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Thrombomodulin-dependent effect of factor VLeiden mutation on factor XIII activation
Thrombosis Research 129 (2012) 508–513
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Thrombomodulin-dependent effect of factor VLeiden mutation on factor XIII activation Zsuzsa Koncz a, Zsuzsa Bagoly a, Gizella Haramura a, Zoltán A. Mezei a, László Muszbek a, b,⁎ a b
Clinical Research Center University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Thrombosis, Hemostasis and Vascular Biology Research Group of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary
a r t i c l e
i n f o
Article history: Received 11 April 2011 Received in revised form 21 June 2011 Accepted 28 June 2011 Available online 20 July 2011 Keywords: factor VLeiden mutation factor XIII fibrin thrombomodulin
a b s t r a c t Introduction: Factor V Leiden mutation (FVLeiden) is associated with impaired down-regulation of activated FV procoagulant activity and loss of FV anticoagulant function that result in an increased risk of venous thromboembolism. As the downstream effects of FVLeiden on clot formation and fibrinolyis have only partially been revealed, we investigated its effect on the activation of factor XIII (FXIII) and the cross-linking of fibrin. Methods: In the plasma samples of fifteen healthy individuals with known FV genotypes coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human thrombomodulin (rhTM). FV deficient plasma supplemented with purified wild type FV or FVLeiden were also investigated. Clots were recovered and analyzed by SDS-PAGE and quantitative densitometric evaluation of Western blots. Results: rhTM considerably delayed the activation of FXIII in the plasma from FV wild type individuals. This effect of rhTM was significantly impaired in the plasma from FVLeiden carriers. The results were confirmed in experiments with FV deficient plasma supplemented by FV prepared from wild type individuals or FVLeiden homozygotes. Fibrin γ-chain dimerization was also considerably delayed by rhTM in plasma samples from individuals without Leiden mutation, but not in plasma samples from FVLeiden heterozygotes or homozygotes. The difference between heterozygotes and homozygotes was not statistically significant. Conclusion: The highly diminished delaying effect of TM on FXIII activation and on the cross-linking of fibrin in FVLeiden carriers might represent a novel mechanism contributing to the increased thrombosis risk of these individuals. © 2011 Elsevier Ltd. All rights reserved.
Introduction p.506R N Q mutation in coagulation factor V (FVLeiden) is common among Caucasians and it is associated with a 5-8-fold increased risk of venous thrombosis in heterozygotes and with a 50-80-fold increased risk in homozygotes [1,2]. The mutation eliminates the primary cleavage site of activated protein C (APC) in FV [3–5] and this way impairs the down-regulation of procoagulant activity of activated FV (FVa). The mutation also compromises FV cofactor activity that accelerates APC-catalyzed inactivation of activated factor VIII [6]. Due to these mechanisms, FVLeiden impairs the down-regulation of thrombin generation by the APC pathway [7,8]. The downstream
Abbreviations: α2-PI, α2-plasmin inhibitor; APC, activated protein C; FVa, activated factor V; FVLeiden, factor V Leiden mutation; FXIII, factor XIII; FXIIIa, active factor XIII; FXIII-A, factor XIII A subunit; FXIII-A’, proteolytically activated factor XIII A subunit; PDP, platelet depleted plasma; PPACK, D-phenylalanyl-L-prolyl-L-arginin chloromethyl ketone; rhTM, recombinant human thrombomodulin; T1/2, time required for half maximal activation of FXIII or fibrin gamma chain dimer formation; TAFI, thrombin activatable fibrinolysis inhibitor; TF, tissue factor; TM, thrombomodulin. ⁎ Corresponding author at: University of Debrecen, Medical and Health Science Center, Clinical Research Center, PO Box 40, H-4012 Debrecen, Hungary. Tel.: + 36 52431956; fax: + 36 52340011. E-mail address: [email protected] (L. Muszbek). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.06.030
consequences of FVLeiden, which might be important in the increased thrombotic risk, have only partially been revealed. An impaired thrombin activatable fibrinolysis inhibitor (TAFI) dependent profibrinolytic response to APC has been observed in subjects with FVLeiden and it was suggested to contribute to the thrombotic tendency caused by this mutation [9]. The effect of FVLeiden, on the activation of TAFI was modulated by thrombomodulin (TM) concentration [10]. Blood coagulation factor XIII (FXIII) is another thrombin substrate with an important role in the regulation of fibrinolysis [11]. FXIII binds to fibrinogen with high affinity (Kd is ~ 10 - 8 M) and circulates in the plasma as fibrinogen-FXIII complex [12]. Thrombin activates plasma FXIII, a tetrameric protein (FXIII-A2B2), by cleaving off an activation peptide from the potentially active A subunit (FXIII-A). In the presence of Ca 2+ the truncated FXIII-A (FXIII-A’) dimer dissociates from the inhibitory B subunits and becomes an active transglutaminase (FXIIIa) [13]. Due to the tight binding of FXIII to fibrin this process occurs on the surface of newly formed fibrin and FXIIIa remains associated with its substrate, fibrin [14,15]. By cross-linking fibrin chains and α2-plasmin inhibitor (α2-PI) to fibrin, FXIIIa makes fibrin more resistant to shear stress and protects it from fibrinolytic degradation [16–19]. Here we investigated the effect of FVLeiden in the presence and absence of TM on the activation of FXIII in a system resembling in vivo activation of coagulation.
Z. Koncz et al. / Thrombosis Research 129 (2012) 508–513
Fig. 1. Proteolytic activation of factor XIII (FXIII) in plasma samples of a FV wild type individual in the absence and presence of recombinant human thrombomodulin (rhTM). (A) Western blots showing the effect of rhTM on the time course of FXIII activation. Washed plasma clots were recovered at various times after the activation of coagulation in the absence or presence of rhTM. FXIII-A and its proteolytically activated truncated form (FXIII-A’) incorporated in the clot were detected by Western blotting and quantified by quantitative densitometry. The Figure is a representative of experiments on plasma samples of five FV wild type individuals. Numbers under the individual bands on the blots indicate the time elapsed after the initiation of coagulation. FXIII-A’: truncated FXIII-A, St.: purified FXIII-A2 standard. (B) Densitometric tracings of Western blots obtained at selected times after the initiation of coagulation. The delaying effect of rhTM on FXIII activation is clearly demonstrated.
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Fig. 2. Proteolytic activation of factor XIII (FXIII) in plasma samples of a FVLeiden homozygote in the absence and presence of recombinant human thrombomodulin (rhTM). (A) Western blots showing the effect of rhTM on the time course of FXIII activation. Washed plasma clots were recovered at various times after the activation of coagulation in the absence or presence of rhTM. FXIII-A and its proteolytically activated truncated form (FXIII-A’) incorporated in the clot were detected by Western blotting and quantified by quantitative densitometry. The Figure is a representative of experiments on plasma samples of five FVLeiden homozygotes. Numbers under the individual bands on the blots indicate the time elapsed after the initiation of coagulation. FXIII-A’: truncated FXIII-A, St.: purified FXIII-A2 standard. (B) Densitometric tracings of Western blots obtained at selected times after the initiation of coagulation. In FVLeiden homozygotes rhTM only slightly delays FXIII activation.
Material and methods Subjects Fifteen apparently healthy individuals of known FV genotypes (5 wild type, 5 FVLeiden heterozygotes and 5 FVLeiden homozygotes) with no history of thrombosis or bleeding disorders were recruited for the study (3 men and 12 women; age 17–67 years). All individuals had normal coagulation screening tests; their fibrinogen, factor XIII activity and antigen values were in the reference interval. None of them were on anticoagulant therapy, and they did not take any medication for at least 2 weeks prior to blood sampling. As the rate of FXIII activation is modified by FXIII-A Val34Leu polymorphism [14,20,21], only subjects being wild type for this polymorphism were selected for the study. Genotyping for FVLeiden mutation and FXIII-A Val34Leu polymorphism was carried out by standard methods [22,23]. The study protocol was approved by the Ethics Committee of the University of Debrecen. Informed consent was given by the subjects.
Preparation of plasma samples 18 mL blood was collected by venipuncture into 2 mL 0.105 M trisodium citrate using 21 gauge, 0.8 × 38 mm needle (Becton Dickinson, Franklin Lakes, NJ). In order to suppress contact activation of coagulation, 50 μg/mL corn trypsin inhibitor (Gentaur, Brussels, Belgium) was added immediately to the blood sample [24]. Citrated blood was centrifuged at 1,400 g for 20 min, then the upper two-third of platelet poor plasma was transferred to another centrifuge tube and centrifuged again at 1,400 g for 20 min. The upper two-third of the second supernatant was considered as platelet-depleted plasma
(PDP). In PDP no platelet was detected by electronic platelet counting, but occasionally a single platelet could be seen in the microscope. Preparation of crude factor V from pooled plasma Partial purification of FV from the pooled plasma of wild type or FVLeiden homozygous individuals was carried out essentially as described by Dahlback [25]. 35 ml blood from each of three individuals of the same genotype was collected in acid-citrate-dextrose Vacutainer tubes (ACD-B, Becton Dickinson, Franklin Lakes, NJ). PDP was prepared as described above. PDP samples of the same genotypes were pooled and one tablet of protease inhibitor cocktail (Complete, Mini, EDTA-free, Roche Diagnostics, Indianapolis, IN) was added to each 10 ml of pooled plasma. Barium citrate adsorption was carried out by the drop-wise addition of 0.8 ml 1 M BaCl2/10 ml plasma. After the mixture had been stirred for 1 h, the barium citrate was removed by centrifugation at 6,000 g for 10 min. Next, PEG-6,000 fractionation was performed. First, solid PEG-6,000 (Sigma-Aldrich, St. Louis, MO) was added to the supernatant to obtain a final concentration of 80 g/l. After stirring for 1 h, the precipitate was removed by centrifugation at 6,000 g for 10 min. Solid PEG-6,000 was added to the supernatant, again, to bring the PEG concentration to 120 g/l. The solution was stirred for 1 h, then, the precipitate was collected by centrifugation (6,000 g, 10 min). The FV containing plasma precipitate was washed twice with 120 g/l PEG-6,000 in dist. water. The whole preparation procedure was carried out at 4 °C. The precipitate containing wild type FV or FVLeiden was dissolved in onetenth of the original plasma volume of immunodepleted FV deficient plasma (Technoclone, Vienna, Austria). The FV activity of the mixture was determined by one-stage clotting assay, and it was set to 90% by further dilution in FV deficient plasma in both cases.
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Fig. 3. The effect of recombinant human thrombomodulin (rhTM) on the time-course of FXIII activation in plasma samples from individuals of different FV genotypes. FXIII-A’ in each sample was expressed as percentage of the maximum amount of activated FXIII-A incorporated into the clot as determined by quantitative densitometry. Results obtained in the presence of rhTM are depicted by solid symbols and broken lines, while empty symbols with solid lines represent results obtained in the absence of rhTM. Horizontal dotted lines demonstrate when 50% of FXIII-A became truncated. Representatives of 5 experiments with similar results are shown for each genotype.
Factor XIII activation in plasma samples As the activation of FXIII takes place exclusively on the surface of fibrin and after its formation FXIIIa remains associated with fibrin [14], the activation of FXIII was measured in recovered fibrin clots. Fibrin clots were generated by the activation of tissue factor (TF) pathway in the presence and absence of recombinant human TM (rhTM, American Diagnostica, Stamford, CT). An rhTM concentration that inhibits thrombin generation in the plasma of FV wild type individuals by approximately 50% was selected as described by Dielis
et al. [26]. Using the selected rhTM concentration the thrombin generation was determined in the plasma of FV wild type individuals and 45.4% mean inhibition (range 40-61%) by rhTM was obtained as calculated from the AUC (area under the curve; also referred to as endogen thrombin potential) values. Aliquots of 150 μl PDP were incubated with 40 μl activator cocktail at 37 °C for various intervals and the reaction was stopped by an equal volume of inhibitor cocktail. To produce an activator cocktail Technothrombin® TGA RC Low (Technoclone GmbH, Vienna, Austria) was dissolved in 62.5 mM CaCl2, with or without 7.5 nM rhTM. The dissolved Technothrombin® TGA RC Low contained ~ 5 pM recombinant human TF, low concentration of phospholipid micelles in Tris-Hepes-NaCl buffer. The concentration of rhTM used in the experiments was established in preliminary experiments. The selected rhTM concentration (final concentration in the sample: 1.5 nM) inhibited activator cocktailinduced thrombin generation in normal pooled plasma by 50%. Thrombin generation was measured by S-2238 chromogenic substrate (Chromogenix, Lexington, MA). The inhibitor cocktail contained 20 mM benzamidine, 0.1 mM D-phenylalanyl-L-prolyl-L-arginin chloromethyl ketone (PPACK), 2 mM iodoacetamide, 50 mM εaminocaproic acid, 100 mM NaCl, 50 mM EDTA in 50 mM HEPES buffer, pH 7.5 to block thrombin and FXIIIa, and to prevent fibrinolysis [14]. The reaction was stopped at various intervals after the initiation of coagulation by the activator cocktail. Polymerized fibrin, if present, was fully recovered by centrifugation of the formed clots; the clots were then exhaustively washed with physiological NaCl and dissolved in SDS PAGE sample buffer.
SDS-PAGE and Western blotting
Fig. 4. The effect of FVLeiden genotype on the lag time required for 50% activation of FXIII (T1/2) in the presence and absence of recombinant human thrombomodulin (rhTM) (A) and on the difference of T1/2 values measured in the presence and absence of rhTM (T1/2rhTM+)-(T1/2rhTM+) (B). The bars represent the mean differences of T1/2 values obtained with (T1/2rhTM+) and without (T1/2rhTM-) rhTM. Values were calculated from the results of 5 individuals in each genotype. Error bars depict SEM, w.t.: FV wild type individuals, het.: FVLeiden heterozygotes , hom.: FVLeiden homozygotes. *p b 0.05, **p b 0.001.
SDS-PAGE of dissolved fibrin samples and Western blotting for FXIII-A were performed as described previously [14]. The extent of FXIII activation was monitored by Western blotting using affinity purified sheep antihuman FXIII-A antibody as primary antibody (Affinity Biologicals, Ancaster, Canada). The immunoreaction was developed by biotinylated rabbit anti-sheep IgG and avidin-biotinylated peroxidase complex (components of Vectastain ABC kit, Vector, Burlingame, CA, USA) and it was visualized by enhanced chemiluminescence detection (ECL Plus+, Amersham, Little Chalfont, UK) according to the manufacturer's instructions. The determination of fibrin bound activated FXIII-A was used as parameter to study the kinetics of FXIII activation. The amount of truncated, proteolytically activated FXIII-A (FXIII-A’) was determined in each sample by quantitative densitometry using GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, USA) and expressed as percentage of the maximal amount of FXIII-A’ formed in the clot. To compare the effect of rhTM on the rate of FXIII activation in different FV genotypes quantitatively, the time required for half maximal activation of FXIII was calculated (T1/2 values).
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Statistical analysis Statistical analysis was performed using GraphPad Prism software (La Jolla, CA). Kruskal-Wallis one-way analysis of variance was used to determine differences among groups. The extent of significance was determined by Mann-Whitney U test; p b 0.05 was considered as significant. Results Figs. 1–3 show the effect of rhTM on FXIII activation on representative plasma samples from selected individuals of different FV genotypes while on Fig. 4 the individual results are summarized. Western blots demonstrating the proteolytic activation of FXIII in the plasma clots of a FV wild type individual and a FVLeiden homozygote are shown on Figs. 1A and 2A. The kinetics of FXIII activation was followed by quantitative densitometry of Western blots (Figs. 1B and 2B) and the results were expressed as percentage of maximal FXIII activation (Fig. 3). To be able to compare the effect of rhTM on FXIII activation in the plasma samples of individuals with different FV genotypes quantitatively, the mean time required for the activation of half of the FXIII molecules present in the plasma (T1/2 FXIII activation) was determined and the difference of T1/2 values measured in the presence and absence of rhTM, (T1/2 rhTM+)-(T1/2 rhTM-), was calculated (Fig. 4). In the absence of rhTM no relevant differences were seen in the rate or in the pattern of FXIII activation in the plasma of individuals with different FV genotype (Figs. 1–3) and the T1/2 of FXIII activation did not differ significantly among genotypes (Fig. 4A). In the plasma of wild type individuals, the activation of FXIII occurred considerably later in the presence of rhTM than in its absence (Figs. 1, 3, 4). The effect of rhTM was considerably less in the case of heterozygous and homozygous individuals (Figs. 2–4). The T1/2 values for FXIII activation measured in the presence of rhTM were more prolonged in the plasma samples of wild type individuals than in the plasma samples of FVLeiden heterozygotes or homozygotes, but, due to considerable individual variation and the low sample number, the differences among genotypes were not statistically significant (Fig. 4A). However, when homozygotes and heterozygotes were grouped, the difference between FVLeiden carriers and wild type individuals became significant. As individual variations in FXIII activation may partially obscure the genotype-dependent effect of rhTM, the delay in T1/2 activation of FXIII, caused by rhTM was calculated for each individual and the effect of rhTM was expressed as the difference between T1/2 values obtained in its presence (T1/2rhTM+) and absence (T1/2rhTM-) (Fig. 4B). The mean difference between T1/2rhTM + and T1/2rhTM- values was significantly more pronounced in
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the case of FV wild type individuals than in the cases of FVLeiden heterozygotes and homozygotes (175 sec, 39 sec and 25 sec, respectively). Although the difference between heterozygotes and homozygotes was noticeable, it was not statistically significant. FVLeiden homozygotes, wild type for FXIII Val34Leu polymorphism and not on anticoagulant treatment, were very hard to recruit and five was the maximal number which we were able to select for the study from over three thousands genotyped individuals. For this reason and in order to eliminate individual variations that influence clot formation we also followed a different approach. FV deficient plasma was supplemented with FV isolated from pooled plasma of individuals either wild type or homozygous for FVLeiden. These supplemented plasma samples were used to study how FV of different genotypes influences the effect of rhTM on FXIII activation in the very same plasmatic environment (Fig. 5). In the absence of rhTM, in the plasma with FVLeiden FXIII activation occurred somewhat earlier than in the plasma with wild type FV, which might be related to some differences in the composition of the two partially purified FV preparations. In FV deficient plasma supplemented with wild type FV rhTM significantly delayed FXIII activation, the difference between T1/2 values was 185 sec. In the plasma supplemented with FVLeiden the effect of rhTM was more moderate (the difference between T1/2 values was only 65 sec). In the followings we tested how rhTM influences the cross-linking of fibrin chains in plasma samples from individuals of different FV genotypes. rhTM considerably delayed the dimerization of fibrin γchains in plasma samples containing wild type FV, but was much less effective in the plasma of FVLeiden homozygotes (Fig. 6A). The effect of rhTM on the formation of fibrin α-chain polymers seemed to follow a similar tendency (see partially polymerized α-chains on the upper part of the gel), however the slow formation of fully polymerized αchain polymers was out of the timeframe of the experiments and quantitative measurement could not be performed. Similarly to the evaluation of FXIII activation, the time intervals required for half maximal γ-chain dimerization were used for quantitative comparisons. In the plasma samples of wild type individuals there was a considerable difference between T1/2 obtained in the presence and absence of rhTM, while in FVLeiden carriers only slight differences could be observed (Fig. 6B). In the plasma samples from FVLeiden homozygotes rhTM delayed γ-chain dimerization to a lesser extent than in the plasma of heterozygotes, but the difference was not statistically significant. Discussion TM could influence FXIII activation by direct and an indirect mechanisms. In the absence of fibrinogen high concentration of thrombin
Fig. 5. Proteolytic activation of FXIII in FV deficient plasma supplemented by crude FV prepared from plasma of FV wild type or FVLeiden homozygous individuals. Washed plasma clots were recovered at various times after the activation of coagulation in the absence or presence of rhTM. The proteolytically activated truncated form of FXIII-A (FXIII-A’) recovered in the clot was detected by Western blotting and quantified by quantitative densitometry. FXIII-A’ in each sample was expressed as percentage of the maximum amount of activated FXIII-A incorporated into the clot. The results are the means of two parallel experiments with practically identical results. Results obtained in the presence of rhTM are depicted by solid symbols and broken lines, while empty symbols with solid lines represent results obtained in the absence of rhTM. Dotted lines demonstrate when 50% of FXIII-A became truncated.
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Fig. 6. The effect of recombinant human thrombomodulin (rhTM) on fibrin crosslinking in plasma from individuals of different FVLeiden genotypes. (A) Western blots demonstrating fibrin cross-linking in plasma samples of a FV wild type individual and a FVLeiden homozygote in the absence and presence of rhTM. Washed plasma clots recovered at various time after the activation of coagulation were analyzed by SDS PAGE. Both gels are representatives of experiments with the plasma samples of five individuals. Numbers under the individual bands on the gels indicate the time elapsed after the initiation of coagulation. Fbg.: fibrinogen standard. Fibrin(ogen) chains and their cross-linked products, γ-chain dimers (γ-γ) and polymerized α-chains (αn) are indicated. (B) The effect of FVLeiden genotype on the difference in the lag time required for the dimerization of 50% of fibrin γ-chain in the presence (T1/2rhTM+) and in the absence (T1/2rhTM-) of rhTM. The percentage of dimerized γ-chains as compared to the total amount of γ-chain dimers was determined by quantitative densitometry Error bars depict SEM, w.t.: wild type, het.: heterozygous, hom.: homozygous for FVLeiden mutation. *p b 0.05, **p b 0.001.
Gly38 and accelerates the thrombin-induced cleavage of FXIII-A [30,33]. Consequently much lower concentration of thrombin is sufficient to activate FXIII in plasmatic conditions. Here TM exerts its effect primarily through different mechanisms [34]. It competes with fibrin for the binding of exosite 1 in thrombin. As the binding of fibrin to certain amino acid side-chains present in exosite 1 is essential to its cofactor activity on FXIII-A truncation by thrombin, TM inhibits the acceleration of FXIII activation on the surface of fibrin [34]. An additional indirect mechanism, by which TM influences FXIII activation, operates through enhancing the thrombin-induced activation of protein C. Activated protein C proteolytically cleaves FVa and FVIIIa and down-regulates the promotion of thrombin generation by these activated clotting factors. Then, impaired thrombin generation might lead to decreased/delayed activation of FXIII. In our experiments the presence of rhTM caused a significant delay of FXIII activation and fibrin cross-linking only in the plasma of individuals wild type for FV. This finding suggests that in whole plasma it is not the direct inhibitory effect of TM on thrombin, but the suppression of thrombin generation and fibrin formation that play the primary role in delaying FXIII activation by TM, i.e., in the plasma from wild type individuals the formation of both the activator protease and the enhancer fibrin becomes impaired. In the case of FVLeiden the down-regulation of thrombin generation and fibrin formation through the APC pathway is lost and rhTM delayed FXIII activation and fibrin cross-linking only to a minimal extent. The effect of TM on FXIII activation and fibrin cross-linking might be important in the regulation of fibrinolysis and the impairment of this regulatory mechanism in FVLeiden carriers might contribute to the increased risk of venous thromboembolism conferred by the mutation. Indeed, one of the prothrombotic effects of FVLeiden is the inhibition of fibrinolysis, which has been demonstrated both in vitro [9] and in vivo in transgenic mice [35]. Two key down-regulators of fibrinolysis are TAFI and FXIII. TAFI, is activated by thrombin, a process that is more than 1,000-fold enhanced in the presence of TM. Activated TAFI cleaves off C-terminal lysines from partially digested fibrin, which would be required for efficient plasminogen activation (see reviewed in reference [36]). TM regulates TAFI by an intricate mechanism [10,37,38]. It increases the rate of TAFI activation by thrombin, but at the same time, through the activation of PC, it decreases the generation of thrombin required for TAFI activation. These effects of TM are concentration dependent, at low TM concentrations the activation of TAFI is predominantly stimulated, whereas at high concentrations, the decreased thrombin generation, due to the effect of APC on FV, results in decreased TAFI activation. Accordingly, at low TM concentrations its antifibrinolytic/prothrombotic, while at high TM concentrations its profibrinolytic/antithrombotic effect prevails. In the case of FVLeiden this antithrombotic effect is abrogated, i.e., TM at high concentration does not influence the activation of TAFI [10]. Similarly, FVLeiden also abrogates the profibrinolytic/antithrombotic effect of TM exerted through FXIII activation and fibrin cross-linking. The diminished delaying effect of TM on FXIII activation in FVLeiden carriers could represent a novel downstream molecular mechanism that, in addition to the effect on TAFI activation, contributes to the increased risk of thrombosis in these individuals. Conflict of Interest Statement
is required for the activation of purified plasma FXIII and complex formation with TM impairs the FXIII activating capability of thrombin [27]. If fibrinogen is also present a different picture emerges. The activation of plasma FXIII is enhanced by polymerizing fibrin approximately 100-fold [28–32]. There is a clear correlation between fibrin formation and FXIII activation and in plasma the onset of fibrin formation is followed by the onset of FXIII activation [14]. It has also been shown that the activation of FXIII in plasma occurs only on the surface of polymerized fibrin [14]. The binding to fibrin makes the orientation of both FXIII and thrombin favorable for the proteolysis of FXIII-A at Arg37-
The authors state that they have no conflict of interest. Acknowledgements This study was supported by grants from the Hungarian National Research Fund (OTKA-NKTH CNK80776), from the Hungarian Academy of Sciences (TKI 227), from the Hungarian Ministry of Health (ETT) and by the TÁMOP 4.2.1./B-09/1/KONV-2010-0007 project. Z.B. was supported by the Bolyai Foundation.
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Thrombomodulin-dependent effect of factor VLeiden mutation on factor XIII activation Zsuzsa Koncz a, Zsuzsa Bagoly a, Gizella Haramura a, Zoltán A. Mezei a, László Muszbek a, b,⁎ a b
Clinical Research Center University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Thrombosis, Hemostasis and Vascular Biology Research Group of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary
a r t i c l e
i n f o
Article history: Received 11 April 2011 Received in revised form 21 June 2011 Accepted 28 June 2011 Available online 20 July 2011 Keywords: factor VLeiden mutation factor XIII fibrin thrombomodulin
a b s t r a c t Introduction: Factor V Leiden mutation (FVLeiden) is associated with impaired down-regulation of activated FV procoagulant activity and loss of FV anticoagulant function that result in an increased risk of venous thromboembolism. As the downstream effects of FVLeiden on clot formation and fibrinolyis have only partially been revealed, we investigated its effect on the activation of factor XIII (FXIII) and the cross-linking of fibrin. Methods: In the plasma samples of fifteen healthy individuals with known FV genotypes coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human thrombomodulin (rhTM). FV deficient plasma supplemented with purified wild type FV or FVLeiden were also investigated. Clots were recovered and analyzed by SDS-PAGE and quantitative densitometric evaluation of Western blots. Results: rhTM considerably delayed the activation of FXIII in the plasma from FV wild type individuals. This effect of rhTM was significantly impaired in the plasma from FVLeiden carriers. The results were confirmed in experiments with FV deficient plasma supplemented by FV prepared from wild type individuals or FVLeiden homozygotes. Fibrin γ-chain dimerization was also considerably delayed by rhTM in plasma samples from individuals without Leiden mutation, but not in plasma samples from FVLeiden heterozygotes or homozygotes. The difference between heterozygotes and homozygotes was not statistically significant. Conclusion: The highly diminished delaying effect of TM on FXIII activation and on the cross-linking of fibrin in FVLeiden carriers might represent a novel mechanism contributing to the increased thrombosis risk of these individuals. © 2011 Elsevier Ltd. All rights reserved.
Introduction p.506R N Q mutation in coagulation factor V (FVLeiden) is common among Caucasians and it is associated with a 5-8-fold increased risk of venous thrombosis in heterozygotes and with a 50-80-fold increased risk in homozygotes [1,2]. The mutation eliminates the primary cleavage site of activated protein C (APC) in FV [3–5] and this way impairs the down-regulation of procoagulant activity of activated FV (FVa). The mutation also compromises FV cofactor activity that accelerates APC-catalyzed inactivation of activated factor VIII [6]. Due to these mechanisms, FVLeiden impairs the down-regulation of thrombin generation by the APC pathway [7,8]. The downstream
Abbreviations: α2-PI, α2-plasmin inhibitor; APC, activated protein C; FVa, activated factor V; FVLeiden, factor V Leiden mutation; FXIII, factor XIII; FXIIIa, active factor XIII; FXIII-A, factor XIII A subunit; FXIII-A’, proteolytically activated factor XIII A subunit; PDP, platelet depleted plasma; PPACK, D-phenylalanyl-L-prolyl-L-arginin chloromethyl ketone; rhTM, recombinant human thrombomodulin; T1/2, time required for half maximal activation of FXIII or fibrin gamma chain dimer formation; TAFI, thrombin activatable fibrinolysis inhibitor; TF, tissue factor; TM, thrombomodulin. ⁎ Corresponding author at: University of Debrecen, Medical and Health Science Center, Clinical Research Center, PO Box 40, H-4012 Debrecen, Hungary. Tel.: + 36 52431956; fax: + 36 52340011. E-mail address: [email protected] (L. Muszbek). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.06.030
consequences of FVLeiden, which might be important in the increased thrombotic risk, have only partially been revealed. An impaired thrombin activatable fibrinolysis inhibitor (TAFI) dependent profibrinolytic response to APC has been observed in subjects with FVLeiden and it was suggested to contribute to the thrombotic tendency caused by this mutation [9]. The effect of FVLeiden, on the activation of TAFI was modulated by thrombomodulin (TM) concentration [10]. Blood coagulation factor XIII (FXIII) is another thrombin substrate with an important role in the regulation of fibrinolysis [11]. FXIII binds to fibrinogen with high affinity (Kd is ~ 10 - 8 M) and circulates in the plasma as fibrinogen-FXIII complex [12]. Thrombin activates plasma FXIII, a tetrameric protein (FXIII-A2B2), by cleaving off an activation peptide from the potentially active A subunit (FXIII-A). In the presence of Ca 2+ the truncated FXIII-A (FXIII-A’) dimer dissociates from the inhibitory B subunits and becomes an active transglutaminase (FXIIIa) [13]. Due to the tight binding of FXIII to fibrin this process occurs on the surface of newly formed fibrin and FXIIIa remains associated with its substrate, fibrin [14,15]. By cross-linking fibrin chains and α2-plasmin inhibitor (α2-PI) to fibrin, FXIIIa makes fibrin more resistant to shear stress and protects it from fibrinolytic degradation [16–19]. Here we investigated the effect of FVLeiden in the presence and absence of TM on the activation of FXIII in a system resembling in vivo activation of coagulation.
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Fig. 1. Proteolytic activation of factor XIII (FXIII) in plasma samples of a FV wild type individual in the absence and presence of recombinant human thrombomodulin (rhTM). (A) Western blots showing the effect of rhTM on the time course of FXIII activation. Washed plasma clots were recovered at various times after the activation of coagulation in the absence or presence of rhTM. FXIII-A and its proteolytically activated truncated form (FXIII-A’) incorporated in the clot were detected by Western blotting and quantified by quantitative densitometry. The Figure is a representative of experiments on plasma samples of five FV wild type individuals. Numbers under the individual bands on the blots indicate the time elapsed after the initiation of coagulation. FXIII-A’: truncated FXIII-A, St.: purified FXIII-A2 standard. (B) Densitometric tracings of Western blots obtained at selected times after the initiation of coagulation. The delaying effect of rhTM on FXIII activation is clearly demonstrated.
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Fig. 2. Proteolytic activation of factor XIII (FXIII) in plasma samples of a FVLeiden homozygote in the absence and presence of recombinant human thrombomodulin (rhTM). (A) Western blots showing the effect of rhTM on the time course of FXIII activation. Washed plasma clots were recovered at various times after the activation of coagulation in the absence or presence of rhTM. FXIII-A and its proteolytically activated truncated form (FXIII-A’) incorporated in the clot were detected by Western blotting and quantified by quantitative densitometry. The Figure is a representative of experiments on plasma samples of five FVLeiden homozygotes. Numbers under the individual bands on the blots indicate the time elapsed after the initiation of coagulation. FXIII-A’: truncated FXIII-A, St.: purified FXIII-A2 standard. (B) Densitometric tracings of Western blots obtained at selected times after the initiation of coagulation. In FVLeiden homozygotes rhTM only slightly delays FXIII activation.
Material and methods Subjects Fifteen apparently healthy individuals of known FV genotypes (5 wild type, 5 FVLeiden heterozygotes and 5 FVLeiden homozygotes) with no history of thrombosis or bleeding disorders were recruited for the study (3 men and 12 women; age 17–67 years). All individuals had normal coagulation screening tests; their fibrinogen, factor XIII activity and antigen values were in the reference interval. None of them were on anticoagulant therapy, and they did not take any medication for at least 2 weeks prior to blood sampling. As the rate of FXIII activation is modified by FXIII-A Val34Leu polymorphism [14,20,21], only subjects being wild type for this polymorphism were selected for the study. Genotyping for FVLeiden mutation and FXIII-A Val34Leu polymorphism was carried out by standard methods [22,23]. The study protocol was approved by the Ethics Committee of the University of Debrecen. Informed consent was given by the subjects.
Preparation of plasma samples 18 mL blood was collected by venipuncture into 2 mL 0.105 M trisodium citrate using 21 gauge, 0.8 × 38 mm needle (Becton Dickinson, Franklin Lakes, NJ). In order to suppress contact activation of coagulation, 50 μg/mL corn trypsin inhibitor (Gentaur, Brussels, Belgium) was added immediately to the blood sample [24]. Citrated blood was centrifuged at 1,400 g for 20 min, then the upper two-third of platelet poor plasma was transferred to another centrifuge tube and centrifuged again at 1,400 g for 20 min. The upper two-third of the second supernatant was considered as platelet-depleted plasma
(PDP). In PDP no platelet was detected by electronic platelet counting, but occasionally a single platelet could be seen in the microscope. Preparation of crude factor V from pooled plasma Partial purification of FV from the pooled plasma of wild type or FVLeiden homozygous individuals was carried out essentially as described by Dahlback [25]. 35 ml blood from each of three individuals of the same genotype was collected in acid-citrate-dextrose Vacutainer tubes (ACD-B, Becton Dickinson, Franklin Lakes, NJ). PDP was prepared as described above. PDP samples of the same genotypes were pooled and one tablet of protease inhibitor cocktail (Complete, Mini, EDTA-free, Roche Diagnostics, Indianapolis, IN) was added to each 10 ml of pooled plasma. Barium citrate adsorption was carried out by the drop-wise addition of 0.8 ml 1 M BaCl2/10 ml plasma. After the mixture had been stirred for 1 h, the barium citrate was removed by centrifugation at 6,000 g for 10 min. Next, PEG-6,000 fractionation was performed. First, solid PEG-6,000 (Sigma-Aldrich, St. Louis, MO) was added to the supernatant to obtain a final concentration of 80 g/l. After stirring for 1 h, the precipitate was removed by centrifugation at 6,000 g for 10 min. Solid PEG-6,000 was added to the supernatant, again, to bring the PEG concentration to 120 g/l. The solution was stirred for 1 h, then, the precipitate was collected by centrifugation (6,000 g, 10 min). The FV containing plasma precipitate was washed twice with 120 g/l PEG-6,000 in dist. water. The whole preparation procedure was carried out at 4 °C. The precipitate containing wild type FV or FVLeiden was dissolved in onetenth of the original plasma volume of immunodepleted FV deficient plasma (Technoclone, Vienna, Austria). The FV activity of the mixture was determined by one-stage clotting assay, and it was set to 90% by further dilution in FV deficient plasma in both cases.
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Fig. 3. The effect of recombinant human thrombomodulin (rhTM) on the time-course of FXIII activation in plasma samples from individuals of different FV genotypes. FXIII-A’ in each sample was expressed as percentage of the maximum amount of activated FXIII-A incorporated into the clot as determined by quantitative densitometry. Results obtained in the presence of rhTM are depicted by solid symbols and broken lines, while empty symbols with solid lines represent results obtained in the absence of rhTM. Horizontal dotted lines demonstrate when 50% of FXIII-A became truncated. Representatives of 5 experiments with similar results are shown for each genotype.
Factor XIII activation in plasma samples As the activation of FXIII takes place exclusively on the surface of fibrin and after its formation FXIIIa remains associated with fibrin [14], the activation of FXIII was measured in recovered fibrin clots. Fibrin clots were generated by the activation of tissue factor (TF) pathway in the presence and absence of recombinant human TM (rhTM, American Diagnostica, Stamford, CT). An rhTM concentration that inhibits thrombin generation in the plasma of FV wild type individuals by approximately 50% was selected as described by Dielis
et al. [26]. Using the selected rhTM concentration the thrombin generation was determined in the plasma of FV wild type individuals and 45.4% mean inhibition (range 40-61%) by rhTM was obtained as calculated from the AUC (area under the curve; also referred to as endogen thrombin potential) values. Aliquots of 150 μl PDP were incubated with 40 μl activator cocktail at 37 °C for various intervals and the reaction was stopped by an equal volume of inhibitor cocktail. To produce an activator cocktail Technothrombin® TGA RC Low (Technoclone GmbH, Vienna, Austria) was dissolved in 62.5 mM CaCl2, with or without 7.5 nM rhTM. The dissolved Technothrombin® TGA RC Low contained ~ 5 pM recombinant human TF, low concentration of phospholipid micelles in Tris-Hepes-NaCl buffer. The concentration of rhTM used in the experiments was established in preliminary experiments. The selected rhTM concentration (final concentration in the sample: 1.5 nM) inhibited activator cocktailinduced thrombin generation in normal pooled plasma by 50%. Thrombin generation was measured by S-2238 chromogenic substrate (Chromogenix, Lexington, MA). The inhibitor cocktail contained 20 mM benzamidine, 0.1 mM D-phenylalanyl-L-prolyl-L-arginin chloromethyl ketone (PPACK), 2 mM iodoacetamide, 50 mM εaminocaproic acid, 100 mM NaCl, 50 mM EDTA in 50 mM HEPES buffer, pH 7.5 to block thrombin and FXIIIa, and to prevent fibrinolysis [14]. The reaction was stopped at various intervals after the initiation of coagulation by the activator cocktail. Polymerized fibrin, if present, was fully recovered by centrifugation of the formed clots; the clots were then exhaustively washed with physiological NaCl and dissolved in SDS PAGE sample buffer.
SDS-PAGE and Western blotting
Fig. 4. The effect of FVLeiden genotype on the lag time required for 50% activation of FXIII (T1/2) in the presence and absence of recombinant human thrombomodulin (rhTM) (A) and on the difference of T1/2 values measured in the presence and absence of rhTM (T1/2rhTM+)-(T1/2rhTM+) (B). The bars represent the mean differences of T1/2 values obtained with (T1/2rhTM+) and without (T1/2rhTM-) rhTM. Values were calculated from the results of 5 individuals in each genotype. Error bars depict SEM, w.t.: FV wild type individuals, het.: FVLeiden heterozygotes , hom.: FVLeiden homozygotes. *p b 0.05, **p b 0.001.
SDS-PAGE of dissolved fibrin samples and Western blotting for FXIII-A were performed as described previously [14]. The extent of FXIII activation was monitored by Western blotting using affinity purified sheep antihuman FXIII-A antibody as primary antibody (Affinity Biologicals, Ancaster, Canada). The immunoreaction was developed by biotinylated rabbit anti-sheep IgG and avidin-biotinylated peroxidase complex (components of Vectastain ABC kit, Vector, Burlingame, CA, USA) and it was visualized by enhanced chemiluminescence detection (ECL Plus+, Amersham, Little Chalfont, UK) according to the manufacturer's instructions. The determination of fibrin bound activated FXIII-A was used as parameter to study the kinetics of FXIII activation. The amount of truncated, proteolytically activated FXIII-A (FXIII-A’) was determined in each sample by quantitative densitometry using GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, USA) and expressed as percentage of the maximal amount of FXIII-A’ formed in the clot. To compare the effect of rhTM on the rate of FXIII activation in different FV genotypes quantitatively, the time required for half maximal activation of FXIII was calculated (T1/2 values).
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Statistical analysis Statistical analysis was performed using GraphPad Prism software (La Jolla, CA). Kruskal-Wallis one-way analysis of variance was used to determine differences among groups. The extent of significance was determined by Mann-Whitney U test; p b 0.05 was considered as significant. Results Figs. 1–3 show the effect of rhTM on FXIII activation on representative plasma samples from selected individuals of different FV genotypes while on Fig. 4 the individual results are summarized. Western blots demonstrating the proteolytic activation of FXIII in the plasma clots of a FV wild type individual and a FVLeiden homozygote are shown on Figs. 1A and 2A. The kinetics of FXIII activation was followed by quantitative densitometry of Western blots (Figs. 1B and 2B) and the results were expressed as percentage of maximal FXIII activation (Fig. 3). To be able to compare the effect of rhTM on FXIII activation in the plasma samples of individuals with different FV genotypes quantitatively, the mean time required for the activation of half of the FXIII molecules present in the plasma (T1/2 FXIII activation) was determined and the difference of T1/2 values measured in the presence and absence of rhTM, (T1/2 rhTM+)-(T1/2 rhTM-), was calculated (Fig. 4). In the absence of rhTM no relevant differences were seen in the rate or in the pattern of FXIII activation in the plasma of individuals with different FV genotype (Figs. 1–3) and the T1/2 of FXIII activation did not differ significantly among genotypes (Fig. 4A). In the plasma of wild type individuals, the activation of FXIII occurred considerably later in the presence of rhTM than in its absence (Figs. 1, 3, 4). The effect of rhTM was considerably less in the case of heterozygous and homozygous individuals (Figs. 2–4). The T1/2 values for FXIII activation measured in the presence of rhTM were more prolonged in the plasma samples of wild type individuals than in the plasma samples of FVLeiden heterozygotes or homozygotes, but, due to considerable individual variation and the low sample number, the differences among genotypes were not statistically significant (Fig. 4A). However, when homozygotes and heterozygotes were grouped, the difference between FVLeiden carriers and wild type individuals became significant. As individual variations in FXIII activation may partially obscure the genotype-dependent effect of rhTM, the delay in T1/2 activation of FXIII, caused by rhTM was calculated for each individual and the effect of rhTM was expressed as the difference between T1/2 values obtained in its presence (T1/2rhTM+) and absence (T1/2rhTM-) (Fig. 4B). The mean difference between T1/2rhTM + and T1/2rhTM- values was significantly more pronounced in
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the case of FV wild type individuals than in the cases of FVLeiden heterozygotes and homozygotes (175 sec, 39 sec and 25 sec, respectively). Although the difference between heterozygotes and homozygotes was noticeable, it was not statistically significant. FVLeiden homozygotes, wild type for FXIII Val34Leu polymorphism and not on anticoagulant treatment, were very hard to recruit and five was the maximal number which we were able to select for the study from over three thousands genotyped individuals. For this reason and in order to eliminate individual variations that influence clot formation we also followed a different approach. FV deficient plasma was supplemented with FV isolated from pooled plasma of individuals either wild type or homozygous for FVLeiden. These supplemented plasma samples were used to study how FV of different genotypes influences the effect of rhTM on FXIII activation in the very same plasmatic environment (Fig. 5). In the absence of rhTM, in the plasma with FVLeiden FXIII activation occurred somewhat earlier than in the plasma with wild type FV, which might be related to some differences in the composition of the two partially purified FV preparations. In FV deficient plasma supplemented with wild type FV rhTM significantly delayed FXIII activation, the difference between T1/2 values was 185 sec. In the plasma supplemented with FVLeiden the effect of rhTM was more moderate (the difference between T1/2 values was only 65 sec). In the followings we tested how rhTM influences the cross-linking of fibrin chains in plasma samples from individuals of different FV genotypes. rhTM considerably delayed the dimerization of fibrin γchains in plasma samples containing wild type FV, but was much less effective in the plasma of FVLeiden homozygotes (Fig. 6A). The effect of rhTM on the formation of fibrin α-chain polymers seemed to follow a similar tendency (see partially polymerized α-chains on the upper part of the gel), however the slow formation of fully polymerized αchain polymers was out of the timeframe of the experiments and quantitative measurement could not be performed. Similarly to the evaluation of FXIII activation, the time intervals required for half maximal γ-chain dimerization were used for quantitative comparisons. In the plasma samples of wild type individuals there was a considerable difference between T1/2 obtained in the presence and absence of rhTM, while in FVLeiden carriers only slight differences could be observed (Fig. 6B). In the plasma samples from FVLeiden homozygotes rhTM delayed γ-chain dimerization to a lesser extent than in the plasma of heterozygotes, but the difference was not statistically significant. Discussion TM could influence FXIII activation by direct and an indirect mechanisms. In the absence of fibrinogen high concentration of thrombin
Fig. 5. Proteolytic activation of FXIII in FV deficient plasma supplemented by crude FV prepared from plasma of FV wild type or FVLeiden homozygous individuals. Washed plasma clots were recovered at various times after the activation of coagulation in the absence or presence of rhTM. The proteolytically activated truncated form of FXIII-A (FXIII-A’) recovered in the clot was detected by Western blotting and quantified by quantitative densitometry. FXIII-A’ in each sample was expressed as percentage of the maximum amount of activated FXIII-A incorporated into the clot. The results are the means of two parallel experiments with practically identical results. Results obtained in the presence of rhTM are depicted by solid symbols and broken lines, while empty symbols with solid lines represent results obtained in the absence of rhTM. Dotted lines demonstrate when 50% of FXIII-A became truncated.
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Fig. 6. The effect of recombinant human thrombomodulin (rhTM) on fibrin crosslinking in plasma from individuals of different FVLeiden genotypes. (A) Western blots demonstrating fibrin cross-linking in plasma samples of a FV wild type individual and a FVLeiden homozygote in the absence and presence of rhTM. Washed plasma clots recovered at various time after the activation of coagulation were analyzed by SDS PAGE. Both gels are representatives of experiments with the plasma samples of five individuals. Numbers under the individual bands on the gels indicate the time elapsed after the initiation of coagulation. Fbg.: fibrinogen standard. Fibrin(ogen) chains and their cross-linked products, γ-chain dimers (γ-γ) and polymerized α-chains (αn) are indicated. (B) The effect of FVLeiden genotype on the difference in the lag time required for the dimerization of 50% of fibrin γ-chain in the presence (T1/2rhTM+) and in the absence (T1/2rhTM-) of rhTM. The percentage of dimerized γ-chains as compared to the total amount of γ-chain dimers was determined by quantitative densitometry Error bars depict SEM, w.t.: wild type, het.: heterozygous, hom.: homozygous for FVLeiden mutation. *p b 0.05, **p b 0.001.
Gly38 and accelerates the thrombin-induced cleavage of FXIII-A [30,33]. Consequently much lower concentration of thrombin is sufficient to activate FXIII in plasmatic conditions. Here TM exerts its effect primarily through different mechanisms [34]. It competes with fibrin for the binding of exosite 1 in thrombin. As the binding of fibrin to certain amino acid side-chains present in exosite 1 is essential to its cofactor activity on FXIII-A truncation by thrombin, TM inhibits the acceleration of FXIII activation on the surface of fibrin [34]. An additional indirect mechanism, by which TM influences FXIII activation, operates through enhancing the thrombin-induced activation of protein C. Activated protein C proteolytically cleaves FVa and FVIIIa and down-regulates the promotion of thrombin generation by these activated clotting factors. Then, impaired thrombin generation might lead to decreased/delayed activation of FXIII. In our experiments the presence of rhTM caused a significant delay of FXIII activation and fibrin cross-linking only in the plasma of individuals wild type for FV. This finding suggests that in whole plasma it is not the direct inhibitory effect of TM on thrombin, but the suppression of thrombin generation and fibrin formation that play the primary role in delaying FXIII activation by TM, i.e., in the plasma from wild type individuals the formation of both the activator protease and the enhancer fibrin becomes impaired. In the case of FVLeiden the down-regulation of thrombin generation and fibrin formation through the APC pathway is lost and rhTM delayed FXIII activation and fibrin cross-linking only to a minimal extent. The effect of TM on FXIII activation and fibrin cross-linking might be important in the regulation of fibrinolysis and the impairment of this regulatory mechanism in FVLeiden carriers might contribute to the increased risk of venous thromboembolism conferred by the mutation. Indeed, one of the prothrombotic effects of FVLeiden is the inhibition of fibrinolysis, which has been demonstrated both in vitro [9] and in vivo in transgenic mice [35]. Two key down-regulators of fibrinolysis are TAFI and FXIII. TAFI, is activated by thrombin, a process that is more than 1,000-fold enhanced in the presence of TM. Activated TAFI cleaves off C-terminal lysines from partially digested fibrin, which would be required for efficient plasminogen activation (see reviewed in reference [36]). TM regulates TAFI by an intricate mechanism [10,37,38]. It increases the rate of TAFI activation by thrombin, but at the same time, through the activation of PC, it decreases the generation of thrombin required for TAFI activation. These effects of TM are concentration dependent, at low TM concentrations the activation of TAFI is predominantly stimulated, whereas at high concentrations, the decreased thrombin generation, due to the effect of APC on FV, results in decreased TAFI activation. Accordingly, at low TM concentrations its antifibrinolytic/prothrombotic, while at high TM concentrations its profibrinolytic/antithrombotic effect prevails. In the case of FVLeiden this antithrombotic effect is abrogated, i.e., TM at high concentration does not influence the activation of TAFI [10]. Similarly, FVLeiden also abrogates the profibrinolytic/antithrombotic effect of TM exerted through FXIII activation and fibrin cross-linking. The diminished delaying effect of TM on FXIII activation in FVLeiden carriers could represent a novel downstream molecular mechanism that, in addition to the effect on TAFI activation, contributes to the increased risk of thrombosis in these individuals. Conflict of Interest Statement
is required for the activation of purified plasma FXIII and complex formation with TM impairs the FXIII activating capability of thrombin [27]. If fibrinogen is also present a different picture emerges. The activation of plasma FXIII is enhanced by polymerizing fibrin approximately 100-fold [28–32]. There is a clear correlation between fibrin formation and FXIII activation and in plasma the onset of fibrin formation is followed by the onset of FXIII activation [14]. It has also been shown that the activation of FXIII in plasma occurs only on the surface of polymerized fibrin [14]. The binding to fibrin makes the orientation of both FXIII and thrombin favorable for the proteolysis of FXIII-A at Arg37-
The authors state that they have no conflict of interest. Acknowledgements This study was supported by grants from the Hungarian National Research Fund (OTKA-NKTH CNK80776), from the Hungarian Academy of Sciences (TKI 227), from the Hungarian Ministry of Health (ETT) and by the TÁMOP 4.2.1./B-09/1/KONV-2010-0007 project. Z.B. was supported by the Bolyai Foundation.
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