % higher than in group 3. In group 1, intravascular platelet activation was more marked than in group 2: the total sum of activated platelets was increased by 16.2 % and the number of platelets involved in aggregation was by 26.3 % higher. Intravascular platelet activity was also increased in group 3, but it was less conspicuous than in patients with FV Leiden and no LA: 10.7 % vs. 16.4 %, respectively. Factor V activity was increased by 25 % in groups 1 and 2, compared to the controls. Groups 1 and 2 demonstrated a statistically significant 2.3- and 2.6-time increase in the activity of coagulation factor VIII, respectively, compared to the controls. Compared to group 3, this activity was by 27 and 30 % higher, respectively. Von Willebrand factor also exhibited increased activity in all three groups of patients with RCO, in contrast to the controls: by 55, 70 and 30 % in groups 1, 2 and 3, respectively. It proves that in retinal vein occlusion, the main role is played by the venous endothelium. However, increased activity of vWF in groups 1 and 2 was more conspicuous compared to group 3: by 16.0 and 23.5 %, respectively.

Protein C activity was more conspicuous in patients from groups 2 and 3 who had RVO in comparison with the controls: by 36.5 and 39.4 %, respectively. APCR was reduced in groups 1 and 2 by 64.0 and 58.9 %, respectively, in comparison with healthy individuals. In group 3 APCR was by 53.6 and 46.4 % higher than in groups 1 and 2, respectively. In patients with FV Leiden, the APCR index value was lower if LA was present.

In all groups, patients with RVO had elevated fibrinogen (inflammation protein) levels in comparison with healthy individuals: they were increased by 56.3, 43.3 and 25.0 % for groups 1, 2 and 3, respectively. In groups 1 and 2 this index was 1.5- and 1.2-times higher, respectively, than in group 3.