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Decreased factor XIII levels in factor XIII A subunit Leu34 homozygous patients with coronary artery disease
Thrombosis Research (2008) 121, 469–476
intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
Decreased factor XIII levels in factor XIII A subunit Leu34 homozygous patients with coronary artery disease Zsuzsanna Bereczky a , Emilia Balogh b , Éva Katona a , István Czuriga b , Levente Kárpáti c , Amir H. Shemirani a , István Édes b , László Muszbek a,c,⁎ a
Clinical Research Center, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary Department of Cardiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary c Haemostasis, Thrombosis and Vascular Biology Research Group of the Hungarian Academy of Sciences at the Medical and Health Science Center, University of Debrecen, Debrecen, Hungary b
Received 9 January 2007; received in revised form 9 May 2007; accepted 22 May 2007 Available online 27 June 2007
KEYWORDS Coronary artery disease; Coronary sclerosis; Factor XIII; Factor XIII V34L polymorphism; Fibrinogen; Myocardial infarction
Abstract Introduction: The effect of factor XIII A subunit (FXIII-A) Val34Leu polymorphism on the risk of coronary artery disease (CAD) has been extensively studied. In this study we investigated how FXIII-A Val34Leu genotypes influence plasma factor XIII levels in patients with coronary sclerosis (CS) and myocardial infarction (MI) and how fibrinogen level modulates this effect. Patients and methods: 955 consecutive patients admitted for coronary angiography were categorized according to the presence or absence of significant CS and the history of MI. The frequency of FXIII-A Val34Leu polymorphism, fibrinogen, FXIII activity and antigen levels were determined. Results and conclusions: CS or MI decreased FXIII levels in patients homozygous for FXIII-A Leu34 allele, but not in heterozygous or wild type patients. In the subgroup of patients with CS, but without the history of MI no significant effect was detected, which suggests that MI has a more prominent role. The specific activity of plasma FXIII was independent of FXIII-A Val34Leu genotype. FXIII and fibrinogen levels significantly correlated in CS+ and MI+ patients. In MI+ patients of Leu/Val or Leu/Leu genotypes and with fibrinogen levels in the lowest quartile, FXIII levels were lower than in the
Abbreviations: ANOVA, analysis of variance; AP-FXIII, factor XIII activation peptide; CAD, coronary artery disease; CS, coronary sclerosis; FXIII, blood coagulation factor XIII; FXIIIa, activated factor XIII; FXIII-A, factor XIII subunit A; FXIII-A2B2, plasma factor XIII; FXIII-B, factor XIII subunit B; MI, myocardial infarction. ⁎ Corresponding author. Clinical Research Center, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary. Tel.: +36 52431956; fax: +36 52340011. E-mail address: [email protected] (L. Muszbek). 0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.05.012
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Z. Bereczky et al. same patient groups, but with higher fibrinogen level. The low-scale continuous activation of blood coagulation in CAD patients could lead to parallel FXIII and fibrinogen consumption. As the same amount of thrombin activates more Leu34 FXIII than Val34 FXIII, increased FXIII consumption might be responsible for the decreased FXIII levels in Leu34 homozygous CAD patients. © 2007 Elsevier Ltd. All rights reserved.
Introduction Blood coagulation factor XIII (FXIII) consists of two potentially active A subunits (FXIII-A) and two inhibitory/carrier B subunits (FXIII-B). In the circulation it is associated with fibrinogen through FXIIIB. FXIII becomes activated in the last phase of the clotting cascade by thrombin and Ca2+. Thrombin cleaves off an activation peptide (AP-FXIII) from the N-terminal end of FXIII-A, then in the presence of Ca2+FXIII-B dissociates and the remaining FXIII-A dimer becomes transformed into an active transglutaminase enzyme (FXIIIa). Transglutaminases catalyze the cross-linking of two polypeptide chains through their glutamine and lysine residues via isopeptide bond. The primary physiological function of FXIIIa is to stabilize fibrin and to protect it from proteolytic degradation by the fibrinolytic system. This task is performed by cross-linking fibrin α-and γ-chains and covalently linking α2 plasmin inhibitor to fibrin. (See reviews in Refs. [1,2].) Val34Leu polymorphism in FXIII-A [3] has been associated with a protective effect against coronary artery disease (CAD) [4–10]. Although, contradictory studies have also been published [11–16], a metaanalysis of the published data demonstrated a moderate, but statistically significant protection by the Leu34 allele [17]. Most recent results suggest that a significant gene–covariate interaction exists between the FXIII-A Val34Leu genotype and fibrinogen levels [18,19] and we have demonstrated that in the high risk Hungarian population the protective effect of Leu34 allele prevails only in the upper quartile of fibrinogen concentration [19]. Part of the biochemical consequences of the polymorphism has been explored. As Val34Leu is located in AP-FXIII, just three amino acid N-terminal from the cleavage site for thrombin, it is not surprising that it influences the rate of thrombin cleavage. The release of AP-FXIII from the Leu34 variant by thrombin proceeds at a significantly higher rate than from the Val34 variant [20–22]. The higher rate of proteolytic truncation of Leu34 FXIII-A results in earlier activation of FXIII and consequently, in accelerated cross-linking of fibrin chains and the cross-linking of α2-PI to fibrin [20–23]. It has been demonstrated that FXIII-AVal34Leu polymorphism also influences the structure of fibrin, probably through
the alteration of fibrin cross-linking kinetic [22,24,25]. However, it is hard to link these mechanisms causally to the protective effect of this polymorphism. The relationship between CAD and factor XIII levels in patients of different FXIII-A Val34Leu genotypes has not been explored. In this study involving a large patient population we investigated how FXIII-A Val34Leu genotypes influence plasma factor XIII levels in patients with coronary sclerosis (CS) and myocardial infarction (MI) and it was also tested if such an effect is modulated by fibrinogen concentration.
Subjects and methods Subjects One thousand and ten consecutive patients admitted for coronary angiography at the Department of Cardiology, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary to investigate suspected CAD were recruited to the study. All enrolled participants had been informed about the study according to the study protocol, and thereafter gave written informed consent. Ethical approval for the study was obtained from the Ethics Committee of the Medical and Health Science Center, University of Debrecen, Hungary. Patients with the presence of ≥ 50% stenosis in at least one of the major coronary arteries or in one of their branches were graded as CS+. The diagnosis of MI (MI+) was established at the time of its onset according to the criteria of the American College of Cardiology and the European Society of Cardiology. Patients without significant CS and without the history of MI were considered as clinical controls (CC). Due to uncertainty of the diagnosis of MI or the lack of the required laboratory parameters, 55 patients were excluded from the study. Detailed characterization of the patients has been provided elsewhere [19].
Laboratory methods Serum cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride, apo AI, apo B, lipoprotein (a) and Creactive protein were determined by routine laboratory methods. Plasma fibrinogen level was measured by a modified Clauss method. Plasma homocysteine
Decreased factor XIII levels in XIII A subunit Leu34 homozygous pts with coronary artery disease concentration was determined by fluorescence polarization immunoassay. Plasma FXIII activity was measured according to Kárpáti et al. [26] using the REA-chrom FXIII assay kit (Reanal-ker, Budapest, Hungary) and the results were expressed as percentage of average normal. When specific activities were calculated FXIII activities expressed as U/L were used for the calculations. The determination of FXIII-A2B2 complex concentration in the plasma was carried out by a one step sandwich enzyme-linked immunosorbent assay (R-ELISA FXIII; Reanal-ker) [27]. To eliminate the influence of acute phase reaction and short-term anticoagulant therapy after the acute coronary event, at least three months were allowed to elapse between the onset of MI and the time obtaining blood samples for laboratory analysis. DNA was isolated from the buffy coat of citrated blood samples by QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). FXIII-A Val34Leu polymorphism was determined by real time PCR with fluorescence resonance energy transfer detection and melting curve analysis [28] on a LightCycler equipment (Roche Diagnostics, Mannheim, Germany).
Statistical analysis As the distribution of fibrinogen level was found nonGaussian the results were expressed as median levels and interquartile ranges. After logarithmic transformation the distribution became normal and the differences in fibrinogen level among the patient groups and FXIII-AVal34Leu genotypes were evaluated by Student's t test. Spearman's correlation coefficients were used to analyze the correlation between FXIII levels and different variables. A multiple linear regression analysis demonstrated that plasma fibrinogen level, gender, current smoking, serum cholesterol level were independently associated with FXIII activity and antigen concentrations and FXIII levels were adjusted for these parameters. Obviously, when the effect of fibrinogen level was studied this parameter was not used for adjustment. One-way Table 1
Val/Val Val/Leu Leu/Leu
ANOVA was used to identify any relationship between FXIII-A Val34Leu genotype and FXIII levels. When oneway ANOVA indicated a significant difference, posthoc pair-wise comparisons were made using the LSD test. A p-value of 0.05 or less was considered to indicate statistical significance. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS 11.5, Chicago, IL).
Results Adjusted FXIII activity and antigen levels of individuals with different FXIII-A Val34Leu genotype are summarized in Table 1. The distribution of FXIII-A Val34Leu genotype in the CC, CS+ and MI+ groups did not differ significantly from each other and from a representative population control group of 1146 Hungarian individuals (Val/Val: 54.9%; Val/Leu: 38.4%, Leu/Leu: 6.7%) [19]. The genotype distributions were in the Hardy–Weinberg equilibrium. Reference intervals for FXIII activity and FXIII-A2B2 antigen in healthy controls have been established by our laboratory [26,27]. As reported elsewhere, neither FXIII activity nor FXIII antigen level of CC group differed significantly from those measured in the healthy reference population [29]. Although there was a slight tendency of decreasing FXIII activity and antigen with increasing number of Leu alleles in the CC group, the differences among FXIII-A genotypes were not significant. In contrast, FXIII activity and antigen level in CS+ patients homozygous for FXIII-A Leu34 allele was significantly (10%) lower than in CS+ patients with Val/Val genotype. The difference between FXIII levels in wild type (Val/Val) MI+ patients and those homozygous for FXIII-A Leu34 allele was quite considerable, almost 20% lower in the latter group. In this patient group the difference between Val/Leu heterozygotes and Leu/Leu homozygotes was also statistically significant. The CS+ group contained patients with and without the history of MI. To find out if the decrease of FXIII levels in Leu/
The effect of FXIII-A Val34Leu genotypes on FXIII levels in different patient groups
Val34Leu genotype CC (n = 302) Val/Val Val/Leu Leu/Leu
471
FXIII activity (%) 105 (101–109) 102 (97–106) 100 (91–109) FXIII antigen (mg/L) 23.1 (21.1–24.1) 22.8 (21.7–23.8) 21.8 (19.8–23.9)
CS+ (n = 619) n 167 (55.3%) 114 (37.7%) 21 (7.0%) 167 (55.3%) 114 (37.7%) 21 (7.0%)
FXIII activity (%) 104 (100–107) 103 (99–107) 94 (86–103)⁎ FXIII antigen (mg/L) 23.2 (22.4–24.1) 22.7 (21.8–23.6) 20.9 (19.0–22.9)⁎
MI+ (n = 341) n 345 (55.7%) 235 (38.0%) 39 (6.3%) 345 (55.7%) 235 (38.0%) 39 (6.3%)
FXIII activity (%) 107 (102–112) 106 (101–111) 88 (76–99)⁎† FXIII antigen (mg/L) 24.0 (22.8–25.1) 23.2 (22.0–24.5) 20.3 (17.6–23.0) ⁎†
n 191 (56.0%) 131 (38.4%) 19 (5.6%) 191 (56.0%) 131 (38.4%) 19 (5.6%)
CC: clinical controls; CS+: patients with coronary sclerosis; MI+: patients with the history of myocardial infarction. FXIII levels were adjusted for gender, smoking, cholesterol and fibrinogen. Values in parenthesis represent 95% confidence interval. ⁎p b 0.05 Leu/Leu versus Val/Val, †p b 0.05 Leu/Leu versus Val/Leu.
472 Table 2
Z. Bereczky et al. The effect of FXIII-A Val34Leu genotypes on the specific activity of FXIIIa in different patient groups
Val34Leu genotype CC (n = 302) Val/Val Val/Leu Leu/Leu
CS+ (n = 619)
MI+ (n = 341)
FXIII specific activity (U/mg) n FXIII specific activity (U/mg) n FXIII specific activity (U/mg) n 7.19 (7.06–7.31) 167 7.25 (7.16–7.34) 345 7.22 (7.10–7.34) 191 7.00 (6.88–7.13) 114 7.27 (7.17–7.38) 235 7.30 (7.16–7.45) 131 7.12 (6.61–7.63) 21 7.32 (7.03–7.61) 39 7.03 (6.56–7.49) 19
CC: clinical controls; CS+: patients with coronary sclerosis; MI+: patients with the history of myocardial infarction. One unit (U) enzyme activity represents 1 μmol substrate conversion per minute. Values in parenthesis represent 95% confidence interval.
Leu homozygotes was also evident in CS+ patients without the history of MI this group of patients (n = 312) were separately assessed. FXIII activities of 103%, 101% and 97% were found in the Val/Val (n = 169), Val/Leu (n = 123) and Leu/Leu (n = 23) groups, respectively. The corresponding antigen levels were 22.9 mg/L, 22.5 mg/L and 20.9 mg/L. Although there was a tendency of decrease with increasing number of Leu34 allele, the differences were not statistically significant and did not seem to differ from those observed in the CC group. There was a small subgroup (n = 34) of patients with the history of MI, but without significant coronary sclerosis. In this subgroup rupture of small plaques and/or coronary vasospasm must have been responsible for the previous MI. The small number of such patients (there were only three Leu34 homozygotes in this subgroup) excluded the possibility of any meaningful statistical evaluation. Theoretically, the decrease of FXIII activity could be due to the decrease in the concentration of FXIII molecules or to the decreased activity of the variant factor. The parallel decrease of FXIII activity and antigen levels support the former possibility. To give a more correct assessment of the problem, specific FXIIIa activities, i.e. activities of a given amount of FXIII protein, were calculated (Table 2). Practically identical specific FXIIIa activities were obtained in all patient groups with any FXIII-A genotypes which demonstrate that the decreased FXIII activity in the Leu/Leu patient groups was due to the decrease of FXIII concentration. As FXIII is closely associated with fibrinogen and the clinical role of FXIII-A Val34Leu polymorphism has been related to fibrinogen levels, the effect of fibrinogen concentration on FXIII levels was studied in different patient groups with different FXIII Val34Leu genotypes. Fibrinogen levels in the clinical control, CS+ and MI+ groups were 3.87 g/L (3.21– 4.43), 3.95 g/L (3.28–4.73) and 4.02 g/L (3.20–4.98), respectively. Although the median values were slightly higher in the CS+ and MI+ groups than in clinical controls, the differences did not reach the level of statistical significance. The fibrinogen levels of wild type, Leu34 heterozygote, and Leu34 homozygote patients were 3.91 g/L (3.23–4.64), 3.92 (3.25–4.63) and 3.54 (3.24–4.43), respectively. The differences were not statistically significant. It has
been shown by Ariens et al. that FXIII activity correlated significantly with fibrinogen levels in healthy individuals [30]. Table 3 demonstrates the relation between fibrinogen and FXIII activity or antigen levels in the patient groups and in groups of FXIII-A Val34Leu genotypes. With the exception of clinical control group, statistically significant correlations were found between FXIII and fibrinogen levels. To test the effect of fibrinogen level on FXIII activity and antigen in the different patient groups and in patients with different FXIII-A Val34Leu genotypes patients with fibrinogen level in the lowest quartile (≤3.23 g/L plasma fibrinogen; low fibrinogen group) were compared with the rest of the patients (high fibrinogen group). In the clinical control group there was no significant difference in FXIII levels between individuals with low and high fibrinogen (Table 4). In the CS+ and MI+ groups in the low fibrinogen group significantly lower FXIII levels were observed. The tendency of decreasing FXIII level with increasing number of Leu34 allele, as already shown in Table 1, was also observed within both the high and low fibrinogen groups. Due to the small number of patients in the Leu/Leu group, statistically significant difference in FXIII activity was observed only when of Leu34 homozygous MI+ patients with low fibrinogen levels and their wild
Table 3 Correlations of fibrinogen levels with FXIII activity and antigen levels in different patient groups and FXIII-A Val34Leu genotypes Patient groups
CC (n = 302)
CS+ (n = 619)
MI+ (n = 341)
FXIII activity FXIII antigen
0.015 (p = 0.797) − 0.026 (p = 0.659)
0.243 (p b 0.001) 0.188 (p = 0.001)
0.176 (p b 0.001) 0.162 (p b 0.001)
Val34Leu genotype
Val/Val (n = 539)
Val/Leu (n = 373)
Leu/Leu (n = 64)
FXIII activity FXIII antigen
0.124 (p = 0.008) 0.068 (p = 0.144)
0.133 (p = 0.016) 0.158 (p = 0.004)
0.262 (p = 0.058) 0.085 (p = 0.543)
Spearman's correlation coefficients are shown in the Table. CC: clinical controls; CS+: patients with coronary sclerosis; MI+: patients with the history of myocardial infarction.
Decreased factor XIII levels in XIII A subunit Leu34 homozygous pts with coronary artery disease
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Table 4 The effect of FXIII-A Val34Leu genotypes and fibrinogen levels on the FXIII activity and antigen in different patient groups CC Val34Leu genotype Val/Val
Val/Leu
Leu/Leu
Total undivided
Fibrinogen FXIII activity (%) High 105 (101–110) Low 104 (98–111) High 100 (95–105) Low 107 (99–115) High 101 (91–110) Low 97 (68–126) High 103 (99–106) Low 105 (100–111)
CS+ FXIII antigen (mg/L) 23.0 (21.9–24.0) 23.6 (22.0–25.1) 22.4 (21.2–23.5) 24.0 (22.1–25.9) 21.8 (19.6–24.0) 22.4 (15.8–29.0) 22.6 (21.8–23.5) 23.7 (22.4–24.9)
n 115 45 81 29 19 2 215 76
MI+
FXIII activity (%) 105 (101–109) 101 (95–107) 105 (100–109) 97⁎ (90–104) 97 (87–107) 88 (73–102) 104 (101–107) 98⁎ (93–103)
FXIII antigen (mg/L) 23.4 (22.6–24.3) 22.6 (21.2–24.1) 23.2 (22.2–24.1) 21.0⁎ (19.4–22.6) 20.9† (18.6–23.3) 20.7 (17.3–24.0) 23.2 (22.4–23.9) 21.8⁎ (20.6–22.9)
n 217 62 149 46 19 9 385 117
FXIII activity (%) 109 (103–114) 102 (93–110) 108 (102–114) 99⁎ (90–107) 96 (83–110) 71⁎ § (52–91) 108 (103–112) 98# (22–105)
FXIII antigen (mg/L) 24.3 (23.1–25.4) 23.0 (21.0–25.0) 23.7 (22.4–25.1) 21.5⁎ (19.4–23.5) 21.3 (18.1–24.5) 18.3 (13.7–23.0) 23.9 (22.8–24.9) 21.9# (20.3–23.5)
n 120 37 83 31 10 5 213 73
CC: clinical controls; CS+: patients with coronary sclerosis; MI+: patients with the history of myocardial infarction. FXIII levels were adjusted for gender, smoking and cholesterol. Values in parenthesis represent 95% confidence interval. Patients with fibrinogen level in the lowest quartile (low fibrinogen) were compared to the rest of the patients (high fibrinogen). ⁎p b 0.05, #p b 0.01 low versus high fibrinogen group within the same genotype or within the undivided group; †p b 0.05 Leu/Leu versus Val/Val genotype in the same (low or high) fibrinogen group; §p b 0.01 Leu/Leu versus Val/Leu in the same (low or high) fibrinogen group.
type counterparts were compared. The difference in FXIII antigen level between Leu34 homozygous and wild type CS+ patients with high fibrinogen levels was also significant. The effect of plasma fibrinogen concentration on FXIII levels was also compared within the same Val34Leu genotypes. In the CS+ and MI+ groups, but not in clinical controls there was a clear tendency of parallel decrease of fibrinogen concentrations and FXIII levels. Differences reached statistical significance in the CS+ and MI+ heterozygous and in the MI+ homozygous groups. The lowest FXIII activity and antigen levels were measured in the MI+ Leu34 homozygous group with low fibrinogen concentration. It is interesting that the frequency of patients homozygous for the Leu34 allele and having fibrinogen concentration in the lowest quartile is higher in the CS+ (1.79%) and in the MI+ (1.75%) groups than in the clinical control group (0.91%). However, the low number of Leu34 homozygous patients with fibrinogen in the lowest quartile (altogether 16 individuals) makes hardly possible to perform a meaningful statistical evaluation of the combined effect of Leu34 homozygosity and low fibrinogen on the risk of CS or MI.
Discussion Due to methodological differences, there is some confusion in the literature concerning plasma FXIII activity in individuals of different FXIII-A Val34Leu
genotypes. In assays using low thrombin concentration and/or short incubation with thrombin FXIII is only partially activated, and the extent of FXIII activation depends on the rate of proteolytic activation. As the rate of the release of AP-FXIII from the Leu34 variant by thrombin is about 2.5fold higher than its release from the Val34 variant, using assays measuring FXIIIa at the early stage of thrombin activation higher FXIII activities were measured in individuals possessing the Leu34 allele in heterozygous form than in wild type individuals and even higher in Leu34 homozygotes [31,32]. The activity results measured by such an assay show poor correlation with results of FXIII antigen assays [33]. In contrast, at full activation of FXIII by thrombin the differences in FXIII activity among FXIII-A Val34Leu genotypes disappear and there are no Val34Leu genotype-dependent differences in the specific activities of FXIIIa [20–22]. For a review on FXIII assay methods and their relation to FXIII-A Val34Leu polymorphism see reference [34]. In this study we used a FXIII assay which measures the activity of fully activated FXIII, the results obtained with this assay reflect the catalytic concentration and show good correlation with estimated plasma FXIII antigen values [26]. The few studies that investigated the effect of coronary artery disease on plasma FXIII antigen levels resulted in mainly negative outcome. No differences in FXIII antigen level were found when patients suffering from coronary artery disease (proven by
474 angiography) were compared with clinical controls (normal angiography) [35]. Comparison of FXIII-A2B2 level in patients with coronary artery disease and in a community control group also failed to detect differences [36]. The history of MI had no influence on plasma FXIII-A antigen level [33,37], only in a small prospective study (n = 63) had patients, who developed MI, lower FXIII-A level at recruitment [38]. In our most recent study, utilizing the same patient populations as the present study, neither the presence of CS nor previous MI influenced the adjusted plasma FXIII activity and FXIII-A2B2 antigen levels significantly. Only in females with a history of MI could a slight increase in both FXIII activity and antigen levels be detected [29]. Here we investigated the influence of CS and MI on FXIII levels in patients with different FXIII-A Val34Leu genotype. It was surprising to detect a genotype specific effect and to reveal statistically significant decrease of both FXIII activity and antigen levels in CS+ and MI+ patients homozygous for the Leu34 allele. It is to be stressed that neither the homozygous mutation alone nor the presence of the disease in non-homozygous mutants or wild type patients altered the level of FXIII significantly. Their concomitant presence was required to decrease FXIII activity and antigen concentration. It was also shown that the history of MI exerted a more considerable effect on the FXIII levels than CS without MI; in the latter case only a tendency of decrease could be detected. From the parallel decrease of FXIII antigen and activity and from the practically identical FXIIIa specific activities in the different patient and genotype groups it became evident that the Val34Leu mutation did not influence the activity of FXIIIa, but the concentration of FXIII was decreased in the Leu34 homozygous CS+ and MI+ groups. The reason for the FXIII-A Val34Leu genotypespecific decrease of FXIII level in the patient groups is not clear. For the time being, the following working hypothesis can be offered. Persistent elevation of prothrombin fragment 1 + 2, thrombin– antithrombin III complex and fibrinopeptide A levels has been reported in CS+, and particularly in MI+ patients [39–42]. The elevation of these markers of thrombin formation suggests a continuous low-scale activation of blood coagulation that leads to the parallel consumption of FXIII and fibrinogen. In addition, the formation of fibrin and the activation of FXIII are intimately associated, fibrin as a cofactor is required for the rapid activation of plasma FXIII [43,44]. Although such activation of blood coagulation occurs independently of FXIII genotypes, its effect on FXIII levels could well be FXIII-A Val34Leu genotype-dependent. We have shown that lower
Z. Bereczky et al. amount of thrombin was required to activate FXIII of Leu/Leu genotype than wild type FXIII [20,21]. It seems logical that the same amount of thrombin formed during the coarse of such low-scale activation of blood coagulation activates higher amount of Leu/Leu FXIII. As FXIIIa is quickly removed from the circulation, such mechanism could lead to decreased FXIII levels in Leu/Leu homozygotes. The interaction among plasma fibrinogen concentration and FXIII-AVal34Leu polymorphism and plasma FXIII levels in affecting the risk of CAD is an intriguing, but not fully clarified question. The effect of FXIII-A Val34Leu polymorphism on the risk of CAD, just like its effect on fibrin polymerization [24], seems to be fibrinogen concentration dependent. In the study population the protective effect of Leu34 allele could be demonstrated only at high fibrinogen level [19]. In a recent study Leu34 homozygosity conferred an almost 3-fold increased risk of CAD to patients with low fibrinogen level, while at high fibrinogen it exerted a protective, although statistically not significant, effect [18]. We have demonstrated that in women increased FXIII level represented a significant risk of MI [29] and the effect was independent of fibrinogen level. In the present study the limited number of Leu34 homozygotes with low fibrinogen concentration did not allow an appropriate risk analysis for CAD. The fact that those homozygous for the Leu34 allele and with low fibrinogen were more common among patients with CS and MI than among clinical controls and showed the lowest FXIII levels is suggestive for further investigations involving higher number of patients.
Acknowledgments This work was supported by grants from the Hungarian National Research Fund (OTKA-NKTH NI69238), from the Hungarian Academy of Sciences (MTA 11003, 2006TKI227) from the Hungarian Ministry of Health (ETT 406/2006) and from the National Office of Research and Technology (NKTH RET-06/ 2004).
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intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
Decreased factor XIII levels in factor XIII A subunit Leu34 homozygous patients with coronary artery disease Zsuzsanna Bereczky a , Emilia Balogh b , Éva Katona a , István Czuriga b , Levente Kárpáti c , Amir H. Shemirani a , István Édes b , László Muszbek a,c,⁎ a
Clinical Research Center, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary Department of Cardiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary c Haemostasis, Thrombosis and Vascular Biology Research Group of the Hungarian Academy of Sciences at the Medical and Health Science Center, University of Debrecen, Debrecen, Hungary b
Received 9 January 2007; received in revised form 9 May 2007; accepted 22 May 2007 Available online 27 June 2007
KEYWORDS Coronary artery disease; Coronary sclerosis; Factor XIII; Factor XIII V34L polymorphism; Fibrinogen; Myocardial infarction
Abstract Introduction: The effect of factor XIII A subunit (FXIII-A) Val34Leu polymorphism on the risk of coronary artery disease (CAD) has been extensively studied. In this study we investigated how FXIII-A Val34Leu genotypes influence plasma factor XIII levels in patients with coronary sclerosis (CS) and myocardial infarction (MI) and how fibrinogen level modulates this effect. Patients and methods: 955 consecutive patients admitted for coronary angiography were categorized according to the presence or absence of significant CS and the history of MI. The frequency of FXIII-A Val34Leu polymorphism, fibrinogen, FXIII activity and antigen levels were determined. Results and conclusions: CS or MI decreased FXIII levels in patients homozygous for FXIII-A Leu34 allele, but not in heterozygous or wild type patients. In the subgroup of patients with CS, but without the history of MI no significant effect was detected, which suggests that MI has a more prominent role. The specific activity of plasma FXIII was independent of FXIII-A Val34Leu genotype. FXIII and fibrinogen levels significantly correlated in CS+ and MI+ patients. In MI+ patients of Leu/Val or Leu/Leu genotypes and with fibrinogen levels in the lowest quartile, FXIII levels were lower than in the
Abbreviations: ANOVA, analysis of variance; AP-FXIII, factor XIII activation peptide; CAD, coronary artery disease; CS, coronary sclerosis; FXIII, blood coagulation factor XIII; FXIIIa, activated factor XIII; FXIII-A, factor XIII subunit A; FXIII-A2B2, plasma factor XIII; FXIII-B, factor XIII subunit B; MI, myocardial infarction. ⁎ Corresponding author. Clinical Research Center, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary. Tel.: +36 52431956; fax: +36 52340011. E-mail address: [email protected] (L. Muszbek). 0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.05.012
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Z. Bereczky et al. same patient groups, but with higher fibrinogen level. The low-scale continuous activation of blood coagulation in CAD patients could lead to parallel FXIII and fibrinogen consumption. As the same amount of thrombin activates more Leu34 FXIII than Val34 FXIII, increased FXIII consumption might be responsible for the decreased FXIII levels in Leu34 homozygous CAD patients. © 2007 Elsevier Ltd. All rights reserved.
Introduction Blood coagulation factor XIII (FXIII) consists of two potentially active A subunits (FXIII-A) and two inhibitory/carrier B subunits (FXIII-B). In the circulation it is associated with fibrinogen through FXIIIB. FXIII becomes activated in the last phase of the clotting cascade by thrombin and Ca2+. Thrombin cleaves off an activation peptide (AP-FXIII) from the N-terminal end of FXIII-A, then in the presence of Ca2+FXIII-B dissociates and the remaining FXIII-A dimer becomes transformed into an active transglutaminase enzyme (FXIIIa). Transglutaminases catalyze the cross-linking of two polypeptide chains through their glutamine and lysine residues via isopeptide bond. The primary physiological function of FXIIIa is to stabilize fibrin and to protect it from proteolytic degradation by the fibrinolytic system. This task is performed by cross-linking fibrin α-and γ-chains and covalently linking α2 plasmin inhibitor to fibrin. (See reviews in Refs. [1,2].) Val34Leu polymorphism in FXIII-A [3] has been associated with a protective effect against coronary artery disease (CAD) [4–10]. Although, contradictory studies have also been published [11–16], a metaanalysis of the published data demonstrated a moderate, but statistically significant protection by the Leu34 allele [17]. Most recent results suggest that a significant gene–covariate interaction exists between the FXIII-A Val34Leu genotype and fibrinogen levels [18,19] and we have demonstrated that in the high risk Hungarian population the protective effect of Leu34 allele prevails only in the upper quartile of fibrinogen concentration [19]. Part of the biochemical consequences of the polymorphism has been explored. As Val34Leu is located in AP-FXIII, just three amino acid N-terminal from the cleavage site for thrombin, it is not surprising that it influences the rate of thrombin cleavage. The release of AP-FXIII from the Leu34 variant by thrombin proceeds at a significantly higher rate than from the Val34 variant [20–22]. The higher rate of proteolytic truncation of Leu34 FXIII-A results in earlier activation of FXIII and consequently, in accelerated cross-linking of fibrin chains and the cross-linking of α2-PI to fibrin [20–23]. It has been demonstrated that FXIII-AVal34Leu polymorphism also influences the structure of fibrin, probably through
the alteration of fibrin cross-linking kinetic [22,24,25]. However, it is hard to link these mechanisms causally to the protective effect of this polymorphism. The relationship between CAD and factor XIII levels in patients of different FXIII-A Val34Leu genotypes has not been explored. In this study involving a large patient population we investigated how FXIII-A Val34Leu genotypes influence plasma factor XIII levels in patients with coronary sclerosis (CS) and myocardial infarction (MI) and it was also tested if such an effect is modulated by fibrinogen concentration.
Subjects and methods Subjects One thousand and ten consecutive patients admitted for coronary angiography at the Department of Cardiology, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary to investigate suspected CAD were recruited to the study. All enrolled participants had been informed about the study according to the study protocol, and thereafter gave written informed consent. Ethical approval for the study was obtained from the Ethics Committee of the Medical and Health Science Center, University of Debrecen, Hungary. Patients with the presence of ≥ 50% stenosis in at least one of the major coronary arteries or in one of their branches were graded as CS+. The diagnosis of MI (MI+) was established at the time of its onset according to the criteria of the American College of Cardiology and the European Society of Cardiology. Patients without significant CS and without the history of MI were considered as clinical controls (CC). Due to uncertainty of the diagnosis of MI or the lack of the required laboratory parameters, 55 patients were excluded from the study. Detailed characterization of the patients has been provided elsewhere [19].
Laboratory methods Serum cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride, apo AI, apo B, lipoprotein (a) and Creactive protein were determined by routine laboratory methods. Plasma fibrinogen level was measured by a modified Clauss method. Plasma homocysteine
Decreased factor XIII levels in XIII A subunit Leu34 homozygous pts with coronary artery disease concentration was determined by fluorescence polarization immunoassay. Plasma FXIII activity was measured according to Kárpáti et al. [26] using the REA-chrom FXIII assay kit (Reanal-ker, Budapest, Hungary) and the results were expressed as percentage of average normal. When specific activities were calculated FXIII activities expressed as U/L were used for the calculations. The determination of FXIII-A2B2 complex concentration in the plasma was carried out by a one step sandwich enzyme-linked immunosorbent assay (R-ELISA FXIII; Reanal-ker) [27]. To eliminate the influence of acute phase reaction and short-term anticoagulant therapy after the acute coronary event, at least three months were allowed to elapse between the onset of MI and the time obtaining blood samples for laboratory analysis. DNA was isolated from the buffy coat of citrated blood samples by QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). FXIII-A Val34Leu polymorphism was determined by real time PCR with fluorescence resonance energy transfer detection and melting curve analysis [28] on a LightCycler equipment (Roche Diagnostics, Mannheim, Germany).
Statistical analysis As the distribution of fibrinogen level was found nonGaussian the results were expressed as median levels and interquartile ranges. After logarithmic transformation the distribution became normal and the differences in fibrinogen level among the patient groups and FXIII-AVal34Leu genotypes were evaluated by Student's t test. Spearman's correlation coefficients were used to analyze the correlation between FXIII levels and different variables. A multiple linear regression analysis demonstrated that plasma fibrinogen level, gender, current smoking, serum cholesterol level were independently associated with FXIII activity and antigen concentrations and FXIII levels were adjusted for these parameters. Obviously, when the effect of fibrinogen level was studied this parameter was not used for adjustment. One-way Table 1
Val/Val Val/Leu Leu/Leu
ANOVA was used to identify any relationship between FXIII-A Val34Leu genotype and FXIII levels. When oneway ANOVA indicated a significant difference, posthoc pair-wise comparisons were made using the LSD test. A p-value of 0.05 or less was considered to indicate statistical significance. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS 11.5, Chicago, IL).
Results Adjusted FXIII activity and antigen levels of individuals with different FXIII-A Val34Leu genotype are summarized in Table 1. The distribution of FXIII-A Val34Leu genotype in the CC, CS+ and MI+ groups did not differ significantly from each other and from a representative population control group of 1146 Hungarian individuals (Val/Val: 54.9%; Val/Leu: 38.4%, Leu/Leu: 6.7%) [19]. The genotype distributions were in the Hardy–Weinberg equilibrium. Reference intervals for FXIII activity and FXIII-A2B2 antigen in healthy controls have been established by our laboratory [26,27]. As reported elsewhere, neither FXIII activity nor FXIII antigen level of CC group differed significantly from those measured in the healthy reference population [29]. Although there was a slight tendency of decreasing FXIII activity and antigen with increasing number of Leu alleles in the CC group, the differences among FXIII-A genotypes were not significant. In contrast, FXIII activity and antigen level in CS+ patients homozygous for FXIII-A Leu34 allele was significantly (10%) lower than in CS+ patients with Val/Val genotype. The difference between FXIII levels in wild type (Val/Val) MI+ patients and those homozygous for FXIII-A Leu34 allele was quite considerable, almost 20% lower in the latter group. In this patient group the difference between Val/Leu heterozygotes and Leu/Leu homozygotes was also statistically significant. The CS+ group contained patients with and without the history of MI. To find out if the decrease of FXIII levels in Leu/
The effect of FXIII-A Val34Leu genotypes on FXIII levels in different patient groups
Val34Leu genotype CC (n = 302) Val/Val Val/Leu Leu/Leu
471
FXIII activity (%) 105 (101–109) 102 (97–106) 100 (91–109) FXIII antigen (mg/L) 23.1 (21.1–24.1) 22.8 (21.7–23.8) 21.8 (19.8–23.9)
CS+ (n = 619) n 167 (55.3%) 114 (37.7%) 21 (7.0%) 167 (55.3%) 114 (37.7%) 21 (7.0%)
FXIII activity (%) 104 (100–107) 103 (99–107) 94 (86–103)⁎ FXIII antigen (mg/L) 23.2 (22.4–24.1) 22.7 (21.8–23.6) 20.9 (19.0–22.9)⁎
MI+ (n = 341) n 345 (55.7%) 235 (38.0%) 39 (6.3%) 345 (55.7%) 235 (38.0%) 39 (6.3%)
FXIII activity (%) 107 (102–112) 106 (101–111) 88 (76–99)⁎† FXIII antigen (mg/L) 24.0 (22.8–25.1) 23.2 (22.0–24.5) 20.3 (17.6–23.0) ⁎†
n 191 (56.0%) 131 (38.4%) 19 (5.6%) 191 (56.0%) 131 (38.4%) 19 (5.6%)
CC: clinical controls; CS+: patients with coronary sclerosis; MI+: patients with the history of myocardial infarction. FXIII levels were adjusted for gender, smoking, cholesterol and fibrinogen. Values in parenthesis represent 95% confidence interval. ⁎p b 0.05 Leu/Leu versus Val/Val, †p b 0.05 Leu/Leu versus Val/Leu.
472 Table 2
Z. Bereczky et al. The effect of FXIII-A Val34Leu genotypes on the specific activity of FXIIIa in different patient groups
Val34Leu genotype CC (n = 302) Val/Val Val/Leu Leu/Leu
CS+ (n = 619)
MI+ (n = 341)
FXIII specific activity (U/mg) n FXIII specific activity (U/mg) n FXIII specific activity (U/mg) n 7.19 (7.06–7.31) 167 7.25 (7.16–7.34) 345 7.22 (7.10–7.34) 191 7.00 (6.88–7.13) 114 7.27 (7.17–7.38) 235 7.30 (7.16–7.45) 131 7.12 (6.61–7.63) 21 7.32 (7.03–7.61) 39 7.03 (6.56–7.49) 19
CC: clinical controls; CS+: patients with coronary sclerosis; MI+: patients with the history of myocardial infarction. One unit (U) enzyme activity represents 1 μmol substrate conversion per minute. Values in parenthesis represent 95% confidence interval.
Leu homozygotes was also evident in CS+ patients without the history of MI this group of patients (n = 312) were separately assessed. FXIII activities of 103%, 101% and 97% were found in the Val/Val (n = 169), Val/Leu (n = 123) and Leu/Leu (n = 23) groups, respectively. The corresponding antigen levels were 22.9 mg/L, 22.5 mg/L and 20.9 mg/L. Although there was a tendency of decrease with increasing number of Leu34 allele, the differences were not statistically significant and did not seem to differ from those observed in the CC group. There was a small subgroup (n = 34) of patients with the history of MI, but without significant coronary sclerosis. In this subgroup rupture of small plaques and/or coronary vasospasm must have been responsible for the previous MI. The small number of such patients (there were only three Leu34 homozygotes in this subgroup) excluded the possibility of any meaningful statistical evaluation. Theoretically, the decrease of FXIII activity could be due to the decrease in the concentration of FXIII molecules or to the decreased activity of the variant factor. The parallel decrease of FXIII activity and antigen levels support the former possibility. To give a more correct assessment of the problem, specific FXIIIa activities, i.e. activities of a given amount of FXIII protein, were calculated (Table 2). Practically identical specific FXIIIa activities were obtained in all patient groups with any FXIII-A genotypes which demonstrate that the decreased FXIII activity in the Leu/Leu patient groups was due to the decrease of FXIII concentration. As FXIII is closely associated with fibrinogen and the clinical role of FXIII-A Val34Leu polymorphism has been related to fibrinogen levels, the effect of fibrinogen concentration on FXIII levels was studied in different patient groups with different FXIII Val34Leu genotypes. Fibrinogen levels in the clinical control, CS+ and MI+ groups were 3.87 g/L (3.21– 4.43), 3.95 g/L (3.28–4.73) and 4.02 g/L (3.20–4.98), respectively. Although the median values were slightly higher in the CS+ and MI+ groups than in clinical controls, the differences did not reach the level of statistical significance. The fibrinogen levels of wild type, Leu34 heterozygote, and Leu34 homozygote patients were 3.91 g/L (3.23–4.64), 3.92 (3.25–4.63) and 3.54 (3.24–4.43), respectively. The differences were not statistically significant. It has
been shown by Ariens et al. that FXIII activity correlated significantly with fibrinogen levels in healthy individuals [30]. Table 3 demonstrates the relation between fibrinogen and FXIII activity or antigen levels in the patient groups and in groups of FXIII-A Val34Leu genotypes. With the exception of clinical control group, statistically significant correlations were found between FXIII and fibrinogen levels. To test the effect of fibrinogen level on FXIII activity and antigen in the different patient groups and in patients with different FXIII-A Val34Leu genotypes patients with fibrinogen level in the lowest quartile (≤3.23 g/L plasma fibrinogen; low fibrinogen group) were compared with the rest of the patients (high fibrinogen group). In the clinical control group there was no significant difference in FXIII levels between individuals with low and high fibrinogen (Table 4). In the CS+ and MI+ groups in the low fibrinogen group significantly lower FXIII levels were observed. The tendency of decreasing FXIII level with increasing number of Leu34 allele, as already shown in Table 1, was also observed within both the high and low fibrinogen groups. Due to the small number of patients in the Leu/Leu group, statistically significant difference in FXIII activity was observed only when of Leu34 homozygous MI+ patients with low fibrinogen levels and their wild
Table 3 Correlations of fibrinogen levels with FXIII activity and antigen levels in different patient groups and FXIII-A Val34Leu genotypes Patient groups
CC (n = 302)
CS+ (n = 619)
MI+ (n = 341)
FXIII activity FXIII antigen
0.015 (p = 0.797) − 0.026 (p = 0.659)
0.243 (p b 0.001) 0.188 (p = 0.001)
0.176 (p b 0.001) 0.162 (p b 0.001)
Val34Leu genotype
Val/Val (n = 539)
Val/Leu (n = 373)
Leu/Leu (n = 64)
FXIII activity FXIII antigen
0.124 (p = 0.008) 0.068 (p = 0.144)
0.133 (p = 0.016) 0.158 (p = 0.004)
0.262 (p = 0.058) 0.085 (p = 0.543)
Spearman's correlation coefficients are shown in the Table. CC: clinical controls; CS+: patients with coronary sclerosis; MI+: patients with the history of myocardial infarction.
Decreased factor XIII levels in XIII A subunit Leu34 homozygous pts with coronary artery disease
473
Table 4 The effect of FXIII-A Val34Leu genotypes and fibrinogen levels on the FXIII activity and antigen in different patient groups CC Val34Leu genotype Val/Val
Val/Leu
Leu/Leu
Total undivided
Fibrinogen FXIII activity (%) High 105 (101–110) Low 104 (98–111) High 100 (95–105) Low 107 (99–115) High 101 (91–110) Low 97 (68–126) High 103 (99–106) Low 105 (100–111)
CS+ FXIII antigen (mg/L) 23.0 (21.9–24.0) 23.6 (22.0–25.1) 22.4 (21.2–23.5) 24.0 (22.1–25.9) 21.8 (19.6–24.0) 22.4 (15.8–29.0) 22.6 (21.8–23.5) 23.7 (22.4–24.9)
n 115 45 81 29 19 2 215 76
MI+
FXIII activity (%) 105 (101–109) 101 (95–107) 105 (100–109) 97⁎ (90–104) 97 (87–107) 88 (73–102) 104 (101–107) 98⁎ (93–103)
FXIII antigen (mg/L) 23.4 (22.6–24.3) 22.6 (21.2–24.1) 23.2 (22.2–24.1) 21.0⁎ (19.4–22.6) 20.9† (18.6–23.3) 20.7 (17.3–24.0) 23.2 (22.4–23.9) 21.8⁎ (20.6–22.9)
n 217 62 149 46 19 9 385 117
FXIII activity (%) 109 (103–114) 102 (93–110) 108 (102–114) 99⁎ (90–107) 96 (83–110) 71⁎ § (52–91) 108 (103–112) 98# (22–105)
FXIII antigen (mg/L) 24.3 (23.1–25.4) 23.0 (21.0–25.0) 23.7 (22.4–25.1) 21.5⁎ (19.4–23.5) 21.3 (18.1–24.5) 18.3 (13.7–23.0) 23.9 (22.8–24.9) 21.9# (20.3–23.5)
n 120 37 83 31 10 5 213 73
CC: clinical controls; CS+: patients with coronary sclerosis; MI+: patients with the history of myocardial infarction. FXIII levels were adjusted for gender, smoking and cholesterol. Values in parenthesis represent 95% confidence interval. Patients with fibrinogen level in the lowest quartile (low fibrinogen) were compared to the rest of the patients (high fibrinogen). ⁎p b 0.05, #p b 0.01 low versus high fibrinogen group within the same genotype or within the undivided group; †p b 0.05 Leu/Leu versus Val/Val genotype in the same (low or high) fibrinogen group; §p b 0.01 Leu/Leu versus Val/Leu in the same (low or high) fibrinogen group.
type counterparts were compared. The difference in FXIII antigen level between Leu34 homozygous and wild type CS+ patients with high fibrinogen levels was also significant. The effect of plasma fibrinogen concentration on FXIII levels was also compared within the same Val34Leu genotypes. In the CS+ and MI+ groups, but not in clinical controls there was a clear tendency of parallel decrease of fibrinogen concentrations and FXIII levels. Differences reached statistical significance in the CS+ and MI+ heterozygous and in the MI+ homozygous groups. The lowest FXIII activity and antigen levels were measured in the MI+ Leu34 homozygous group with low fibrinogen concentration. It is interesting that the frequency of patients homozygous for the Leu34 allele and having fibrinogen concentration in the lowest quartile is higher in the CS+ (1.79%) and in the MI+ (1.75%) groups than in the clinical control group (0.91%). However, the low number of Leu34 homozygous patients with fibrinogen in the lowest quartile (altogether 16 individuals) makes hardly possible to perform a meaningful statistical evaluation of the combined effect of Leu34 homozygosity and low fibrinogen on the risk of CS or MI.
Discussion Due to methodological differences, there is some confusion in the literature concerning plasma FXIII activity in individuals of different FXIII-A Val34Leu
genotypes. In assays using low thrombin concentration and/or short incubation with thrombin FXIII is only partially activated, and the extent of FXIII activation depends on the rate of proteolytic activation. As the rate of the release of AP-FXIII from the Leu34 variant by thrombin is about 2.5fold higher than its release from the Val34 variant, using assays measuring FXIIIa at the early stage of thrombin activation higher FXIII activities were measured in individuals possessing the Leu34 allele in heterozygous form than in wild type individuals and even higher in Leu34 homozygotes [31,32]. The activity results measured by such an assay show poor correlation with results of FXIII antigen assays [33]. In contrast, at full activation of FXIII by thrombin the differences in FXIII activity among FXIII-A Val34Leu genotypes disappear and there are no Val34Leu genotype-dependent differences in the specific activities of FXIIIa [20–22]. For a review on FXIII assay methods and their relation to FXIII-A Val34Leu polymorphism see reference [34]. In this study we used a FXIII assay which measures the activity of fully activated FXIII, the results obtained with this assay reflect the catalytic concentration and show good correlation with estimated plasma FXIII antigen values [26]. The few studies that investigated the effect of coronary artery disease on plasma FXIII antigen levels resulted in mainly negative outcome. No differences in FXIII antigen level were found when patients suffering from coronary artery disease (proven by
474 angiography) were compared with clinical controls (normal angiography) [35]. Comparison of FXIII-A2B2 level in patients with coronary artery disease and in a community control group also failed to detect differences [36]. The history of MI had no influence on plasma FXIII-A antigen level [33,37], only in a small prospective study (n = 63) had patients, who developed MI, lower FXIII-A level at recruitment [38]. In our most recent study, utilizing the same patient populations as the present study, neither the presence of CS nor previous MI influenced the adjusted plasma FXIII activity and FXIII-A2B2 antigen levels significantly. Only in females with a history of MI could a slight increase in both FXIII activity and antigen levels be detected [29]. Here we investigated the influence of CS and MI on FXIII levels in patients with different FXIII-A Val34Leu genotype. It was surprising to detect a genotype specific effect and to reveal statistically significant decrease of both FXIII activity and antigen levels in CS+ and MI+ patients homozygous for the Leu34 allele. It is to be stressed that neither the homozygous mutation alone nor the presence of the disease in non-homozygous mutants or wild type patients altered the level of FXIII significantly. Their concomitant presence was required to decrease FXIII activity and antigen concentration. It was also shown that the history of MI exerted a more considerable effect on the FXIII levels than CS without MI; in the latter case only a tendency of decrease could be detected. From the parallel decrease of FXIII antigen and activity and from the practically identical FXIIIa specific activities in the different patient and genotype groups it became evident that the Val34Leu mutation did not influence the activity of FXIIIa, but the concentration of FXIII was decreased in the Leu34 homozygous CS+ and MI+ groups. The reason for the FXIII-A Val34Leu genotypespecific decrease of FXIII level in the patient groups is not clear. For the time being, the following working hypothesis can be offered. Persistent elevation of prothrombin fragment 1 + 2, thrombin– antithrombin III complex and fibrinopeptide A levels has been reported in CS+, and particularly in MI+ patients [39–42]. The elevation of these markers of thrombin formation suggests a continuous low-scale activation of blood coagulation that leads to the parallel consumption of FXIII and fibrinogen. In addition, the formation of fibrin and the activation of FXIII are intimately associated, fibrin as a cofactor is required for the rapid activation of plasma FXIII [43,44]. Although such activation of blood coagulation occurs independently of FXIII genotypes, its effect on FXIII levels could well be FXIII-A Val34Leu genotype-dependent. We have shown that lower
Z. Bereczky et al. amount of thrombin was required to activate FXIII of Leu/Leu genotype than wild type FXIII [20,21]. It seems logical that the same amount of thrombin formed during the coarse of such low-scale activation of blood coagulation activates higher amount of Leu/Leu FXIII. As FXIIIa is quickly removed from the circulation, such mechanism could lead to decreased FXIII levels in Leu/Leu homozygotes. The interaction among plasma fibrinogen concentration and FXIII-AVal34Leu polymorphism and plasma FXIII levels in affecting the risk of CAD is an intriguing, but not fully clarified question. The effect of FXIII-A Val34Leu polymorphism on the risk of CAD, just like its effect on fibrin polymerization [24], seems to be fibrinogen concentration dependent. In the study population the protective effect of Leu34 allele could be demonstrated only at high fibrinogen level [19]. In a recent study Leu34 homozygosity conferred an almost 3-fold increased risk of CAD to patients with low fibrinogen level, while at high fibrinogen it exerted a protective, although statistically not significant, effect [18]. We have demonstrated that in women increased FXIII level represented a significant risk of MI [29] and the effect was independent of fibrinogen level. In the present study the limited number of Leu34 homozygotes with low fibrinogen concentration did not allow an appropriate risk analysis for CAD. The fact that those homozygous for the Leu34 allele and with low fibrinogen were more common among patients with CS and MI than among clinical controls and showed the lowest FXIII levels is suggestive for further investigations involving higher number of patients.
Acknowledgments This work was supported by grants from the Hungarian National Research Fund (OTKA-NKTH NI69238), from the Hungarian Academy of Sciences (MTA 11003, 2006TKI227) from the Hungarian Ministry of Health (ETT 406/2006) and from the National Office of Research and Technology (NKTH RET-06/ 2004).
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