Abstract
Background: Coagulation abnormalities have been reported in familial Mediterranean fever (FMF) patients with amyloidosis and nephrotic syndrome; but there is not enough data about the continuity of the thrombogenic activity in FMF patients in clinical remission. The purpose of this study was to assess thrombin activatable fibrinolysis inhibitor (TAFI) levels and its relationship with fibrinolytic activity and also evaluate relationships between mutations and clinical signs in attack-free patients without amyloidosis. Methods: Seventy-nine FMF patients and 40 healthy adults were included. The study group was divided into five groups as follows: first group, homozygote M694V; second group, homozygote M680I; third group, M694V in one allele, the other allele have other mutations or not; fourth group, other mutations; and fifth group, no mutation. Results: Serum TAFI levels were significantly increased in patients compared with healthy individuals (116.64 ± 21.8 vs. 78.48 ± 19.7 μg/mL, p < 0.001) and a positive correlation was detected between TAFI antigen level and erythrocyte sedimentation rate and C-reactive protein levels (r = 0.247, p = 0.029 and r = 0.252, p = 0.032, respectively). Mean fibrinogen and TAFI levels were significantly higher in Group 1 than the other groups (p = 0.04 and p = 0.001, respectively) and in Group 3 it was higher than Groups 2, 4 and 5 (p = 0.04 and p = 0.001, respectively). Conclusions: High level of TAFI antigen in attack-free period of FMF disease shows ongoing subclinical inflammation and hypercoagulability. Clinicians should be careful about thrombosis even in patients at clinical remission. Also, genetic tests must be considered to predict clinical outcome and to reduce complications of FMF disease.
Introduction
Familial Mediterranean fever (FMF) is an autosomal recessive disease which is characterized by acute, self-limiting fever and polyserositis attacks. FMF is the most prevalent periodic fever syndrome, predominately affects people from the Mediterranean basin, including Turks, Arabs, Armenians and Sephardic Jews.Citation1
The responsible gene for FMF is MEFV gene that regulates inflammation by repressing a proinflammatory molecule or transcriptional inducing of an anti-inflammatory process.Citation2 This molecular modification leads to persistence of leukocytes and prevention of apoptosis. Persistence of leukocytes and their capacity to release large amounts of cytokine leads to amplification of the inflammation. During and between the attacks, acute phase reactants, such as C-reactive protein (CRP), IL-1, IL-6, IL-8, and serum amyloid A (SAA) are increased. Some extracellular matrix proteins such as fibronectin and thrombospondin are increased and may flare up the inflammation.Citation3 Since they are also thrombotic factors, inflammation plays an important role on thrombotic complications. Coagulation abnormalities have been reported in FMF patients with amyloidosis and nephrotic syndrome; but there is not enough data about the continuity of the thrombogenic activity in FMF patients who are not exposed to nephrotic syndrome and in clinical remission.
Thrombin activated fibrinolysis inhibitor molecule is a zymogen which is determined as an important molecule at the hemostatic mechanisms. This molecule, activated by thrombin, shows its effect by breaking the arginine and lysine residues of the fibrin polymers from their carboxy terminal. By this way, it inhibits the interaction between plasminogen and fibrin.Citation4 This causes deceleration of fibrinolytic system and leads predisposition to thrombosis.
The aim of this study was to determine thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels and its relationship with fibrinolytic activity in FMF patients without amyloidosis who are at clinical remission under colchicine therapy. Also, we aimed to show relationships between genetic variations and clinical signs.
Subjects and methods
The study was conducted the Turgut Ozal University Medical Faculty, Gazi University Medical Faculty and Ankara Education and Research Hospital out-patient clinics. Seventy-nine consecutive patients diagnosed as FMF according to Tel-Hashomer criteria and 40 healthy controls were included. Informed consent was obtained from each patient and the study was carried out in accordance with the ethical guidelines of the Declaration of Helsinki. The local ethic committee approved the study.
The study group was divided into five groups: first group, homozygote M694V mutation; second group, homozygote M680I mutation; third group, M694V mutation in one allele, the other allele have other mutations or not; fourth group, other mutations (V726A, M680I, R761H, P369S, A744S, K695R); fifth group, patients who did not carry any mutation.
All patients were at clinical remission with an average 1.33 ± 0.36 g/day colchicine treatment. Blood and urine samples of all subjects were collected after an overnight fasting. Biochemical analysis was measured by standard laboratory methods on a biochemistry autoanalyzer (Hitachi 912; Roche, Japan) with the company’s original kits. DNA samples were isolated from peripheral blood materials from each patient. Second, 3rd, 5th and 10th exons of MEFV gene were amplified by polymerized chain reaction (PCR) method. PCR samples were studied for mutation analysis by hybridization method.
Data including creatinine, albumin, fibrinogen concentrations, complete blood count, sedimentation, and CRP were measured by standard methods. Proteinuria was evaluated by urine protein-to-creatinine ratio in morning urine specimens.
TAFI levels were measured by using commercial kits via ELISA method (Imunclone, American Diagnostica Inc., Stamford, CT). Blood samples for TAFI antigen were centrifuged within 30 min of collection at 4 °C for 20 min at 3000 rpm. The supernatant plasma samples obtained were transferred into polypropylene tubes and stored for up to 1 month at −30 °C until assayed.
Burden score of the disease wasalso calculated for each patient with Tel-Hashomer criteria. Patients with scores ≤5 was evaluated as mild disease, 5–10 scores as moderate, >10 as advanced disease.
SPSS for Windows 15.0 (IBM Cooperation, Boston, MA) statistical packet program was used for evaluating the data. Distribution of variables was tested with Shapiro–Eilk test. Parametric tests were used for normally distributing variables, nonparametric tests [ANOVA test (Kruskal–Wallis)] were used for the variables that were not normally distributed. Student’s t test was applied to make comparison between the patient and control group laboratory parameters. For detecting the differences between the groups, Tukey HSD test from the POSTHOC tests was used. The coherence of variables was evaluated using the Pearson correlation test. p Values of less than 0.05 were accepted as statistically significant.
Results
A total of 79 consecutive patients diagnosed according to Tel-Hashomer criteria were included in the study. Results were compared with those of 40 healthy controls. There was no significant difference between patients and controls with regard to age, gender, complete blood count analyses, and routine biochemical parameters (p > 0.05) ().
Table 1. Demographic and laboratory results of patients and controls.
Proteinuria was significantly higher in patients when compared to controls (268.19 ± 74.87 mg/day and 92.9 ± 62.75 mg/day, respectively; p < 0.001). TAFI antigen level was significantly higher in attack-free FMF patients when compared to control subjects (116.64 ± 21.8 vs. 78.48 ± 19; p < 0.001, ) and a positive correlation was detected between TAFI antigen level and erythrocyte sedimentation rate (ESR), CRP levels (r = 0.247, p = 0.029 and r = 0.252, p = 0.032, respectively). Also a positive correlation was found between proteinuria and ESR, fibrinogen, creatinine levels (r = 0.384, p = 0.001; r = 0.391, p = 0.001; r = 0.345, p = 0.002, respectively) and a negative correlation between proteinuria and albumin levels (r = −0648, p = 0.001). Platelet count was positively correlated with the ESR and fibrinogen levels (r = 0.348, p = 0.002 and r = 0.239, p = 0.033, respectively), and negatively correlated with albumin levels (r = −0.383, p = 0.001). There was also a positive correlation between creatinine and fibrinogen levels (r = 337, p = 0.002) ().
Figure 1. Comparison of TAFI between the patient and control groups.
Table 2. Correlation between TAFI and other laboratory parameters.
When genetic mutations were assessed; the most common was M694V/M694V mutation (27 patients, 34.18%). Ten patients were heterozygote for M694V/− mutation (12.66%), seven patients were M680I/M694V heterozygote (8.86%) and six patients were M694V/V726A heterozygote (7.59%). The remaining 21 patients have other mutations (36.58%) and 8 patients did not have any mutation (10.13%). When allele frequency was assessed; M694V mutation was seen in 81 of 158 allele (51.3%), M680I in 19 (12%) and V726A in 12 (7.6%).
When disease burden score was calculated for each patient, most of our patients had moderate disease activity (39%); mild disease activity group was about 37% and 3% of patients had advanced disease according to burden scores. Mean burden score of the disease was the highest in group 1 (6.81 ± 0.57). Burden score of group 1 was significantly higher when compared to group 4 (3.46 ± 1.91; p < 0.01) and group 5 (5.61 ± 2.38; p = 0.001). Clinical condition and burden score according to groups are shown in and and .
Figure 2. Burden scores of patients according to genetic group.
Table 3. Laboratory parameters in genetic groups.
When the genetic groups were compared according to laboratory parameters, mean fibrinogen and TAFI levels were significantly higher in group 1 than the other groups (p = 0.04 and p = 0.001, respectively) and in group 3 it was higher than groups 2, 4 and 5 (p = 0.04 and p = 0.001, respectively) ().
Discussion
In our study, it is shown for the first time that TAFI Ag levels were significantly higher in FMF patients treated with colchicine without amyloidosis when compared to controls and these results were correlated with inflammatory markers. In many FMF patients, inflammation can persist in attack-free periods, as shown by high levels of acute-phase proteins, cytokines and inflammation-induced proteins. This condition may cause the risk of complications such as tendency to thrombosis, anemia, splenomegaly, decreased bone mineral density and life-threatening amyloid A amyloidosis.
Table 4. Clinical features according to genotype.
TAFI is a molecule that has a negative effect on fibrinolysis via plasminogen–plasmin system. There is also a relationship between atherogenesis–thrombogenesis risk and TAFI antigen levels. In animal studies, reperfusion delay in thrombolysis is reported to have association with TAFI antigen levels.Citation5 It is also observed that increased TAFI level is an important risk factor for coronary artery disease and ischemic stroke.Citation6,Citation7
It is known that hypercoagulability increases after amyloidosis and nephrotic syndrome have developed in FMF patients. Our study group without amyloidosis or nephrotic syndrome had higher levels of TAFI antigen that show hypofibrinolytic activity and predisposition to thrombosis, even in attack-free period. There is a study declaring baseline inflammation and hypercoagubility state in FMF patients in clinical remission.Citation8
There may be several mechanisms to explain elevated TAFI levels in attack-free FMF patients. TAFI is expressed as an acute phase protein at animal studies.Citation9 Also, this antigen causes inflammation and coagulation response.Citation10,Citation11 Natural anticoagulation pathways help to reduce inflammation and inflammatory state can trigger thrombosis. Significant correlation between TAFI levels and ESR and CRP levels strengthen this hypothesis. Also it is an established data that elevated TAFI antigen is associated with thrombin–antithrombin complex and thrombomodulin levels which are markers of endothelial cell damage.Citation12 Inflammation mediated endothelial dysfunction also causes increased tendency to thrombosis in patients with FMF disease.Citation13 Endothelial-source TAFI may be increased via endothelial damage and can cause hypercoagubility state.
The type and frequency of mutations in FMF disease may differ in different populations. M694V mutation is the most common form in all ethnic groups. E148Q is a rare genetic mutation.Citation14 In a study by Turkish FMF Study Group, genetic analysis of 1090 FMF patients has revealed that M694V was the most frequent mutation (51.4%), followed by M680I (14.4%) and V726A (8.6%),Citation15 which are consistent with our results.
The first study evaluating phenotype–genotype relationship was performed by Pras et al. in 1997. They have found at least one allele of M694 mutation in 80 of 83 FMF patients, homozygote positivity in 70 patients and 12 of homozygote patients had amyloidosis.Citation16 Also Dewalle et al. has expressed that M694V mutation had a relationship with higher burden score and worse outcome.Citation17 After these first reports, many studies were performed and showed that M694V mutation was associated with beginning of active disease in early ages, worse prognosis, arthritis, and erysipelas-like erythema.Citation18,Citation19 In our study, burden score of patients were higher in M694V homozygote group when compared to patients who did not carry M694V mutations (p = 0.001). Also fibrinogen levels were higher in M694V homozygote group than the other groups (p = 0.04). In the study of Tunca et al., they have observed higher levels of SAA and CRP in FMF patients and their first degree relatives.Citation20 Also, SAA levels were significantly higher in M694V homozygote patients in another report.Citation21 These strengthen the hypothesis that inflammation persists in attack-free period that is significant in M694V carrying patients.
In conclusion, high levels of TAFI antigen in attack-free period of FMF disease were shown ongoing subclinical inflammation and hypercoagulability especially in MEFV homozygote patients. Clinicians should be careful about thrombosis even in patients at clinical remission. As it is expected, the most common mutation was MEFV mutation and burden score was increased apparently in homozygote form. In light of the foregoing, it can be considered that to know the type of mutation and take into consideration in treatment can reduce complications of the disease. Large scale studies also evaluating TAFI level in attack period and in patients with amyloidosis are needed.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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