Factor XIII levels and factor XIII B subunit polymorphisms in patients with venous thromboembolism

Thrombosis Research 158 (2017) 93–97

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Full Length Article

Factor XIII levels and factor XIII B subunit polymorphisms in patients with venous thromboembolism

MARK

Zoltán A. Mezeia, Éva Katonaa, Judit Kállaia, Zsuzsanna Bereczkya, Laura Somodia, Éva Molnára, Bettina Kovácsa,b, Tünde Miklósc, Éva Ajznerc, László Muszbeka,d,⁎ a

Division of Clinical Laboratory Science, Department of Laboratory Medicine, University of Debrecen, Faculty of Medicine, Debrecen, Hungary Borsod-Abaúj-Zemplén County Hospital and University Teaching Hospital, Miskolc, Hungary c András Jósa Szabolcs-Szatmár-Bereg County Hospital and University Teaching Hospital, Nyíregyháza, Hungary d Vascular Biology, Thrombosis and Hemostasis Research Group of the Hungarian Academy of Sciences, University of Debrecen, Hungary b

A R T I C L E I N F O

A B S T R A C T

Keywords: Factor XIII Factor XIII-B subunit Polymorphisms Venous thromboembolism

Background: The association of plasma factor XIII (FXIII) level with venous thromboembolism (VTE) is still controversial and the effect of sex and FXIII B subunit (FXIII-B) polymorphisms in this respect have not been explored. Objectives: 1/ To determine FXIII activity and antigen levels in patients with a history of VTE and how they are influenced by sex and FXIII-B polymorphisms. 2/ To explore the association of FXIII levels and FXIII-B polymorphisms with the risk of VTE. Methods: 218 VTE patients and equal number of age and sex matched controls were enrolled in the study. FXIII activity was measured by ammonia release assay; FXIII-A2B2 and FXIII-B levels were determined by ELISAs. FXIII-B polymorphisms were identified by RT-PCR using melting point analysis. Results: Adjusted FXIII activity and FXIII-A2B2 antigen levels were significantly higher in females with a history of VTE than in the respective controls. FXIII-B levels were significantly lower in male VTE patients than in controls. FXIII-A2B2 antigen levels in the upper tertile increased the risk of VTE in females (adjusted OR: 2.52; CI: 1.18–5.38). Elevated FXIII-B antigen level had a protective effect only in males (adjusted OR: 0.19; CI: 0.08–0.46). FXIII-B Intron K c.1952+ 144 C > G polymorphism significantly lowered FXIII activity, FXIII-A2B2 and FXIII-B antigen levels in both groups. FXIII-B polymorphisms did not influence the risk of VTE. Conclusions: In VTE patients the changes of FXIII level and their effect on the risk of VTE show considerable sexspecific differences. Intron K polymorphism results in decreased FXIII levels, but does not influence the risk of VTE.

1. Introduction Blood coagulation factor XIII (FXIII) is a tetrameric complex (FXIIIA2B2) consisting of two potentially active catalytic A subunits (FXIII-A) and two carrier/inhibitory B subunits (FXIII-B). FXIII-B is in excess; about 50% of it circulates as free non-complexed subunit. The interaction with FXIII-B is highly important for keeping the catalytic FXIII-A dimer in circulation. In plasma FXIII-A2B2 and FXIII-B2 circulate in association with fibrinogen. FXIII is a pro-transglutaminase, it is transformed into an active transglutaminase (FXIIIa) in the final phase of the clotting cascade by thrombin and Ca2 +. FXIIIa cross-links fibrin

γ-, and α-chains and α2 plasmin inhibitor to fibrin. By this mechanism it protects newly formed fibrin from the shear stress of circulating blood and from the degradation by the fibrinolytic enzyme, plasmin (reviewed in references [1–3]). FXIII-A has five common single nucleotide polymorphisms (SNPs) which results in amino acid replacements. Among them, FXIII-A p.Val34Leu (c.103 G > T; SNP ID: rs5985) occurs with the highest frequency in the Caucasian population. The minor allele accelerates the rate of FXIII activation [4–6] and also influences the structure of fibrin network [4]. Two major polymorphisms have been described in the F13B gene. An A to G transversion within exon 3 (c.344G > A; SNP ID:

Abbreviations: BMI, body mass index; CI, confidence interval; DVT, deep vein thrombosis; FII, factor II (prothrombin); FVL, factor V Leiden mutation; FXIII, plasma factor XIII; FXIIIa, activated FXIII; FXIII-A, FXIII A subunit; FXIII-B, FXIII B subunit; OR, odds ratio; PE, pulmonary embolism; VTE, venous thromboembolism ⁎ Corresponding author at: Division of Clinical Laboratory Science, Department of Laboratory Medicine, University of Debrecen, Faculty of Medicine, 98 Nagyerdei Krt., 4032 Debrecen, Hungary. E-mail address: [email protected] (L. Muszbek). http://dx.doi.org/10.1016/j.thromres.2017.08.018 Received 6 March 2017; Received in revised form 30 June 2017; Accepted 25 August 2017 Available online 26 August 2017 0049-3848/ © 2017 Published by Elsevier Ltd.

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rs6003) results in His to Arg amino acid exchange at position 95 in the protein [7]. Arg95 is a relatively rare (9%) among Caucasians, but it represents the major allele (68%) among Black Africans [8]. Recently, a C-to-G change at nucleotide position 29756 in intron K (c.1952 +144 C > G; SNP ID: rs12134960) was described in the F13B gene [9,10]. This polymorphism results in a novel splice acceptor site and consequently in an allele-specific splicing product. In this isoform the last 10 C-terminal amino acids are replaced by an alternative sequence consisting of 25 amino acids. This sequence includes charged amino acid residues, two lysines and one glutamic acid and changes the isoelectric point of the protein. The polymorphism occurs in Asians with high frequency; the allele frequency in the Caucasian population is 17% [8]. Studies focused on the association of FXIII levels and polymorphisms with thrombotic diseases were reviewed in references [11,12]. To our knowledge only two reports concerned FXIII activity and antigen levels in patients with venous thromboembolism (VTE) and their association with thrombosis risk [13,14]. Sex and age related differences were not investigated in these studies. Studies on the effect of FXIII polymorphisms on the risk of VTE mainly concerned FXIII-A Leu34 allele; its moderate protective effect was confirmed in meta-analyses of published reports [15,16]. The association of FXIII-B polymorphisms with VTE was addressed only in a single former study, in which the p.His95Arg polymorphism was found to increase the risk of VTE [7]. The effect of FXIII-B intron K c.1952 + 144 C > G polymorphism on the risk of VTE has not been investigated. The aims of the present case-control study were the following:

Table 1 Laboratory and clinical variables of controls and VTE patients.

Sex (female/male) Age Current smoker (yes/ no) BMI Fibrinogen (g/L) FV Leiden p.Arg506Gln Wild type n Heterozygote n Homozygote n Gln506 carrier frequency Gln506 allele frequency FII 20210 G > A Wild type n Heterozygote n Homozygote n A carrier frequency A allele frequency FXIII activity (%) Non-adjusted Adjusted FXIII-A2B2 antigen (%) Non-adjusted Adjusted FXIII-B antigen (%) Non-adjusted Adjusted

1. To study FXIII activity, FXIII-A2B2 and FXIII-B levels in patients with the history of VTE and to reveal how sex and FXIII-B polymorphism influence FXIII levels in these patients. 2. To reveal if FXIII levels and FXIII-B polymorphisms influence the risk of VTE.

Controls (n = 218)

VTE (n = 218)

Significance (p)

113/105 40 (30–50) 58/157

113/105 40 (30–53) 32/186

0.419 0.002

25.2 (21.8–28.7) 3.41 ± 0.58

29.0 (25.9–32.9) 3.68 ± 0.66

< 0.001 < 0.001

193 25 0 5.7%

144 62 12 19.7%

< 0.001

11.5%

33.9%

< 0.001

210 8 0 1.8% 3.7%

204 14 0 3.2% 6.4%

0.274 0.274

109.3 ± 25.0 112.9 ± 25.0

119.6 ± 27.4 120.3 ± 27.4

< 0.001 0.003

107.1 ± 23.7 108.5 ± 23.7

117.8 ± 27.4 116.4 ± 27.4

< 0.001 0.001

109.3 ± 17.3 112.9 ± 17.3

108.4 ± 22.3 106.9 ± 22.3

0.635 0.003

Variables showing non-normal distribution (age and BMI) are represented by median and interquartile range, while in the case of variables with normal distribution mean ± SD are shown. FXIII activity was adjusted for smoking and fibrinogen concentration, FXIIIA2B2 antigen for fibrinogen concentration, FXIII-B antigen for smoking, BMI and fibrinogen concentration.

2. Methods

concentration. Factor V Leiden mutation (FVL) and prothrombin (FII) 20210 G > A mutation were analyzed by standard molecular genetic methods [21,22]. FXIII-B p.His95Arg and FXIII-B Intron K c.1952 + 144 C > G polymorphism were determined according to protocols developed in our laboratory [23].

2.1. Patients 218 consecutive non-related VTE patients who were admitted to the Thrombosis Center of the University of Debrecen during the year of 2014 were enrolled in the current study, along with the same number of age and sex matched healthy controls (Table 1). Patients with malignant disease were excluded. Deep vein thrombosis (DVT) was confirmed by color Doppler ultrasonography or venography, pulmonary embolism (PE) was diagnosed according to the guidelines of European Society of Cardiology [17]. Blood samples were taken at least 3 months after the acute event. All enrolled individuals were informed about the study according to the study protocol, and gave written informed consent. The study fully complied with the Declaration of Helsinki, ethical approval was obtained from the Regional Ethics Committee at the University of Debrecen, Hungary.

2.3. Statistical analysis Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS 22.0, Chicago IL). The distribution of parameters was examined by the Kolmogorov-Smirnov and Shapiro-Wilk tests. Variables with normal distribution were expressed as mean ± SD, while variables not showing normal distribution were expressed as median and interquartile range. Differences between groups were analyzed by Student's t-test when normally distributed and by Mann-Whitney test when the distribution was non-normal. Multiple linear regression analysis was performed to determine parameters independently associated with FXIII levels. These parameters were used as covariates when the significance of differences in mean FXIII levels was tested by analysis of variance (ANOVA). Bonferroni correction was applied for multiple comparisons. Differences in category frequencies were evaluated by χ2 test. The effect of both polymorphisms and the effect of FXIII levels in the upper tertile were analyzed in logistic regression models and expressed as odds ratio (OR) and 95% confidence interval (CI). Adjusted ORs were obtained by the use of a model that included the respective parameter and all independently associated variables. A p value of < 0.05 was considered as statistically significant.

2.2. Laboratory methods Fasting blood samples were collected from the antecubital vein into vacutainer tubes (Beckton Dickinson, Franklin Lakes, NJ) with anticoagulant (1/10 volume of 0.109 M citrate). Plasma was separated by centrifugation at 1500 g for 20 min, and 500 μL aliquots were stored at − 70 °C until determination. DNA was isolated from the buffy coat of citrated blood samples by QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). FXIII activity was measured by ammonia release assay [18] using REA-chrom FXIII kit (Reanal-ker, Budapest, Hungary). FXIII-A2B2 antigen and FXIII-B antigen concentration was determined by sandwich ELISA as described earlier [19,20]. FXIII activity and antigen levels showed normal distribution both in controls and patients and they were expressed as percentages of the levels measured in pooled normal plasma. The Clauss method was used for the measurement of fibrinogen 94

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3. Results

Table 3 The association of FXIII levels with the risk of VTE.

3.1. Characterization of the study population The sex and age matched control and patient groups consisted of relatively young individuals (median age 40) and the distribution of males and females was close to equal (Table 1). 9 patients had DVT in the upper, 200 patients in the lower extremities. In 62% of the latter group the DVT was of proximal, in 38% it was of distal localization. 53 patients suffered PE, in 9 of them DVT could not be detected. The number of current smokers was significantly higher in the control group (27% versus 15%), which is due to the fact that a number of patients quitted smoking following the thrombotic event. No reliable data could be collected about the former smoking habit of enrolled individuals. As expected the body mass index (BMI) of VTE patients was significantly higher than that of the controls. Plasma fibrinogen level was also significantly elevated in the patient group. Patients and controls were screened for FVL mutation and FII20210A allele (Table 1). The frequency of heterozygous FVL mutation was 11.5% in the control group, which corresponds to the high prevalence of this mutation in the general Hungarian population [24]. No FVL homozygote was found in the control group. 28.4% of VTE patients were heterozygous and 5.5% were homozygous for FVL. As expected the carrier and allele frequencies were significantly higher in the patient group than in controls. Heterozygous occurrence of FII20210A allele was 3.7% and 6.4% among controls and patients, respectively. Due to the relatively low number of A allele carriers the difference did not reach the level of statistical significance. During the enrollment period 6 antithrombin deficient, 3 protein C deficient and 3 protein S deficient VTE patients were diagnosed, but they were not included in the study group.

Elevated FXIII activity non-adjusted Females Males Elevated FXIII activity adjusted Females Males Elevated FXIII-A2B2 antigen non-adjusted Females Males Elevated FXIII-A2B2 antigen adjusted Females Males Elevated FXIII-B antigen non-adjusted Females Males Elevated FXIII-B antigen adjusted Females Males

95% CI

Significance (p)

2.80 1.98

1.43–5.53 1.02–3.84

0.003 0.045

2.27 2.13

0.99–5.16 0.99–4.52

0.052 0.050

3.00 2.53

1.53–5.87 1.28–5.03

0.001 0.008

2.77 2.14

1.26–6.07 0.98–4.66

0.011 0.056

1.48 0.49

0.79–2.75 0.24–0.99

0.221 0.046

0.93 0.23

0.43–2.02 0.09–0.56

0.861 0.001

Individuals with FXIII levels in the upper tertile (> 124.9%, > 122.8%, > 117.0% for FXIII activity, FXIII A2B2 antigen, FXIII-B antigen, respectively) were compared to individuals with FXIII levels in the lowest tertile (< 102.6%, < 99.8%, < 101.1% for FXIII activity, FXIII A2B2 antigen, FXIII-B antigen, respectively). Adjustment was performed for BMI and factor V Leiden carriership.

elevation of FXIII activity and FXIII-A2B2 antigen in VTE patients was more prominent in females, while a highly significant decrease of FXIIIB levels was observed only in male VTE patients. FVL and FII20210A carrierships had no significant effect on FXIII activity and antigen levels in either sex (results are not shown).

3.2. FXIII levels in VTE patients

3.3. The association of FXIII levels with the risk of VTE

FXIII activity and FXIII-A2B2 antigen level were significantly elevated in VTE patients as compared to controls (Table 1). FXIII-B antigen concentration in patients was significantly lower than in controls, but the difference became evident only after adjustment for smoking, BMI and fibrinogen concentration. Table 2 demonstrates that the effect of VTE on FXIII levels considerably differs according to sexes. The

The effect of elevated FXIII activity and antigen levels on the risk of VTE was analyzed in both sexes (Table 3). Individuals with FXIII levels in the upper tertile were compared to those in the lowest tertile and the ORs were calculated. In females elevated non-adjusted FXIII activity and FXIII-A2B2 antigen significantly increased the risk of VTE; ORs were around 3.0. After adjustment the effect became more moderate, and it reached the level of significance only in the case of FXIII-A2B2 antigen (OR: 2.77; CI: 1.26–6.07). In males there was also a tendency of increasing the risk of VTE by the elevation of FXIII activity and FXIII-A2B2 antigen, but the effects became only of borderline significance after adjustment for BMI and FVL carriership. The elevation of FXIII-B antigen levels was without effect on the risk of VTE in females. In contrast, it conferred a significant protective effect on males, which became particularly evident after adjustment.

Table 2 FXIII levels in female and male controls and VTE patients.

Females (n) FXIII activity non-adjusted (%) FXIII activity adjusted (%) FXIII-A2B2 antigen nonadjusted (%) FXIII-A2B2 antigen adjusted (%) FXIII-B antigen non-adjusted (%) FXIII-B antigen adjusted (%) Males (n) FXIII activity non-adjusted (%) FXIII activity adjusted (%) FXIII-A2B2 antigen nonadjusted (%) FXIII-A2B2 antigen adjusted (%) FXIII-B antigen non-adjusted (%) FXIII-B antigen adjusted (%)

OR

Control

VTE

Significance (p)

113 111.5 ± 23.9

113 123.0 ± 25.8

0.001

115.9 ± 23.7 109.4 ± 22.1

125.6 ± 25.8 121.5 ± 26.4

0.004 < 0.001

110.6 ± 22.1

120.3 ± 26.4

0.003

106.3 ± 18.0

109.6 ± 22.6

0.226

109.8 ± 18.0 105 107.0 ± 25.9

109.7 ± 22.5 105 115.9 ± 28.6

0.978 0.020

110.1 ± 26.0 104.7 ± 25.2

115.6 ± 28.6 113.8 ± 28.1

0.137 0.014

106.1 ± 25.2

112.4 ± 28.1

0.087

112.5 ± 16.1

107.1 ± 22.0

0.042

115.8 ± 16.2

105.0 ± 22.2

< 0.001

3.4. The effect of FXIII-B polymorphisms on FXIII levels The effect of two major FXIII-B polymorphisms (p.His95Arg and Intron K c.1952 + 144 C > G) on FXIII activity, FXIII-A2B2 antigen and FXIII-B antigen levels were analyzed in VTE patients and in the control group (Table 4). All three FXIII parameters were elevated in Arg95 carriers, however the extent of elevation reached statistical significance only in the control group and only in the case of FXIII-A2B2 and FXIII-B antigen. The intron K polymorphism has an opposite and more robust effect. Carriership for the c.1952 + 144 G allele resulted in a statistically significant decrease of all FXIII parameters both in controls and in VTE patients and both in non-adjusted and adjusted set-ups. The effect of this polymorphism on the FXIII-B antigen level of VTE patients was somewhat less robust, but statistically still significant.

FXIII activity was adjusted for smoking and fibrinogen concentration, FXIII-A2B2 antigen for fibrinogen concentration and FXIII-B antigen for smoking, BMI and fibrinogen concentration.

95

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Table 4 Effect of FXIII-B p.His95Arg and FXIII-B intron K c.1952 + 144 C > G polymorphisms on FXIII levels in controls and VTE patients. FXIII activity (%)

Controls FXIII-B p.His95Arg Wild type (n = 177) Arg95 carrier (n = 41) Significance (p) FXIII-B intron K c.1952 + 144 C > G Wild type (n = 168) G carrier (n = 50) Significance (p) VTE patients FXIII-B p.His95Arg Wild type (n = 186) Arg95 carrier (n = 32) Significance (p) FXIII-B intron K c.1952 + 144 C > G Wild type (n = 161) G carrier (n = 57) Significance (p)

FXIII-A2B2 antigen (%)

FXIII-B antigen (%)

Non-adjusted

Adjusted

Non-adjusted

Adjusted

Non-adjusted

Adjusted

108.3 ± 24.8 114.0 ± 25.4 0.188

109.3 ± 24.8 116.8 ± 25.4 0.059

105.2 ± 23.0 115.6 ± 25.0 0.011

104.8 ± 23.0 117.5 ± 25.0 0.001

108.1 ± 17.4 114.5 ± 16.3 0.034

108.4 ± 17.5 117.0 ± 16.3 0.001

113.0 ± 25.0 97.1 ± 20.4 < 0.001

114.0 ± 25.1 100.4 ± 20.3 < 0.001

110.4 ± 24.1 96.1 ± 18.6 < 0.001

110.0 ± 24.1 97.6 ± 18.6 < 0.001

112.8 ± 16.3 97.4 ± 15.3 < 0.001

113.2 ± 16.4 100.3 ± 15.3 < 0.001

118.7 ± 27.0 124.7 ± 29.3 0.255

122.0 ± 27.0 126.3 ± 29.3 0.411

116.4 ± 27.2 125.7 ± 28.0 0.078

116.6 ± 27.2 124.5 ± 28.0 0.136

107.5 ± 21.3 113.6 ± 27.0 0.152

108.6 ± 21.5 113.7 ± 27.0 0.251

123.5 ± 26.6 108.4 ± 26.7 < 0.001

126.1 ± 26.6 111.6 ± 26.7 < 0.001

121.7 ± 26.4 106.6 ± 27.6 < 0.001

121.7 ± 26.4 106.8 ± 27.6 < 0.001

110.8 ± 22.9 101.6 ± 19.1 0.007

111.1 ± 23.2 103.6 ± 18.6 0.030

FXIII activity was adjusted for smoking and fibrinogen concentration, FXIII-A2B2 antigen for fibrinogen concentration and FXIII-B antigen for smoking, BMI and fibrinogen concentration.

concentration of FXIII-A in individuals without VTE was unusually high (141 ± 39%), which might be due to certain non-explored conditions that elevated FXIII-A level. For this reason the results of the two studies are not comparable. FXIII-B levels were significantly lower in VTE patients only after adjustment for smoking BMI and fibrinogen. This result suggests that the decrease of FXIII-B antigen level in VTE patients was counteracted by the effect of increased BMI and elevated fibrinogen concentration. Indeed, the latter variables have been shown to elevate FXIII-B level in healthy individuals [25]. The differences between sexes in the effect of VTE on FXIII levels are intriguing. After adjustment the elevation of FXIII activity and FXIII-A2B2 antigen level in VTE patients was significant only in females, while the decrease in FXIII-B level prevailed only in males suffered VTE (Table 2). Further studies are warranted to explore the reasons for such sex specific differences in the relation of VTE with FXIII levels. Results in Table 3 demonstrate that elevated FXIII-A2B2 levels increase the risk of VTE, particularly in females, while elevated FXIII-B levels markedly decrease the risk of VTE in males. Interestingly, elevated FXIII activity and FXIII-A2B2 levels also increased the risk of coronary artery disease and peripheral artery disease in women, but not in men [26,27]. In a previous study the association of highly elevated FXIII activity, FXIII-A and FXIII-B antigens levels (above 90th percentiles measured in the control subjects) with the risk of DVT was investigated [13]. Only FXIII-B concentrations above 90th percentile were associated with a statistically significant slight increase in the risk of DVT. Sex-related differences were not reported in this study. In our control group, just like in a former study on healthy individuals [25], carriership for the FXIII-B Arg95 allele was associated with elevated adjusted FXIII activity, FXIII-A2B2 and FXIII-B levels, while in the VTE group no significant effect of the Arg95 allele was observed (Table 4). In a combined group of patients with coronary artery disease and controls no effect of FXIII-B His95Arg polymorphism on FXIII levels was revealed [7]. In the latter report it was shown that the Arg95 variant was associated with increased dissociation of the FXIII subunits in plasma, however in steady-state condition the interaction of purified FXIII subunits was not affected by the polymorphism. It is interesting that the binding epitope of an anti-FXIII-B monoclonal antibody that prevents the complex formation between the two subunits involves this polymorphic site [28]. A robust effect of FXIII-B intron K c.1952 + 144 C > G polymorphism on all three FXIII parameters was demonstrated in the present study. The presence of G allele significantly decreased FXIII activity, FXIII-A2B2 and FXIII-B levels both

3.5. FXIII-B polymorphisms and the risk of VTE Table 5 demonstrates the number of hetero-, and homozygotes, the carrier and rare allele frequencies in the control and VTE groups. From these values the risk of VTE as represented by OR values was calculated. As expected, the number of homozygotes for either polymorphism was very low in both groups. The frequency of Arg95 and intron K c.1952 +144 G carriers did not differ significantly in the control and VTE groups. Neither of the two FXIII-B polymorphisms conferred significant risk of VTE or exerted a protective effect against VTE.

4. Discussion In our study FXIII activity and FXIII-A2B2 antigen level were significantly higher in patients with the history of VTE than in controls (Table 1). Cushman et al. could not reveal changes in FXIII-A antigen level in VTE patients [14]. However, in their study the mean Table 5 The distribution of FXIII-B p.His95Arg and FXIII-B intron K c.1952 + 144 C > G genotypes in patients and controls and their effect on the risk of VTE.

FXIII-B p.His95Arg Wild type n Heterozygote n Homozygote n Arg95 carrier frequency Arg95 allele frequency OR for Arg95 carriers nonadjusted OR for Arg95 carriers adjusted FXIII-B intron K c.1952 + 144 C>G Wild type n Heterozygote n Homozygote n G carrier frequency G allele frequency OR for G carriers non-adjusted OR for G carriers adjusted

Controls

VTE

177 36 5 18.8% 10.6%

186 31 1 14.7% 7.6% 0.74 (0.45–1.23) 0.56 (0.30–1.05)

168 46 4 22.9% 12.4%

161 56 1 26.1% 13.3% 1.19 (0.77–1.84) 1.29 (0.78–2.14)

Significance (p)

0.249 0.069

0.436 0.325

Adjusted for BMI, fibrinogen concentration and factor V Leiden carriership.

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in controls and VTE patients. Similar results were obtained in the study on healthy individuals [25]. It is very likely that the drastic change on the C-terminal end of FXIII-B is not without functional consequences. The C-terminal 10th sushi domain has been implicated in the binding of FXIII-B to fibrinogen [29]. In any case, the biochemical consequences of the two polymorphic B subunits remain to be elucidated. In our study neither p.His95Arg nor intron K C > G polymorphism exerted any significant effect on the risk of VTE. Only a single previous study concerned the association of p.His95Arg polymorphism with the risk of VTE, according to which the Arg95 variant conferred a moderately increased risk of VTE (OR: 1.5; CI: 1.1–2.0) [7]. No previous study investigated the effect of intron K c.1952 + 144 C > G on the risk of VTE. The findings that decreased FXIII activity and FXIII-A2B2 levels were associated with a protective effect against VTE are seemingly contradictory to the lack of effect of intron K G allele which also decreases these FXIII parameters. However, the fact that intron K polymorphism decreases FXIII levels both in controls and in VTE patients could explain the results. The study has the general limitations of case-control studies. In addition, we failed to analyze the association of FXIII-B polymorphisms with the risk of VTE in females and males separately and the effect of polymorphisms at high fibrinogen level was not evaluated, either. The low number of carriers in these subgroups would have considerably decreased the statistical power compromising the validity of the results. 5. Conclusion FXIII activity and FXIII-A2B2 antigen levels were significantly higher in females with the history of VTE than in the respective controls, while FXIII-B levels were significantly lower in males with VTE than in the control group. FXIII-A2B2 antigen levels in the upper tertile increased the risk of VTE in females; elevated FXIII-B antigen level had a protective effect in males. FXIII-B Intron K c.1952 +144 G allele significantly lowered FXIII activity, FXIII-A2B2 and FXIII-B antigen levels in both the control and VTE groups. The FXIII-B Arg95 allele elevated FXIII levels only in controls. FXIII-B polymorphisms did not influence the risk of VTE. Contribution Z.A.M. performed genetic studies, was involved in study design, data analysis and manuscript preparation, É.K. performed FXIII antigen measurement, J.K. carried out genotyping, Z.B. was involved in designing the study and in interpretation of the results, L.S. and É.M. performed FXIII activity measurements, É.K., Z.B., B.K., É.A. and T.M. collected samples, analyzed data and critically reviewed the manuscript, L.M. was responsible for concept, study design, interpretation of results and writing the manuscript. Disclosures and funding The study was supported by the GINOP-2.3.2-15-2016-00050 project which was co-financed by the European Union and the European Regional Development Fund. Additional support was received from the National Research, Development and Innovation Fund, Hungary (grants K 113097, K 116228, K 120633) and from the Hungarian Academy of Sciences (MTA 11003 TKI417). The authors declare that they have no conflict of interest. References [1] I. Komaromi, Z. Bagoly, L. Muszbek, Factor XIII: novel structural and functional aspects, J. Thromb. Haemost. 9 (1) (2011) 9–20. [2] L. Muszbek, Z. Bereczky, Z. Bagoly, I. Komaromi, E. Katona, Factor XIII: a coagulation factor with multiple plasmatic and cellular functions, Physiol. Rev. 91 (3) (2011) 931–972.

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