Identification of amino acid residues that are crucial for FXIII-A intersubunit interactions and stability.

Coagulation factor XIII (FXIII) is the main stabilizer of the fibrin clot. It circulates in plasma as a tetramer of two A-subunits and two B-subunits. Under physiological conditions, FXIII-A exists as a dimer (FXIII-A2). The interactions between the FXIII-A-subunits that stabilize the FXIII-A2 dimer are not fully understood. We therefore designed a systematic approach to identify amino acid residues crucial for the expression and stability of FXIII-A2. Based on the available FXIII-A2 crystal structure, we identified 12 amino acid residues forming intersubunit salt bridges and 21 amino acid residues forming hydrogen bonds between the two A-subunits. We chose 10 amino acid residues that form 5 particularly strong interactions, performed site-directed mutagenesis, and expressed the mutants in CHO cells. Disruption of these interactions by single mutation of Lys257, Lys113, Asp343, Glu401, or Asp404 abolished the expression of properly folded, soluble, and functional FXIII-A in CHO cells. On the contrary, mutation of Glu111, Arg100, or Asn112 had no significant effect on FXIII-A expression. Our results suggest that 4 intersubunit interactions (Arg11-Asp343, Lys113-Asp367, Lys257-Glu401, and Arg260-Asp404) are essential for the stability of FXIII-A2. Our findings are supported by reported mutations at Lys257, Arg260, and Asp404 found in patients with congenital FXIII-A deficiency.

Factor XIII deficiency diagnosis: Challenges and tools.

Factor XIII deficiency (FXIIID) is a rare hereditary bleeding disorder arising from heterogeneous mutations, which can lead to life-threatening hemorrhage. The diagnosis of FXIIID is challenging due to normal standard coagulation assays requiring specific FXIII assays for diagnosis, which is especially difficult in developing countries. This report presents an overview of FXIIID diagnosis and laboratory methods and suggests an algorithm to improve diagnostic efficiency and prevent missed or delayed FXIIID diagnosis. Assays measuring FXIII activity: The currently available assays utilized to diagnose FXIIID, including an overview of their complexity, reliability, sensitivity, and specificity, as well as mutational analysis are reviewed. The use of a FXIII inhibitor assay is described. Diagnostic tools in FXIIID: Many laboratories are not equipped with quantitative FXIII activity assays, and if available, limitations in lower activity ranges are important to consider. Clot solubility tests are not standardized, have a low sensitivity, and are therefore not recommended as routine screening test; however, they are the first screening test in almost all coagulation laboratories in developing countries. To minimize the number of patients with undiagnosed FXIIID, test quality should be improved in less well-equipped laboratories. Common country-specific mutations may facilitate diagnosis through targeted genetic analysis in reference laboratories in suspected cases. However, genetic analysis may not be feasible in every country and may miss spontaneous mutations. Centralized FXIII activity measurements should also be considered. An algorithm for diagnosis of FXIIID including different approaches dependent upon laboratory capability is proposed.

Assessment of Factor XIII.

Blood coagulation factor XIII (FXIII) is essential for maintaining hemostasis. The absence of FXIII results in severe bleeding diathesis, which without prophylaxis frequently leads to fatal bleeding. As the usual hemostasis screening tests remain normal, the diagnosis of FXIII deficiency needs specific tests. Here, we describe FXIII activity determination by the ammonia release assay, which is the first-line test in the diagnostic algorithm for FXIII deficiency. The method for another activity test, the undeservedly rarely used fibrin cross-linking assay, is also presented. Further tests used for the classification of FXIII deficiencies, measurement of FXIII activity in platelets, ELISAs for the measurement of complex plasma FXIII (FXIII-A2B2) antigen and for FXIII-A2 in plasma and platelets are also included. Detailed description of the methods for the detection and measurement of neutralizing auto- and alloantibodies is also provided.

Factor XIII deficiency enhances thrombin generation due to impaired fibrin polymerization - An effect corrected by Factor XIII replacement.

INTRODUCTION: Factor XIII (FXIII) cross-links fibrin, completing blood coagulation. Congenital FXIII deficiency is managed with plasma-derived FXIII (pdFXIII) or recombinant FXIII (rFXIII) concentrates. AIM: As the mechanisms protecting patients with low FXIII levels (<5IU/dL) from spontaneous bleeds remain unknown we assessed the interplay between thrombin generation (TG), fibrin formation and clot kinetics before and after FXIII administration in three patients with FXIII deficiency. METHODS: Patients received initially rFXIII (35IU/kg, A-subunit) following with pdFXIII at 1250IU or 2500IU (12-30IU/kg) monthly. TG (CAT), thromboelastometry (ROTEM), prothrombin fragments F1+2, fibrinogen and FXIII activity (FXIII:C) were measured at baseline and one-hour recovery. RESULTS: FXIII was at the target level of 20±6IU/dL at the 4-week trough. rFXIII corrected FXIII to 98±15 and high-dose pdFXIII to a level of 90±6, whereas low-dose/half dose pdFXIII reached 45±4IU/dL. Although fibrinogen (Clauss Method) was normal, coagulation in FIBTEM was impaired, which FXIII administration tended to correct. CAT implied 1.6- to 1.9-fold enhanced TG, which FXIII administration normalized. Inhibition of fibrin polymerization by Gly-Pro-Arg-Pro peptide mimicked FXIII deficiency in CAT by enhancing TG both in control and FXIII recovery plasma. Antithrombin, α2-macroblobulin-thrombin complex and prothrombin were normal, whereas F1+2 were elevated compatible with in vivo TG. DISCUSSION: FXIII deficiency impairs fibrinogen function and fibrin formation simultaneously enhancing TG on the poorly polymerizing fibrin strands, when fibrin's antithrombin I -like function is absent. Our study suggests an inverse link between low FXIII levels and enhanced TG modifying structure-function relationship of fibrin to support hemostasis.

Factor XIII A-Subunit V34L Variant Affects Thrombus Cross-Linking in a Murine Model of Thrombosis.

OBJECTIVE: Factor XIII (FXIII) cross-links fibrin upon activation by thrombin. Activation involves cleavage at residue 37 by thrombin, releasing an activation peptide. A common polymorphism (valine to leucine variant at residue 34, V34L), located in the activation peptide, has been associated with increased activation rates and paradoxically a protective effect in cardiovascular disease. There is, currently, no data available on the effects of V34L from in vivo models of thrombosis. We examined the effect of FXIII V34L on clot formation and cross-linking in vivo. APPROACH AND RESULTS: We generated a panel of full-length recombinant human FXIII-A2 variants with amino acid substitutions in the activation peptide to investigate the effect of these variants on activation rate, and we used wild-type, V34L, and alanine to glycine variant at residue 33 variants to study the effects of varying FXIII activation rate on thrombus formation in a murine model of FeCl3 injury. FXIII activation assay showed that residues 29, 30, 33, and 34 play a critical role in thrombin interaction. Full-length recombinant human FXIII-A2 V34L has significant effects on clot formation, structure, and lysis in vitro, using turbidity assay. This variant influenced fibrin cross-linking but not size of the thrombus in vivo. CONCLUSIONS: Mutations in the activation peptide of full-length recombinant FXIII regulate activation rates by thrombin, and V34L influences in vivo thrombus formation by increased cross-linking of the clot.

Factor XIII: congenital deficiency factor XIII, acquired deficiency, factor XIII A-subunit, and factor XIII B-subunit.

Factor XIII (FXIII) is a transglutaminase consisting of 2 catalytic A subunits and 2 noncatalytic B subunits in plasma. The noncatalytic B subunits protect the catalytic A subunits from clearance. Congenital FXIII deficiency may manifest as a lifelong bleeding tendency, abnormal wound healing, and recurrent miscarriage. Acquired FXIII deficiency, with significant reductions in FXIII levels, has been reported in several medical conditions. The routine screening tests for coagulopathies-prothrombin time, activated partial thromboplastin time, and thrombin time-do not show abnormalities in cases of FXIII deficiency. A quantitative, functional, FXIII activity assay that detects all forms of FXIII deficiency should be used as a first-line screening test. Treatment consists of recombinant FXIII or FXIII concentrate. If these are unavailable, then fresh-frozen plasma and cryoprecipitates may be used. Factor XIII has a long half-life; therefore, the patients can lead near-normal lives with regular replacements. Patients with acquired FXIII deficiency with inhibitors need immunosuppressive therapy in addition to factor replacements.

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.

Molecular modeling predicts structural changes in the A subunit of factor XIII caused by two novel mutations identified in a neonate with severe congenital factor XIII deficiency.

INTRODUCTION: Coagulation factor XIII (FXIII) is a fibrin-stabilizing factor, which contributes to hemostasis, wound healing, and maintenance of pregnancy. Accordingly, patients with congenital FXIII deficiency manifest a life-long bleeding tendency, abnormal wound healing and recurrent miscarriage. In order to understand the molecular mechanisms of congenital FXIII deficiency, genetic analysis and molecular modeling were carried out in a Japanese male neonate with severe FXIII deficiency. METHODS AND RESULTS: Two novel mutations, Y204Stop (or Y204X, TAT to TAA) and S708R (AGC to AGG), were heterozygously identified by nucleotide sequencing analysis in exons V and XV of the gene for the A subunit of FXIII (FXIII-A). Y204X and S708R would lead to nonsense mediated mRNA decay and misfolding of the FXIII-A molecule, respectively. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, the presence of these mutations was confirmed both together in the proband and one each separately in either the maternal or paternal sides of his family. In addition, moderately decreased FXIII activity was associated with the presence of either mutation. Molecular modeling predicted that the mutant molecule of S708R would be structurally compromised by the substitution of the Ser with the larger extended bulky and positively charged Arg side-chain. CONCLUSION: It is probable that the impaired tertiary structure of the mutant S708R molecule leads to its instability, which is at least in part responsible for the FXIII deficiency of this patient. This is consistent with the fact that the mutations and the reduced FXIII activities co-segregate among the patient's family members.

Factor XIII-A is involved in the regulation of gene expression in alternatively activated human macrophages.

Factor XIII subunit A (FXIII-A) is one of the most overrepresented genes that is expressed during the alternative activation of macrophages. Based on its substrate profile and its cellular localisation, FXIII-A is thought to function as an intracellular/intranuclear transglutaminase. Our aim was to find role for the intracellular FXIII-A by comparing the microarray profiles of alternatively activated monocyte-derived macrophages. Microarray analyses of FXIII-A-deficient patients and healthy controls were evaluated, followed by functional clustering of the differentially expressed genes. After a 48-hour differentiation in the presence of interleukin 4 (IL4), 1,017 probes out of the 24,398 expressed in macrophages from FXIII-A- deficient samples were IL4 sensitive, while only 596 probes were IL4 sensitive in wild-type samples. Of these genes, 307 were induced in both the deficient and the wild-type macrophages. Our results revealed that FXIII-A has important role(s) in mediating gene expression changes in macrophages during alternative activation. Functional clustering of the target genes carried out using Cytoscape/BiNGO and Ingenuity Pathways Analysis programs showed that, in the absence of FXIII-A, the most prominent differences are related to immune functions and to wound response. Our findings suggest that functional impairment of macrophages at the level of gene expression regulation plays a role in the wound healing defects of FXIII-A-deficient patients.

Factor XIII deficiency as a potential cause of supratentorial haemorrhage after posterior fossa surgery.

BACKGROUND: Postoperative intracranial haemorrhage can be a dramatic event, carrying significant morbidity and mortality. Bleeding at sites remote from the operation area represents a small percentage of haemorrhages whose aetiology remains unclear (Harders et al. Acta Neurochir (Wien) 74(1-2):57-60, 1985). AIM: We present the case of a 60-year-old patient who underwent posterior fossa craniotomy for the removal of a space-occupying lesion and suffered supratentorial haemorrhage soon after the operation. RESULTS: A thorough postoperative investigation revealed low levels of factor XIII (FXIII), the factor mainly responsible for fibrin clot stabilisation. CONCLUSION: We suggest that reduced FXIII activity may be an important but preventable predisposing factor to remote postoperative haemorrhage in neurosurgical patients.

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.

Effect of four missense mutations in the factor XIII A-subunit gene on protein stability: studies with recombinant proteins.

Four missense mutations in the factor XIII A-subunit gene, Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg, were previously reported by us in patients with severe factor XIII deficiency. The objective of our study was to discern the effect of all four mutations on the stability and intracellular localization of the factor XIII A-subunit by their expression in COS cells. In-vitro mutagenesis, transient expression of the mutants in COS cells and subsequent pulse-chase analyses were carried out. Intracellular localization of wild-type and mutant proteins was analyzed by immunohistochemistry using a monoclonal antibody against factor XIII A-subunit. Pulse-chase analyses of metabolically labeled proteins demonstrated rapid intracellular degradation of each mutant protein as compared with wild type. Immunocytochemical and immunofluorescence analyses disclosed that wild-type and all four mutant factor XIII A-subunit proteins were diffusely distributed within the cytoplasm but not in the endoplasmic reticulum of the COS-7 cells. The Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg mutations in FXIII A-subunit cause rapid intracellular degradation of the corresponding mutated protein.

Maternal blood coagulation factor XIII is associated with the development of cytotrophoblastic shell.

We analysed the early implantation tissues of normal women and of a patient with congenital factor XIII deficiency in order to study the role of maternal subunit A of factor XIII (XIIIA) in the development of extravillous cytotrophoblast. The patient had received adequate administration of factor XIIIA concentrate only up to 7 weeks of gestation (wG). Her pregnancy was maintained until the latter half of 8 wG, but was terminated by intrauterine fetal death at 9 wG. Immunohistochemical staining of cytokeratin, XIIIA and subunit S of factor XIII was performed in the early implantation tissues of normal women and of this patient. Numerous well-formed cytotrophoblastic shells and Nitabuch's layers were detected in implantation tissues at 7-8 wG in normal women, and XIIIA was present in the intercellular space in well-formed cytotrophoblastic shells, while the cytotrophoblastic shells and Nitabuch's layers in this patient's implantation tissue were poorly-formed. Furthermore, XIIIA was not detected around them. It is suggested that when the maternal plasma activity of factor XIII is low, the concentration of XIIIA at the placental bed is also low, leading to the insufficient formation of cytotrophoblastic shell and therefore an increased probability of miscarriage in patients with congenital factor XIII deficiency.

Molecular and genetic mechanisms of factor XIII A subunit deficiency.

Factor XIII is a proenzyme for a plasma transglutaminase. Factor XIII in plasma is a tetramer (A2B2) held together by noncovalent bonds, and the A subunit contains the active site. Recently, the three-dimensional structure of the A subunit has been determined by x-ray crystallography. To understand the structure-function relationships of the factor XIII molecule and its clinical implications in factor XIII deficiency, we characterized its genetic defects and closely examined its gene products, including mRNA and protein levels. A variety of missense and nonsense mutations (Arg260-Cys, Tyr283-Cys, Gly562-Arg) and deletions/insertions with or without out-of-frame shift/premature termination and splicing abnormalities (4-bp deletion with 464Stop, T insertion at the exon IV/intron D boundary with exon IV-skipping, 20-bp deletion at the exon I/intron A boundary) has been identified in cases demonstrating A subunit deficiency. In some cases, the A subunit mRNA levels were severely reduced. Their molecular and cellular bases have also been explored by expression experiments in mammalian cells and by molecular modeling. In most cases, impaired folding and/or conformational changes of the mutant A subunits lead to both intra- and extracellular instability, which is responsible for the A subunit deficiency in the patients.

Molecular mechanisms of mutations in factor XIII A-subunit deficiency: in vitro expression in COS-cells demonstrates intracellular degradation of the mutant proteins.

Factor XIII deficiency is an autosomal recessive bleeding disorder that is largely caused by various mutations in FXIII A-subunit gene. Characteristically, the patients lack both A-subunit activity and antigen in the circulation. Here we have analysed the consequences of four missense mutations (Met242-->Thr, Arg252-->Ile, Arg326-->Gln, Leu498 to Pro) and one stop mutation (Arg661-->Stop) in the FXIII A-subunit gene by expression in COS-cells. After transient transfection each mutant cDNA expressed mRNA at an equal level to the wild type FXIII. However, the mutant polypeptides accumulated in the cells in significantly reduced quantities and demonstrated only very low enzymatic activity. Analysis of immunoprecipitated metabolically labelled polypeptides demonstrated remarkable instability and intracellular degradation of all mutant FXIII proteins. These results verify the deleterious nature of the individual amino acid changes and confirm that protein instability and susceptibility to proteolysis are consequences of the mutations, as predicted from the three-dimensional model of crystallised FXIII A-subunit.

The half life of factor XIII in the management of inherited deficiency.

Following the injection of a large dose of factor XIII concentrate the in vivo half life of factor XIII was estimated in a patient with inherited deficiency. Factor XIII activity and enzyme concentration were measured quantitatively, and a qualitative assessment of the crosslinking of fibrin was also made for upto 6 weeks after the injection. The half life was found to be about 9--10 days. This is longer than most previous reports suggest. An explantation for this finding is offered. The relevance of the long half life of factor XIII to the prophylactic treatment of patients with inherited deficiency is demonstrated.

[Severe factor XIII-deficiency. Studies on subunits and turnover of the fibrin stabilizing factor (author's transl)]. / Ein neuer Fall von schwerem Faktor XIII-Mangel. Untersuchungen zum Verhalten der Untereinheiten und des Turnover des Fibrinstabilisierenden Faktors.

An infant with congenital homozygous factor XIII deficiency demonstrated a severe retroperitoneal and intracerebral bleeding with development of a posthemorrhagic hydrocephalus in the first months of life. Factor XIII activity was not measurable by means of enzymatic method and the antiserum inhibition test. Quantitative immunoelectrophoresis according to Laurell presented absence of the subunit A, whereas the concentration of subunit S was reduced to 47% the normal value. After replacement therapy factor XIII activity was estimated at 23% and corresponded to the concentration of the subunit A, concentration of subunit S increased by 20%. The turnover rate of fibrin stabilizing factor could be observed over a period of 39 days. The half life was estimated at 4,7 days. The child developed normally after continous substitution with 250 units of factor XIII concentrate every 6 weeks.