Transglutaminase 2 limits the extravasation and the resultant myocardial fibrosis associated with factor XIII-A deficiency.

BACKGROUND AND AIMS: Transglutaminase (TG) 2 and Factor (F) XIII-A have both been implicated in cardiovascular protection and repair. This study was designed to differentiate between two competing hypotheses: that TG2 and FXIII-A mediate these functions in mice by fulfilling separate roles, or that they act redundantly in this respect. METHODS: Atherosclerosis was assessed in brachiocephalic artery plaques of fat-fed mixed strain apolipoprotein (Apo)e deficient mice that lacked either or both transglutaminases. Cardiac fibrosis was assessed both in the mixed strain mice and also in C57BL/6J Apoe expressing mice lacking either or both transglutaminases. RESULTS: No difference was found in the density of buried fibrous caps within brachiocephalic plaques from mice expressing or lacking these transglutaminases. Cardiac fibrosis developed in both Apoe/F13a1 double knockout and F13a1 single knockout mice, but not in Tgm2 knockout mice. However, concomitant Tgm2 knockout markedly increased fibrosis, as apparent in both Apoe/Tgm2/F13a1 knockout and Tgm2/F13a1 knockout mice. Amongst F13a1 knockout and Tgm2/F13a1 knockout mice, the extent of fibrosis correlated with hemosiderin deposition, suggesting that TG2 limits the extravasation of blood in the myocardium, which in turn reduces the pro-fibrotic stimulus. The resulting fibrosis was interstitial in nature and caused only minor changes in cardiac function. CONCLUSIONS: These studies confirm that FXIII-A and TG2 fulfil different roles in the mouse myocardium. FXIII-A protects against vascular leakage while TG2 contributes to the stability or repair of the vasculature. The protective function of TG2 must be considered when designing clinical anti-fibrotic therapies based upon FXIII-A or TG2 inhibition.

Functional factor XIII-A is exposed on the stimulated platelet surface.

Factor XIII (FXIII) stabilizes thrombi against fibrinolysis by cross-linking α2-antiplasmin (α2AP) to fibrin. Cellular FXIII (FXIII-A) is abundant in platelets, but the extracellular functions of this pool are unclear because it is not released by classical secretion mechanisms. We examined the function of platelet FXIII-A using Chandler model thrombi formed from FXIII-depleted plasma. Platelets stabilized FXIII-depleted thrombi in a transglutaminase-dependent manner. FXIII-A activity on activated platelets was unstable and was rapidly lost over 1 hour. Inhibiting platelet activation abrogated the ability of platelets to stabilize thrombi. Incorporating a neutralizing antibody to α2AP into FXIII-depleted thrombi revealed that the stabilizing effect of platelet FXIII-A on lysis was α2AP dependent. Platelet FXIII-A activity and antigen were associated with the cytoplasm and membrane fraction of unstimulated platelets, and these fractions were functional in stabilizing FXIII-depleted thrombi against lysis. Fluorescence confocal microscopy and flow cytometry revealed exposure of FXIII-A on activated membranes, with maximal signal detected with thrombin and collagen stimulation. FXIII-A was evident in protruding caps on the surface of phosphatidylserine-positive platelets. Our data show a functional role for platelet FXIII-A through exposure on the activated platelet membrane where it exerts antifibrinolytic function by cross-linking α2AP to fibrin.

Substrates of Factor XIII-A: roles in thrombosis and wound healing.

FXIII (Factor XIII) is a Ca²+-dependent enzyme which forms covalent ϵ-(γ-glutamyl)lysine cross-links between the γ-carboxy-amine group of a glutamine residue and the ϵ-amino group of a lysine residue. FXIII was originally identified as a protein involved in fibrin clot stabilization; however, additional extracellular and intracellular roles for FXIII have been identified which influence thrombus resolution and tissue repair. The present review discusses the substrates of FXIIIa (activated FXIII) involved in thrombosis and wound healing with a particular focus on: (i) the influence of plasma FXIIIa on the formation of stable fibrin clots able to withstand mechanical and enzymatic breakdown through fibrin-fibrin cross-linking and cross-linking of fibrinolysis inhibitors, in particular α2-antiplasmin; (ii) the role of intracellular FXIIIa in clot retraction through cross-linking of platelet cytoskeleton proteins, including actin, myosin, filamin and vinculin; (iii) the role of intracellular FXIIIa in cross-linking the cytoplasmic tails of monocyte AT1Rs (angiotensin type 1 receptors) and potential effects on the development of atherosclerosis; and (iv) the role of FXIIIa on matrix deposition and tissue repair, including cross-linking of extracellular matrix proteins, such as fibronectin, collagen and von Willebrand factor, and the effects on matrix deposition and cell-matrix interactions. The review highlights the central role of FXIIIa in the regulation of thrombus stability, thrombus regulation, cell-matrix interactions and wound healing, which is supported by observations in FXIII-deficient humans and animals.

Critical role of factor XIII in the initial stages of carbon tetrachloride-induced adult liver remodeling.

The transglutaminase-mediated, covalent cross-linking of proteins is an essential step in tissue remodeling after injury. This process provides tissues with extra rigidity and resistance against proteolytic degradation. Plasma coagulation factor XIII (FXIII) is a transglutaminase that promotes cross-linking of the extracellular matrix (ECM) components fibrin and fibronectin to form a provisional matrix in response to tissue damage. However, the functional requirement for this FXIII-mediated cross-linked provisional matrix in adult tissue remodeling remains to be defined. Although it has been proposed that the formation FXIII-mediated fibrin-fibronectin provisional matrix is a critical step for ECM remodeling, we show in an FXIII subunit A-deficient murine model of acute liver injury that the lack of FXIII subunit A did not interfere with collagen reconstruction and resolution after liver injury. Furthermore, FXIIIA deficiency caused significantly increased hepatocyte apoptosis and a delay in hepatocyte regeneration after injury, which were accompanied by a significantly high induction of p53 expression. These findings suggest novel functions of FXIII that the FXIII-mediated covalently cross-linked matrix could promote survival signals for hepatocytes in adult tissue remodeling.

Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase.

In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin-pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction may influence the TSG-6/heavy Chain 2-mediated transfer of heavy chains observed during inflammation.

Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A.

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood.

Evidence that αC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers.

Fibrin fibers form the structural scaffold of blood clots and perform the mechanical task of stemming blood flow. Several decades of investigation of fibrin fiber networks using macroscopic techniques have revealed remarkable mechanical properties. More recently, the microscopic origins of fibrin's mechanics have been probed through direct measurements on single fibrin fibers and individual fibrinogen molecules. Using a nanomanipulation system, we investigated the mechanical properties of individual fibrin fibers. The fibers were stretched with the atomic force microscope, and stress-versus-strain data was collected for fibers formed with and without ligation by the activated transglutaminase factor XIII (FXIIIa). We observed that ligation with FXIIIa nearly doubled the stiffness of the fibers. The stress-versus-strain behavior indicates that fibrin fibers exhibit properties similar to other elastomeric biopolymers. We propose a mechanical model that fits our observed force extension data, is consistent with the results of the ligation data, and suggests that the large observed extensibility in fibrin fibers is mediated by the natively unfolded regions of the molecule. Although some models attribute fibrin's force-versus-extension behavior to unfolding of structured regions within the monomer, our analysis argues that these models are inconsistent with the measured extensibility and elastic modulus.

Impaired clot retraction in factor XIII A subunit-deficient mice.

Factor XIII (FXIII) is a plasma transglutaminase that cross-links fibrin monomers, alpha(2)-plasmin inhibitor, and so forth. Congenital FXIII deficiency causes lifelong bleeding symptoms. To understand the molecular pathology of FXIII deficiency in vivo, its knockout mice have been functionally analyzed. Because prolonged bleeding times, a sign of defective/abnormal primary hemostasis, were commonly observed in 2 separate lines of FXIII A subunit (FXIII-A) knockout mice, a possible role or roles of FXIII in platelet-related function was investigated in the present study. Although platelet aggregation induced by adenosine diphosphate or collagen was normal, clot retraction (CR) was lost in the platelet-rich plasma (PRP) of FXIII-A knockout mice. In contrast, there was no CR impairment in the PRP of tissue transglutaminase-knockout mice compared with that of wild-type mice. Furthermore, a transglutaminase inhibitor, cystamine, halted CR in the PRP of wild-type mice. These results indicate that the enzymatic activity of FXIII is necessary for CR, at least in mice.

New insights into the expression and role of platelet factor XIII-A.

BACKGROUND: The A subunit of factor XIII (FXIII-A) functions as an intracellular transglutaminase (TG) in the megakaryocyte/platelet lineage, where it probably participates in the cytoskeletal remodeling associated with cell activation. However, so far, the precise role of cellular FXIII (cFXIII) and the functional consequences of its absence in FXIII-A-deficient patients are unknown. OBJECTIVES AND METHODS: In this study, we used platelets from four patients with congenital deficiency of FXIII-A to study the role of cFXIII in platelet functions. RESULTS: We found that FXIII-A represents the only detectable source of TG activity in platelets and that the binding of fibrinogen in response to thrombin receptor agonist peptide (TRAP) stimulation was significantly reduced in platelets from the patients. In agreement with this, in control platelets, monodansyl-cadaverine (MDC), a competitive amino-donor for TGs, inhibited fibrinogen binding induced by TRAP in a dose-dependent manner. Moreover, upon adhesion to fibrinogen, normal platelets incubated with MDC as well as FXIII-A-deficient platelets showed a distinct extension pattern with reduced lamellipodia and increased filopodia formation, suggesting a delay in spreading. CONCLUSIONS: These findings provide evidence for the direct involvement of cFXIII-dependent TG activity in the regulation of platelet functions.

Coagulation factor XIII-A. A flow cytometric intracellular marker in the classification of acute myeloid leukemias.

The association of coagulation factors with leukocytes have been demonstrated in several previous studies. This study was designed to study the sensitivity and specificity of factor XIII subunit A (FXIII-A) labelling in cultured myeloblastic and monoblastic cell lines and to investigate the intracytoplasmic expression of FXIII-A in de novo acute myeloid leukemia (AML) samples. Myeloblastic and a monoblastic cell lines were cultured and investigated for lineage specific maturation markers and FXIII-A expression. Furthermore, FXIII-A expression was investigated in 12 normal samples (7 bone marrow and 5 peripheral blood), 86 de novo AML samples and 6 chronic myelomonocytic leukemia (CMML) samples. In the monoblastic MonoMac6 cell line the appearance of FXIII-A preceded that of CD14 while it remained negative in the myeloblastic PLB-985 cell line throughout its maturation period. Among the AML samples the average frequency of FXIII-A positive cells in myeloblastic leukemia samples was below 10%, while in M4 and M5AML samples it was above 50% and was significantly higher than the generally used CD14 marker (p < 0.0001). In the AML M4 and M5 cases, FXIII-A proved sensitive for the identification of monoblasts. FXIII-A can be considered as a reliable intracytoplasmic marker for the monocytic and megakaryocytic series and its presence is highly predictive for mono- and megakaryocytic AML and for CMML.

Congenital blood coagulation factor XIII deficiency and perinatal management.

Transglutaminases are at least 9 enzymes which cross-link a number of proteins. This type of reaction not only enhances the original functions of substrate proteins, but also adds new functions to them. Factor XIII (FXIII) is a plasma transglutaminase circulating in blood as a heterotetramer and consisting of two catalytic A subunits and two non-catalytic B subunits. It is a proenzyme activated by thrombin in the blood coagulation cascade. It plays an important role(s) in hemostasis, wound healing, and maintenance of pregnancy. Accordingly, a lifelong bleeding tendency as well as abnormal wound healing and recurrent spontaneous miscarriage are common symptoms of FXIII deficiency. Genetic and molecular mechanisms of congenital deficiencies have been analyzed in vitro. The mechanisms of these defects have also been studied in detail by using FXIII gene knock-out mice in vivo. We analyzed eight successful outcomes of pregnancy in patients with congenital deficiency of FXIIIA, in which the plasmatic level of maternal FXIIIA and/or the precise substitute therapies were mentioned. Then we propose the following guidelines for the perinatal management: (i) decidual bleeding usually begins from 5 weeks of gestation and spontaneous abortion always occurs subsequently without substitute therapy; (ii) the plasma level of FXIIIA must be at least 2 approximately 3%, however, if possible, higher than 10% to prevent bleeding and miscarriage; (iii) the administration of 250 IU of FXIIIA concentrate each 7 days is enough to keep the level of plasma FXIIIA more than 10% in the early gestation, however 500 IU each 7 days is adequate in the later period to keep that level; (iv) during labor, the desired level is higher than 20%, if possible, higher than 30% to avoid any risk of strong obstetrical bleeding.

Pharmacokinetic studies of a novel 1,2,4-thiadiazole derivative, inhibitor of Factor XIIIa, in the rabbit by a validated HPLC method.

Activated Factor XIII (FXIIIa) stabilizes fibrin clot by covalent cross-linking of fibrin strands in the fibrin, making it resistant to physiological and pharmacologically induced fibrinolysis. Inhibition of Factor XIIIa offers a novel approach to treatment of thrombosis. Selected derivatives of 1,2,4-thiadiazoles, presently in discovery and development, may offer new treatment strategies as inhibitors of Factor XIIIa. In order to evaluate its pharmacokinetic (PK) profile and to facilitate the selection of drug candidates for drug discovery and development process, we developed and validated a simple and selective reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection for the determination of N-[6-(imidazo[1,2-d][1,2,4]thiadiazol-3-ylamino)hexyl]-2-nitrobenzensulfonamide (5624) in rabbit plasma. The plasma protein precipitation and sample preparation was achieved by using acetonitrile, followed by organic phase evaporation to dryness and the residue reconstitution in the mobile phase. The 5624 recovery from the plasma was about 90%. Chromatography was performed on a C18 column using a gradient of acetonitrile in water as a mobile phase. A chemically related compound, N-[6-(imidazo[1,2-d][1,2,4]thiadiazol-3-ylamino)hexyl]naphthalene-1-sulfonamide (5422), was used as an internal standard. Limit of detection (LOD), based on signal to noise ratio>3, was 0.2 microM (on-column amount of about 7 ng), while limit of quantification (LOQ), based on signal to noise ratio>10, was 0.5 microM (on-column amount of about 20 ng). The plasma samples for the PK study were collected at defined time points during and after 5624 slow intravenous infusion (25 mg/kg) to male White New Zealand rabbits and analyzed by RP-HPLC method. The PK parameters, such as half-life, volume of distribution, total clearance, elimination rate constant etc., were determined. The PK profile of 5624 offered insights in the design and development of additional new compounds, derivatives of 1,2,4-thiadiazole, with desired PK properties.

The molecular physiology and pathology of fibrin structure/function.

The formation of a fibrin clot is a pivotal event in atherothrombotic vascular disease and there is mounting evidence that the structure of clots is of importance in the development of disease. This review describes the crucial events in the formation and dissolution of a clot, with particular focus on genetic and environmental factors that have been identified as determinants of fibrin structure in vivo, and discusses the substantiation of the relationship between fibrin structure and disease in conjunction with a review of the current literature.

Factor XIII A subunit-deficient mice developed severe uterine bleeding events and subsequent spontaneous miscarriages.

To understand the molecular pathology of factor XIII (FXIII) deficiency in vivo, its A subunit (FXIIIA)-knockout (KO) mice were functionally analyzed. Although homozygous FXIIIA female KO mice were capable of becoming pregnant, most of them died due to excessive vaginal bleeding during gestation. Abdominal incisions revealed that the uteri of the dead mice were filled with blood and that some embryos were much smaller than others within a single uterus. A series of histologic examinations of the pregnant animals suggested that massive placental hemorrhage and subsequent necrosis developed in the uteri of the FXIIIA KO mice on day 10 of gestation. This was true regardless of the genotypes of fetuses. These results are reminiscent of spontaneous miscarriage in pregnant humans with FXIII deficiency and indicate that maternal FXIII plays a critical role in uterine hemostasis and maintenance of the placenta during gestation.

Factor XIII mediates adhesion of platelets to endothelial cells through alpha(v)beta(3) and glycoprotein IIb/IIIa integrins.

Coagulation factor XIII (FXIII) is a transglutaminase that catalyzes crosslink formation in fibrin clots. Endothelial cells (EC) were demonstrated to bind FXIII via their alpha(v)beta3 integrin receptor. FXIII was also shown to bind platelet glycoprotein IIb/IIIa receptor. In the present study, we analyzed if FXIII can mediate platelet-EC interaction. Both FXIII and activated FXIII (FXIIIa) bound to EC monolayers; this binding was enhanced by the addition of Mn2+ and was inhibited by the monoclonal antibody L609 against alpha(v)beta3 integrin. Normal washed platelets also bound surface-immobilized or soluble FXIII and FXIIIa, and the binding was GPIIb/IIIa dependent. The effect of FXIII concentrate (Fibrogammin-P) treatment on the interaction of ECs with platelets from six FXIII-deficient patients was studied. Patients' platelets were radiolabeled with 3H-Adenine, washed, resuspended in autologous plasma and allowed to adhere to immortalized EC line EAhy926. Adhesion of platelets from FXIII-deficient patients to ECs increased 1.7+/-0.4-fold (P=.01) following intravenous infusion of FXIII concentrate. Similarly, addition of 1 U/ml of FXIII concentrate to the patients' PRP in vitro increased the adhesion 1.8+/-0.5-fold (P=.008). Preincubation of the EC monolayers with increasing concentrations of either FXIII or FXIIIa augmented the adhesion of normal washed platelets to ECs in a dose-dependent manner. At 10 U/ml of EC-bound FXIII or FXIIIa, platelet adhesion enhanced 1.7+/-0.25-fold (P=.03) and 2.5+/-0.5-fold (P=.02), respectively. The increase in platelet adhesion was completely abolished by pretreatment of ECs with the anti-alpha(v)beta3 antibody L609 or by preincubation of the platelets with the GPIIb/IIIa inhibitor Abciximab. Taken together, our data indicate that FXIII mediates the interaction of platelets with ECs by bridging between endothelial alpha(v)beta3 and platelet GPIIb/IIIa integrins. This interaction may be relevant for tissue remodeling and wound repair after vascular injury in FXIII-deficient patients.

A hamster antibody to the mouse fibrinogen gamma chain inhibits platelet-fibrinogen interactions and FXIIIa-mediated fibrin cross-linking, and facilitates thrombolysis.

Murine models employing genetically altered mice have the potential to provide important new information about the hemostatic system, but before such data can be extrapolated to humans it is necessary to define the similarities and differences between murine and human hemostasis. After establishing the similarities of murine fibrinogen to human fibrinogen in its pattern of proteolysis in response to plasmin and its cross-linking by factor XIIIa, we studied a new hamster monoclonal antibody (mAb) 7E9 that reacts with the gamma chain of mouse fibrinogen. This antibody inhibits platelet adhesion to fibrinogen, platelet-mediated clot retraction, platelet aggregation, and FXIIIa-mediated cross-linking of fibrin; it also facilitates tissue plasminogen activator (tPA)-mediated lysis of fibrin formed either in the absence or presence of platelets. These data provide evidence that the C-terminus of mouse fibrinogen gamma chain, like that of human fibrinogen, is involved in fibrinogen binding to platelets and FXIIIa-mediated cross-linking of fibrin. Our data raise the possibility that a therapeutic agent that targets the C-terminus of the gamma chain in human fibrinogen might have broad antithrombotic and profibrinolytic effects.

Factor XIII of blood coagulation as a nuclear crosslinking enzyme.

Intracellular localization and distribution of Factor XIII subunit A (FXIIIA) was investigated in association with monocyte-macrophage differentiation in a long term culture of human monocytes by light- and electron microscopical as well as biochemical and immunobiochemical techniques. To allow the detection of FXIIIA in cells with well-preserved ultrustructure, immunosera against glutaraldehyde-derivatized recombinant FXIIIA were developed in rabbits, then characterized and used in this study. In the early phase of macrophage differentiation intranuclear accumulation of FXIIIA was detected as a transient phenomenon in cells of the 2nd day culture by optical sectioning with 0,7 microm steps in laser scanning confocal microscopy and immunoblotting technique. FXIIIA could be detected by immunoelectron microscopic postembedding staining over electrodense DNA-containing areas. Fluoresceinated monodansylcadaverine incorporation assay was used to demonstrate that FXIIIA is not only present in the nuclei, but also expresses its transglutaminase activity. Our finding of the nuclear accumulation of FXIIIA in differentiating human macrophages is also unique in that a blood clotting factor has, for the first time, been localized in nuclei and has been shown to be an intracellular crosslinking enzyme. The possible role of nuclear FXIIIA in association with cellular processes involving chromatin structure remodeling, such as cell death, cell differentiation or cellular proliferation requires further in-depth investigation.