Genetic polymorphism of coagulation factor XIII B subunit in the Japanese population: description of three new rare alleles

Polyacrylamide gel isoelectric focusing (PAGIEF) of neuraminidase-treated EDTA plasma samples followed by electroblotting with enzyme immunoassay was performed to further investigate coagulation factor XIII B subunit (FXIII B) polymorphism. In 435 Japanese subjects PAGIEF patterns of FXIII B were classified into five common and three rare allotypes. This suggested that the FXIII B*2 allele existed in the Japanese population in the same manner as in Caucasians. Three new rare allotypes were considered to be controlled by three rare alleles which were designated FXIII B*13, FXIII B*14, and FXIII B*15, respectively. The gene frequencies calculated from 435 Japanese subjects were FXIII B*1 = 0.2977, FXIII B*2 = 0.0184, FXIII B*3 = 0.6805, FXIII B*13 = 0.0011, FXIII B*14 = 0.0011, and FXIII B*15 = 0.0011, respectively.     

Mapping of factor XIII solvent accessibility as a function of activation state using chemical modification methods

The transglutaminase Factor XIII (FXIII) catalyzes the formation of covalent cross-links between adjacent noncovalently associated fibrin chains in blood coagulation. The resulting covalently cross-linked hard clot is much more mechanically stable and resistant to proteolytic degradation. FXIII is activated by the serine protease thrombin in the presence of calcium ions. Protein modification experiments involving the labeling of cysteine and lysine side chains of the enzyme were performed before and after activation of the enzyme in an effort to gain further insight into structural changes occurring during the activation of FXIII. The experiments revealed differences in the labeling patterns of nonactivated and activated FXIII. These differences result from the exposure or sequestration of specific cysteine or lysine residues when the enzyme is activated, either physiologically with thrombin or nonproteolytically by exposure to calcium. Of note is the acetylation of Lys 73 and Lys 221 upon activation. Both of these residues lie within possible substrate recognition regions of FXIII. The active site Cys 314 is consistently alkylated in the activated enzyme, as is Cys 409, located near the dimer interface. Within the beta-barrel 2 domain of FXIII, Cys 695 becomes alkylated in activated FXIII. Within the same domain, an acetylated Lys (677 or 678), which is observed in the zymogen, cannot be found in the activated enzyme. The results provide a more extensive view of FXIII activation than has been previously available.     

Reversible activation of cellular factor XIII by calcium

Factor XIII (FXIII) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets and macrophages. Plasma FXIII is activated by thrombin cleavage (FXIIIa*) and acts in the final stages of blood coagulation cascade. In contrast, the function and activation of cellular FXIII are less characterized. Cellular FXIII relies on a conformational activation of the protein. The nonproteolytic activation of FXIII to FXIIIa° induced by Ca(2+) alone is well known, but up until now it has been discussed under which conditions the process can be induced and whether it can be reversed. Here, we study the nature of the Ca(2+)-induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIa° because they rely on occurrence of enzyme activity or on active site Cys-314 solvent accessibility. Therefore, an analytical HPLC method was developed that separates zymogen recombinant FXIII (rFXIII) from rFXIIIa°. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca(2+) concentration, buffer, and salt components. Moreover, it is established that Ca(2+) activation of rFXIII is fully reversible, and only 2-5 mm CaCl(2) is sufficient to retain full rFXIIIa° activity. However, below 2 mm CaCl(2) the rFXIIIa° molecule deactivates. The deactivated molecule can subsequently undergo a new activation round. Furthermore, it is demonstrated that thermal stress of freeze-dried rFXIII can induce a new predisposed form that activates faster than nonstressed rFXIII.     

Inhibition of Clotting Factor XIII Activity by Nitric Oxide

The plasma factor XIII (FXIII) is a transglutaminase which catalyzes the cross-linking of fibrin monomers during blood coagulation. S-nitrosylation of protein sulfhydryl groups has been shown to regulate protein function. Therefore, to establish whether nitric oxide (NO) affects the enzymatic activity of FXIII, we studied the effect of the NO-donorS-nitroso-N-acetylpenicillamine (SNAP) in a blood coagulation testin vitro. High concentrations of SNAP were found to have inhibitory effects on clot formation. Moreover, specific formation of γ-dimers through the action of FXIII is selectively inhibited by high concentrations of SNAP, as revealed by Western blot. Purified activated FXIII and plasma preparations were then exposed to NO-donor compounds and the enzyme activity was assayed by measuring the incorporation of [³H] putrescine into dimethylcasein. The NO donors, SNAP, spermine-NO (SPER-NO) and 3-morpholinosydnonimine (SIN-1), and the NO-carrier, S-nitrosoglutathione (GSNO), inhibited FXIII activity in a dose-dependent manner, in both purified enzyme and plasma preparations. Titration of -SH groups of FXIII with [¹⁴C] iodoacetamide has shown that the number of titratable cysteines per monomer of FXIII decreased from 1 (in absence of NO donors) to 0 (in the presence of NO donors). These results demonstrate that blood coagulation FXIII is a target for NO bothin vitroandin vivo,and that inhibition occurs by S-nitrosylation of a highly reactive cysteine residue. In conclusion, we show that inhibition of FXIII activity by NO may represent an additional regulatory mechanism for the formation of blood clot with physio-pathological implications.     

Inhibition of factor XIII activation by an anti-peptide monoclonal antibody.

As the final enzyme in the coagulation cascade, activated fibrin stabilizing factor or factor XIII catalyzes the intermolecular cross-linking of fibrin chains. To study this enzyme in plasma, we derived a monoclonal antibody (MAb 309) against a peptide sequence (NH2-G-V-N-L-Q-E-F-C-COOH) in the thrombin activation site of factor XIII. Radioimmunoassays indicate that MAb 309 binds specifically to both platelet and plasma factor XIII. Peptide inhibition studies demonstrate that the MAb binds equally well to the factor XIII (FXIII) zymogen and the active form of FXIII (FXIIIa). In immunoblots of whole platelet lysates, MAb 309 binds only to FXIII and does not cross-react with other proteins. In saturation binding studies, the antibody shows a binding avidity of (1.75 +/- 0.35) x 10(9) M-1. MAb 309 also inhibited 99% of apparent FXIIIa activity in a standard transglutaminase assay. SDS-PAGE analysis of fibrin clots showed that MAb 309 inhibited fibrin gamma-gamma cross-linking. Moreover, MAb 309 accelerated the lysis of plasma clots, consistent with inhibition of fibrin-fibrin and fibrin-alpha 2-antiplasmin cross-linking. Immunoblotting experiments revealed that MAb 309 affected apparent FXIIIa activity by inhibiting the thrombin activation of the FXIII zymogen. In addition to its utility as a specific probe for the FXIII a-subunit, the strategy used to obtain MAb 309 may be used to generate MAbs that inhibit the activation of other coagulation factor zymogens.     

Modeling of factor XIII activation peptide (28-41) V34L mutant bound to thrombin

Factor XIII (FXIII) is a transglutaminase involved in blood coagulation. The enzyme is activated by thrombin cleaving the peptide bond R(37)-G(38). A common mutation V34L found in FXIII has been correlated with protection from myocardial infarction. Also FXIII V34L is activated more quickly than the wild type. In the present study, FXIII (28-41) V34L mutant peptide bound to thrombin has been modeled and molecular dynamics simulations were carried out using Insight II. An average structure was calculated after simulation. The structure showed significant difference from the crystal structure of the wild type FXIII (28-37) peptide bound to thrombin. In the crystal structure the peptide adopts a folded conformation in such a way that the hydrophobic side chains of V(29) and V(34) occupy the apolar binding site of thrombin. The modeled V34L peptide adopts a significantly different conformation and only the bulkier L(34) occupies the apolar binding site while V(29) side chain is exposed to the bulk solvent. Hence, this may speed up the release of FXIII from thrombin after its activation.     

FXIII polymorphisms, fibrin clot structure and thrombotic risk

Fibrin clot structure is highly dependent on factor XIII activity. Activated FXIII catalyzes the formation of the peptide bonds between the gamma and alpha chains in noncovalently bound fibrin polymers and incorporates various adhesive and antifibrinolytic proteins into the final fibrin clot. In the absence of activated FXIII, clots are unstable and susceptible to fibrinolysis. Several studies have examined the effects of FXIII polymorphisms on final fibrin clot structure and clinical thrombotic risk. The Val34Leu FXIII polymorphism is associated with increased activation by thrombin. In the presence of saturating thrombin concentrations, however, FXIIIa specific enzyme activity is not affected by genetic polymorphisms. Fibrin clots formed in the presence of the FXIII 34Leu polymorphisms do tend to be thinner and less porous, however. The effects of prothrombin concentrations on clot structure have suggested that thinner clots are more resistant to fibrinolysis and associated with increased thrombotic risk. Most clinical studies of 34Leu FXIII carriers, however, have demonstrated a lower incidence of both venous and arterial thrombosis in carriers of the mutant allele compared to Val/Val carriers. One recent study has suggested that the interactions between FXIII phenotype and plasma fibrinogen concentrations significantly influence clinical thrombotic risk.     

Probing interactions between the coagulants thrombin, Factor XIII, and fibrin(ogen)

Thrombin cleaves fibrinopeptides A and B from fibrinogen leading to the formation of a fibrin network that is later covalently crosslinked by Factor XIII (FXIII). Thrombin helps activate FXIII by catalyzing hydrolysis of the FXIII activation peptides (AP). In the current work, the role of exosites in the ternary thrombin-FXIII-fibrin(ogen) complex was further explored. Hydrolysis studies indicate that thrombin predominantly utilizes its active site region to bind extended Factor XIII AP (FXIII AP 33-64 and 28-56) leaving the anion-binding exosites for fibrin(ogen) binding. The presence of fibrin-I leads to improvements in the K(m) for hydrolysis of FXIII AP (28-41), whereas peptides based on the cardioprotective FXIII V34L sequence exhibit less reliance on this cofactor. Surface plasmon resonance measurements reveal that d-Phe-Pro-Arg-chloromethylketone-thrombin binds to fibrinogen faster than to FXIII a(2) and dissociates from fibrinogen more slowly than from FXIII a(2). This system of thrombin exosite interactions with differing affinities promotes efficient clot formation.     

Probing thrombin's ability to accommodate a V34F substitution within the factor XIII activation peptide segment (28-41).

In blood coagulation, thrombin helps to activate factor XIII (FXIII) by cleaving the activation peptide (AP) at the R37-G38 peptide bond. The common polymorphism V34L yields a FXIII that is more easily activated than the wild type enzyme. Peptides based on the FXIII (28-41) (28TVELQGVVPRGVNL41) sequence serve as an important model system to evaluate the substrate specificity of thrombin and thus how to regulate FXIII activation. Our previous kinetic and nuclear magnetic resonance (NMR) studies have suggested that the P4-P1 amino acids on this FXIII segment provide key anchors to the thrombin active site surface. Furthermore, the most effective amino acid to have at the P4 position is a leucine. In the current work, a peptide containing V34F was examined to probe the ability to accommodate an aromatic residue at this position. Kinetic parameters for thrombin-catalyzed hydrolysis of FXIII AP (28-41) V34F are comparable with that of the wild type V34. One-dimensional proton line-broadening studies reveal that the 34FVPR37 segment encompassing the P4-P1 positions makes the most contact with the thrombin surface. Two-dimensional transferred-nuclear overhauser effect spectroscopy (NOESY) studies indicate that when the peptide is bound to thrombin, the F34 aromatic ring is oriented to promote P4-P2 interactions with P36. This characteristic has been viewed as a hallmark for V34L. An ability to generate this interaction may promote the ability of FXIII AP (28-41) V34F to remain a viable substrate for thrombin.     

Immunohistochemical detection of factor XIII subunit a in histiocytes of human uterus

As spontaneous abortion is a frequent finding in females with Factor XIII (FXIII) deficiency it has been presumed that this clotting factor is essential to normal pregnancy. FXIII subunit a (FXIII A) has been demonstrated in the homogenate of human uterus, but no information on its cellular distribution has been published, so far. In the present study first FXIII A was detected in paraformaldehyde-fixed, paraffin-embedded sections of human uterus by immunoperoxidase technique. Cells containing FXIII A were localized between collagen fibrils stained by Picrosirius Red F3B in the connective tissue. To characterize them the immunofluorescent detection of FXIII A was combined by the visualization of different marker antigens of monocytes and macrophages recognized by Leu-M3, RFD7, anti-HLA-DR and DAKO-anti-macrophage monoclonal antibodies on frozen sections. The coexpression of FXIII A with monocyte and macrophage differentiation marker antigens clearly proves that cells containing FXIII A in the uterus are monocyte-derived tissue macrophages. The results well agree with our previous findings demonstrating FXIII A in human monocytes and different types of macrophages. On the basis of these results, the presence of FXIII A does not seem to be a specificity of the uterus but a characteristic of monocyte/macrophage cell line including tissue macrophages, in general.     

Thrombin hydrolysis of V29F and V34L mutants of factor XIII (28-41) reveals roles of the P(9) and P(4) positions in factor XIII activation

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The more easily activated factor XIII V34L has been correlated with protection from myocardial infarction. V34L and V29F factor XIII mutant peptides were designed to further characterize substrate binding to thrombin. HPLC kinetic studies have been carried out on thrombin hydrolysis of FXIII activation peptide (28-41), FXIII (28-41) V34L, FXIII (28-41) V29F, and FXIII (28-41) V29F V34L. The V34L mutations lead to improvements in both K(m) and k(cat) whereas the V29F mutation primarily affects K(m). Interactions of the peptides with thrombin have been monitored by 1D proton line broadening NMR and 2D transferred NOESY studies. The results were compared with previously published X-ray crystal structures of thrombin-bound fibrinogen Aalpha (7-16), thrombin receptor PAR1 (38-60), and factor XIII (28-37). In solution, the (34)VVPR(37) and (34)LVPR(37) segments of the factor XIII activation peptide serve as the major anchor points onto thrombin. The N-terminal segments are proposed to interact transiently with the enzyme surface. Long-range NOEs from FXIII V29 or F29 toward (34)V/LVPR(37) have not been observed by NMR studies. Overall, the kinetic and NMR results suggest that the factor XIII activation peptide binds to thrombin in a manner more similar to the thrombin receptor PAR1 than to fibrinogen Aalpha. The V29 and V34 positions affect, in different ways, the ability of thrombin to effectively hydrolyze the activation peptide. Mutations at these sites may prove useful in controlling factor XIII activation.     

Immunohistochemical detection of factor XIII subunit a in histiocytes of human uterus

Summary As spontaneous abortion is a frequent finding in females with Factor XIII (FXIII) deficiency it has been presumed that this clotting factor is essential to normal pregnancy. FXIII subunit a (FXIII A) has been demonstrated in the homogenate of human uterus, but no information on its cellular distribution has been published, so far. In the present study first FXIII A was detected in paraformaldehyde-fixed, paraffin-embedded sections of human uterus by immunoperoxidase technique. Cells containing FXIII A were localized between collagen fibrils stained by Picrosirius Red F3B in the connective tissue. To characterize them the immunofluorescent detection of FXIII a was combined by the visualization of different marker antigens of monocytes and macrophages recognized by Leu-M3, RFD7, anti-HLA-DR and DAKO-anti-macrophage monoclonal antibodies on frozen sections. The coexpression of FXIII A with monocyte and macrophage differentiation marker antigens clearly proves that cells containing FXIII A in the uterus are monocyte-derived tissue macrophages. The results well agree with our previous findings demonstrating FXIII A in human monocytes and different types of macrophages. On the basis of these results, the presence of FXIII A does not seem to be a specificity of the uterus but a characteristic of monocyte/macrophage cell line including tissue macrophages, in general.     

Genetic and phenotypic polymorphisms of the A subunit of Coagulation factor XIII in Japanese population

To clarify whether genetic polymorphisms in exon 14 of Coagulation factor XIII A-subunit gene (FXIIIA) affect phenotype expressions, we studied genetic polymorphisms in exon 14 of FXIIIA in a Japanese population and the relationship between the genetic polymorphisms and phenotype expression. Genetic polymorphisms in exon 14 of FXIIIA of 144 unrelated Japanese were analyzed by single-strand conformation polymorphism. Plasma FXIIIA antigen concentrations, FXIII activities, and phenotype were also determined by two-dimensional electrophoresis. The frequencies of the three genotypes, the homozygote (AD), the homozygote (BC) and the heterozygote (AD/BC), were 77.1, 0.7, and 22.2%, respectively. The gene frequencies of AD and BC were 0.88 and 0.12. We detected AD (GTT x GAG) and BC (ATT x CAG) at codon 650 and 651 of exon 14. There were no significant differences of FXIIIA antigen concentrations and FXIII activities between these genotypes. We detected three pl differences among them as being pls of 5.3, 5.6, 5.8 in the homozygote (AD) and the heterozygote (AD/BC), and a pl of 5.8 in the homozygote (BC). These polymorphisms affected isoelectric mobility, but did not affect protein levels, enzyme activities, or the molecular weight of FXIII.     

Modulation of cellular biosynthetic activity in the retracting collagen lattice

When included in a free floating collagen lattice, several types of cells and fibroblasts attach to the collagen polymers, retract the gel, and their biosynthetic activity is repressed. Under similar conditions transformed pulmonary epithelial rat (PER) cells are unable to attach to the fibers and to significantly retract the lattice. Retraction can be induced by adding fibronectin (fn) and factor XIII (FXIII) together. This effect is fibronectin dose dependent and observed with a maximum efficiency for FXIII concentrations of 0.1 U/ml and above. Fibronectin or FXIII alone has only a limited effect on retraction. This experimental model allowed us to study the biosynthetic activity of PER cells under various degrees of cell interaction (control less than FXIII less than fn less than fn + FXIII) with their three-dimensional collagen support. The more the cells interacted with their support and retracted the gel, the more protein and collagen synthesis were reduced. This effect was observed for the products deposited in the cell layer and for those released in the medium. Increasing collagen concentration in a nonretracting lattice to a final density obtained in a maximally retracted lattice resulted in a much lower repression of biosynthetic activity. Fn and FXIII added at the same concentrations in monolayer cultures did not produce significant modification in biosynthetic activities. We propose that the regulation of the biosynthetic activity of adherent cells contracting the lattice is related to mechanical information resulting from the interactions between the cells and their support.     

Design of Factor XIII V34X activation peptides to control ability to interact with thrombin mutants

Thrombin helps to activate Factor XIII (FXIII) by hydrolyzing the R37-G38 peptide bond. The resultant transglutaminase introduces cross-links into the fibrin clot. With the development of therapeutic coagulation factors, there is a need to better understand interactions involving FXIII. Such knowledge will help predict ability to activate FXIII and thus ability to promote/hinder the generation of transglutaminase activity. Kinetic parameters have been determined for a series of thrombin species hydrolyzing the FXIII (28-41) V34X activation peptides (V34, V34L, V34F, and V34P). The V34P substitution introduces PAR4 character into the FXIII, and the V34F exhibits important similarities to the cardioprotective V34L. FXIII activation peptides containing V34, V34L, or V34P could each be accommodated by alanine mutants of thrombin lacking either the W60d or Y60a residue in the 60-insertion loop. By contrast, FXIII V34F AP could be cleaved by thrombin W60dA but not by Y60aA. FXIII V34P is highly reliant on the thrombin W215 platform for its strong substrate properties whereas FXIII V34F AP becomes the first segment that can maintain its K(m) upon loss of the critical thrombin W215 residue. Interestingly, FXIII V34F AP could also be readily accommodated by thrombin L99A and E217A. Hydrolysis of FXIII V34F AP by thrombin W217A/E217A (WE) was similar to that of FXIII V34L AP whereas WE could not effectively cleave FXIII V34P AP. FXIII V34F and V34P AP show promise for designing FXIII activation systems that are either tolerant of or greatly hindered by the presence of anticoagulant thrombins.     

A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma

Based on the iso-peptidase activity of human plasma FXIII, a novel fluorometric assay that determines FXIII concentrations in human plasma below 0.05 IU/ml is introduced. We considered a peptide sequence derived from alpha(2)-antiplasmin (n =12) to yield high sensitivity. Peptide Abz-NE(Cad-Dnp)EQVSPLTLLK exhibits a K(m) value of 19.8+/-2.8 microM and is used in a concentration of 50 microM. The assay design is suitable for measurements in cuvettes (1 ml volume) as well as for the microtiter plate (MTP) format (0.2 ml volume). It provides linear dose-response curves over a wide range of FXIII concentrations (0.05-8.8 IU/ml). The assay was validated with respect to precision, detection and quantitation limits, accuracy/specificity, linearity, and range. A comparison of the fluorometric assay with the photometric assay for FXIII determinations in plasma pools as well as single donor plasma revealed suitability of the fluorometric assay for FXIII determination in plasma of healthy individuals. FXIII concentrations in plasma samples of patients with severe FXIII deficiency are discussed in the context of FXIII antigen levels. These assays correlate well in the critical range below 0.1 IU/ml, whereas the photometric assay may overestimate residual FXIII activity in severe FXIII-deficient patients.     

Accumulation of cells containing factor XIII subunit a around the foci of intense fibrosis in human epulides

Summary On the basis of clinical and biochemical findings, Factor XIII subunita (FXIII A) has been conjectured to play an important role in fibrotic processes. Epulis samples at different stages of fibrotic tissue formation were used as a model system for studying the localization and tissue distribution of FXIII A during the course of connective tissue generation. Marker characteristics of cells containing FXIII A (FXIII A⁺ cells) were determined as well. In double immunofluorescent labelling systems, FXIII A was localized in monocyte-derived (CD-14⁺), activated (HLA-DR⁺), and phagocytosing (Ki-M7⁺) tissue macrophages, which are widely distributed homogeneously in granulation tissues, but start to accumulate around foci of fibrosis as soon as the foci appear. During the relatively long process of fibrosis, FXIII A⁺ macrophages continuously decrease in number, and their morphological appearance changes from stellate to spindle-shaped. The nuclei of these cells were not labelled by Ki-67 monoclonal antibody; this indicating that they represent a non-proliferating cell population in the connective tissue stroma. The present findings may help to link theories concerning the role of FXIII A and those of macrophages in the connective tissue formation so far found separately in the literature.     

ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats

The affinities of Factor XIII (FXIII), Factor XIIIa (FXIIla), and cellular transglutaminase (Tg) for fibrinogen (Fgn), fibrin (Fbn), and fibronectin (Fn) were compared using a solid-phase binding assay. Initial rates of binding were as follows: FXIII bound Fbn 3-fold more than Fgn. FXIII did not bind Fn till 20 min. Increasing the ligands concentrations and binding time, resulted in weak binding of FXIII to Fn. FXIIla bound Fbn 2-fold more than Fgn and 28-fold more than Fn. Tg bound Fn 130-fold more than either Fgn or Fbn. At equilibrium, the extent of binding was determined to be as follows: FXIII bound Fbn 3–15-fold more than Fgn and 8-fold more than Fn. FXIIIa bound Fgn and Fbn equally and 12–25-fold more than Fn. FXIIla bound Fgn or Fbn 2-fold and 25-fold greater than FXIII-Fbn and FXIII-Fgn interactions, respectively. Tg bound about equally to Fgn and Fbn and 10–20-fold less than Fn. The K _d s for FXIIla binding to Fn, Fgn, and Fbn were 100, 23, and 19 nM, respectively. The K _d for Tg binding to Fn was 6.5 nM. The binding hierarchies are: [Tg-Fn]>[FXIIIa-Fgn]=[FXIIIa-Fbn]>[FXIII-Fbn]>[FXIIIFgn]=[FXIIIa-Fn]>[Tg-Fbn]=[Tg-Fgn]>[FXIII-Fn]. Such hierarchies could regulate the cross-linkings by FXIIIa and Tg during hemostasis, wound healing, and cell adhesion.Abbreviations Tg cellular transglutaminase - FXIII coagulation factor XIII - FXIIla factor XIIIa (thrombin-activated FXIII) - Fgn human plasma fibrinogen - Fn human plasma fibronectin - Fbn human plasma fibrin (thrombin-cleaved fibrinogen) - ECM extracellular matrix     

The combined effect of fibrin formation and factor XIII A subunit Val34Leu polymorphism on the activation of factor XIII in whole plasma

The first step in the activation of blood coagulation factor XIII (FXIII) is the proteolytic cleavage of the potentially active A subunit (FXIII-A) by thrombin at Arg37-Gly38. Both fibrin formation and FXIII-A Val34Leu polymorphism influence the rate of proteolytic activation of purified factor XIII, however their relative importance and interaction in determining the time of onset and the rate of FXIII activation in whole plasma have not yet been explored. In the present study it was shown that in plasma, fibrin formation preceded the truncation of FXIII-A by thrombin, the activation process took place exclusively on the surface of newly formed fibrin and activated FXIII remained associated with the fibrin clot. The time of fibrin formation closely correlated with the time of FXIII activation, while there was no significant relationship between the time of FXIII activation and FXIII-A Val34Leu genotype. However, in the case of Leu34 variant the lag phase between fibrin formation and FXIII-A truncation was significantly shorter than in the case of Val34 variant. The results suggest that in whole plasma the onset of FXIII activation is determined by fibrin formation, while the rate of activation is modulated by Val34Leu polymorphism.     

Characterization of cells containing factor XIII subunita in benign and malignant buccal lesions

Summary In the present study, the distribution pattern and characteristics of cells containing Factor XIII subunita (FXIII A) have been studied in benign and malignant lesions of human buccal mucosa. Tissues from four irritation fibromas and three squamous cell carcinomas were studied by means of double immunofluorescent staining techniques in which the detection of FXIII A was combined with a reaction with CD14 (recognizing a monocyte/macrophage differentiation marker antigen), Mac 387 (reacting with a special subset of macrophages), anti-HLA-DR, Ki-M7 (labelling phagocytosing macrophages) or Ki-67 (visualizing a nuclear antigen associated with cell proliferation) monoclonal antibodies. FXIII A was detected in cells of the connective tissue stroma in both benign and malignant buccal lesions. The number of these FXIII A-reactive cells (FXIII A⁺ cells) increased considerably in the tumour tissues, in particular in those surrounding tumour cell clusters. FXIII A⁺ cells scattered in the fibromatous tissues were spindle-shaped, whereas in the tumour stroma, large stellate cells predominated, and round cells were likewise labelled around blood vessels. FXIII A⁺ cells were labelled with CD14 and Ki-M7 in both fibromatous and tumoural buccal mucosa; however, they failed to show any reaction with Ki-67. FXIII A⁺ cells accumulated in the tumour stroma reacted for HLA-DR as well. These results indicate that in both the benign and malignant buccal lesions FXIII A is contained in a subpopulation of tissue macrophages, which represents a monocyte-derived (CD14⁺) and phagocytosing (KiM7⁺) cell population. The accumulation of the FXIII A⁺ cells in the tumour stroma is believed to be a result of direct migration from the circulating blood. The FXIII A⁺ cells of the tumour stroma may be actively involved in both antigen presentation and matrix remodelling during tumour progression.