Primary structure of blood coagulation factor XIIIa (fibrinoligase, transglutaminase) from human placenta.

We have determined the primary structure of human placental factor XIIIa, an enzyme [fibrinoligase, transglutaminase, fibrin-stabilizing factor, EC 2.3.2.13 (protein-glutamine:amine gamma-glutamyltransferase)] that forms intermolecular isopeptide bonds between fibrin molecules as the last step in blood coagulation. Placental factor XIIIa is an unglycosylated polypeptide chain of 730 amino acid residues (Mr = 83,005) that appears to be identical to the a subunit of the plasma zymogen factor XIII. Ca2+-dependent activation of factor XIIIa by thrombin removes a blocked amino-terminal peptide and unmasks a reactive thiol group at Cys-314. A second specific cleavage after Lys-513 by thrombin inactivates factor XIIIa and produces an amino-terminal 56-kDa fragment and a 24-kDa fragment. The amino acid sequence of factor XIIIa is unique and does not exhibit internal homology, but its active center is similar to that of the thiol proteases. The probable Ca2+-binding site of factor XIIIa has been identified by homology to the high-affinity sites in calmodulins. Knowledge of the primary structure of factor XIIIa will aid elucidation of the mechanism of its enzymatic action and that of the many tissue transglutaminases of which it is the prototype. This will also facilitate production of factor XIIIa by recombinant DNA technology for use in treatment of congenital factor XIII deficiencies and in the postoperative healing of wounds.     

Cross-linked A alpha.gamma chain hybrids serve as unique markers for fibrinogen polymerized by tissue transglutaminase.

Notwithstanding the high degree of amino acid sequence homologies between human factor XIIIa on the one hand and intracellular transglutaminases (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) from guinea pig liver or human erythrocytes on the other, we find that the two sets of enzymes differ remarkably in the mode of cross-linking the same protein substrate--i.e., human fibrinogen. In the program of polymerization with factor XIIIa, production of the known gamma-gamma' homologous chain pairs is the dominant feature, whereas with either intracellular transglutaminase, a series of hitherto unidentified A alpha.gamma hybrid chain combinations, designated A alpha p gamma q (p and q = 1, 2, 3...), is generated and practically no gamma-gamma' dimers are formed. Two-dimensional electrophoresis is particularly useful for demonstrating the production of A alpha p gamma q structures by protein staining as well as by immunoblotting against specific antibodies to the A alpha and gamma chains of fibrinogen. These findings should aid in deciding whether the direct cross-linking of fibrinogen by transglutaminase might contribute to thrombotic processes in addition to the thrombin- and factor XIIIa-dependent pathway of clot formation.     

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca²⁺.     

Complete amino acid sequence and homologies of human erythrocyte membrane protein band 4.2.

The complete amino acid sequence for human erythrocyte band 4.2 has been derived from the nucleotide sequence of a full-length 2.35-kilobase (kb) cDNA. The 2.35-kb cDNA was isolated from a human reticulocyte cDNA library made in the expression vector lambda gt11. Of the 2348 base pairs (bp), 2073 bp encode 691 amino acids representing 76.9 kDa (the SDS/PAGE molecular mass is 72 kDa). RNA blot analysis of human reticulocyte total RNA gives a message size for band 4.2 of 2.4 kb. The amino acid sequence of band 4.2 has homology with two closely related Ca2(+)-dependent cross-linking proteins, guinea pig liver transglutaminase (protein-glutamine gamma-glutamyltransferase; protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) (32% identity in a 446-amino acid overlap) and the a subunit of human coagulation factor XIII (27% identity in a 639-amino acid overlap), a transglutaminase that forms intermolecular gamma-glutamyl-epsilon-lysine bonds between fibrin molecules. The region of greatest identity includes a 49-amino acid stretch of band 4.2, which is 69% and 51% identical with guinea pig liver transglutaminase and the a subunit of factor XIII, respectively, within the regions that contain the active sites of these enzymes. Significantly, within the five contiguous consensus residues of the transglutaminase active site, Gly-Gln-Cys-Trp-Val, band 4.2 has an alanine substituted for cysteine (which is apparently essential for activity). Consistent with this active site substitution, erythrocyte membranes or inside-out vesicles, which contain band 4.2, show no evidence of transglutaminase activity by two types of in vitro assay.     

Factor XIII improves gastric stress lesions in rats.

BACKGROUND/AIMS: Tissue transglutaminase has been reported to be involved in the healing of experimental gastric ulcer; nevertheless, other type(s) of transglutaminase could be involved. The present experiments aimed at examining whether plasma transglutaminase (factor XIIIa) contributes to such healing and at evaluating whether factor XIII supplementation improves gastric mucosal lesions. METHODS: The healing effect of 200 U/kg of factor XIII administered intravenously was examined using a water immersion restraint rat model of stress gastric damage. The rats were sacrified 0, 2, 4, and 12 h after stress. The gastric mucosa was examined macroscopically and microscopically, and the transglutaminase activities were assayed in serum and gastric mucosa. Factor XIIIa and tissue transglutaminase protein levels in the gastric mucosa were analyzed by immunoblot. Immunohistochemistry was used to identify the location of tissue transglutaminase, factor XIIIa, and fibronectin in the gastric mucosa. RESULTS: The transglutaminase activity, reduced by stress in the gastric mucosa, increased up to 12 h after stress, peaking at 4 h, when the ulcer index significantly decreased. The serum transglutaminase level was low at all time points. Exogenous administration of factor XIII allowed a faster reduction of the ulcer index that was coincident with an increased transglutaminase activity in the mucosa. Both tissue transglutaminase and factor XIIIa protein levels were reduced by 6 h of stress and increased after factor XIII administration. Immunohistochemistry showed a colocalization of both factor XIIIa and tissue transglutaminase with fibronectin in the extracellular matrix of the damaged area. CONCLUSIONS: Two forms of transglutaminase are involved in the healing of stress-induced gastric erosions, and factor XIII administration allows faster gastric mucosa healing.     

Characterization of cDNA coding for human factor XIIIa.

A cDNA library prepared from human placenta has been screened for sequences coding for factor XIIIa, the enzymatically active subunit of the factor XIII complex that stabilizes blood clots through crosslinking of fibrin molecules. Two oligonucleotides, based on the amino acid sequences of tryptic peptides of factor XIIIa, were used as hybridization probes. Of 0.36 X 10(6) independent recombinants, 1 clone was identified that hybridized to both probes. The insert of 1704 base pairs coded for the amino-terminal 541 amino acid residues of the mature factor XIIIa molecule. Blot-hybridization analysis using this cDNA as a probe showed that the factor XIIIa mRNA from placenta has a size of approximately 4000 bases. The insert was used to rescreen cDNA libraries and to identify further factor XIIIa-specific sequences. The total length of the isolated factor XIIIa cDNA is 3905 bases, and it codes for a protein of 732 amino acids. In spite of the presence of factor XIII in blood plasma, we could not identify a leader sequence typical for secreted proteins.     

Transglutaminases in Crohn's disease.

Transglutaminases are a family of Ca-dependent enzymes involved in various biological events. Circulating transglutaminase (factor XIIIa) is decreased in blood of patients with inflammatory bowel diseases. There is evidence that factor XIIIa and tissue type transglutaminase, present in cell cytosol, bind to various proteins of the extracellular matrix. This study examined the value of serum transglutaminase assay in the treatment and follow up of Crohn's disease and then investigated the intestinal location of both forms of transglutaminases by immunohistochemistry in normal and abnormal tissues. Serum transglutaminase activity was assayed in 36 patients with active Crohn's disease (CDAI > 150). Eighteen patients were studied prospectively from relapse into remission. A significant inverse correlation (p < 0.001) was found between circulating transglutaminase and Crohn's disease activity index; a correlation was also found between serum transglutaminase and serum orosomucoid (p < 0.01) and C reactive protein (p < 0.01). Patients were prospectively studied until clinical remission showed improvement in both their CDAI score mean (SD) (230 (46) to 72 (34), p < 0.01) and transglutaminase activity mean (SD) (0.61 (0.12) to 0.93 (0.13) mU/ml, p < 0.01). The immunohistochemistry assessment showed a colocalisation of factor XIIIa and tissue transglutaminase to the extracellular matrix of damaged tissues. In conclusion, these data confirm the value of serum transglutaminase assay as marker of Crohn's disease activity, extend the utility of serum transglutaminase assay to follow up of the disease, and emphasised the role of different types of transglutaminases in extracellular matrix assembly in the damaged tissues.     

Factor XIIIa supports microvascular endothelial cell adhesion and inhibits capillary tube formation in fibrin

Coagulation factor XIIIa is a transglutaminase that catalyzes covalent cross-link formation in fibrin clots. In this report, we demonstrate that factor XIIIa also mediates adhesion of endothelial cells and inhibits capillary tube formation in fibrin. The adhesive activity of factor XIIIa was not dependent on the transglutaminase activity, and did not involve the factor XIIIb-subunits. The adhesion was inhibited by 99% using a combination of monoclonal antibodies directed against integrin alpha(v)beta(3) and beta(1)-containing integrins, and was dependent on Mg(2+) or Mn(2+). Soluble factor XIIIa also bound to endothelial cells in solution, as detected by flow cytometry. In addition, factor XIIIa inhibited endothelial cell capillary tube formation in fibrin in a dose-dependent manner. Furthermore, the extent of inhibition differed in 2 types of fibrin. The addition of 10 to 100 microg/mL factor XIIIa produced a dose-dependent reduction in capillary tube formation of 60% to 100% in gammaA/gammaA fibrin, but only a 10% to 37% decrease in gammaA/gamma' fibrin. These results show that factor XIIIa supports endothelial cell adhesion in an integrin-dependent manner and inhibits capillary tube formation. (Blood. 2000;95:2586-2592)     

Primary structure of keratinocyte transglutaminase.

The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine gamma-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of approximately 90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unrelated activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.     

Alternative Pathways for the Activation of Factor XIII

Factor XIII is present in plasma as a proenzyme, which when activated catalyses the formation of epsilon(gamma-glutamyl)lysyl bonds in fibrin. In this study the activation of purified plasma factor XIII was examined quantitatively with the fluorescent amine incorporation assay. Activation products were examined by polyacrylamide gel electrophoresis. The serin proteases, thrombin, trypsin, chymotrypsin, and factor Xa, and also Reptilase were tested for their ability to activate factor XIII. Highly purified thrombins activated purified factor XIII; this reaction was not calcium dependent. Trypsin was also a potent activator, but no transglutaminase activity was found with chymotrypsin. The most highly purified preparations of Reptilase had no effect on factor XIII activity. Less purified Reptilase preparations activated factor XIII, which suggests the presence of another enzyme in these Reptilase preparations. Highly purified factor Xa was found to be an effective activator of purified factor XIII. In contrast to thrombin activation, this reaction required calcium. It may be that under certain circumstances factor XIIIa could be formed in vivo directly by the alternative pathway of factor Xa. Factor XIIIa could then crosslink fibrinogen, which would also provide an alternative pathway for thrombus formation. Also, the activation of factor XIII by both factor Xa and thrombin provides a further point of control in the blood coagulation process.     

Isolation of transglutaminase-reactive sequences from complex biological systems: a prominent lysine donor sequence in bovine lens.

The transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13)-catalyzed cross-linking of proteins in biological systems can often be inhibited by inclusion of small primary amines or glutamine-containing peptides, which act as site-specific blockers of the relevant acceptor (i.e., glutamine) and donor (i.e., lysine) functionalities of the natural substrates. Compounds such as dansylcadaverine and dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val are particularly useful in sorting out acceptor-donor relationships among lens crystallins. Apart from its fluorescent properties, the dansyl hapten offered special advantages as a "handle" for the rapid isolation of transglutaminase targets even in the complex system of lens cortical homogenate. The dansylated peptide was incorporated into bovine lens proteins under the influence of the Ca(2+)-activated intrinsic transglutaminase and, after digestion by endoproteinase Glu-C, the tracer-containing fragments were isolated by affinity chromatography on an anti-dansyl antibody column. The major fluorescent peak was isolated by HPLC and sequenced by Edman degradation, which yielded phenylthiohydantoin amino acid derivatives for the first 10 cycles, EKPAVTAAPK, and none for the next 2. The sequence, corresponding to residues 165-174 of alpha B-crystallin, unambiguously identifies the known carboxyl-terminal domain, EK-PAVTAAPKK, as the prominent lysine-donating fragment in bovine lens.     

Serum Transglutaminase Correlates with Endoscopic and Histopathologic Grading in Patients with Ulcerative Colitis

Factor XIIIa, a circulating form of transglutaminase, plays a key role in intestinal mucosal repair. We found that transglutaminase levels are decreased in serum of patients with inflammatory bowel diseases and demonstrated in a rat model of chronic colitis that serum transglutaminase is closely related to the severity of intestinal damage. We aimed, therefore, to correlate serum transglutaminase levels with standard endoscopic and histopathologic grading systems in patients affected by ulcerative colitis (UC). In 249 patients with UC, we assayed serum transglutaminase activity by a radioenzymatic method and measured clinical activity index (CAI) according to modified Rachmilewitz's criteria. In a subset of 82 patients undergoing colonoscopy, endoscopic and histologic indices were studied. Biopsy specimens were also taken from 28 patients to measure myeloperoxidase (MPO) as a marker of mucosa inflammation. Serum transglutaminase levels significantly correlated with the CAI scoring (r = –0.63; P < 0.01); likewise serum transglutaminase showed the best correlation with endoscopic (r = –0.71; P < 0.001) and histologic (r = –0.79; P < 0.001) scores. Myeloperoxidase activity was significantly higher in patients with active UC than those in remission (P < 0.01), showing a significant correlation with serum transglutaminase levels (r = –0.68; P < 0.01). Immunohistochemistry showed factor XIIIa localization in the extracellular matrix of damaged mucosa. In conclusion, these results suggest that transglutaminase assay can be useful in managing UC as a serological, noninvasive indicator of intestinal mucosal status.     

Detection of factor XIIIa (active fibrin-stabilizing factor) in normal plasma

Factor XIIIa (active fibrin-stabilizing factor) generated in heat- defibrinated plasma by the addition of thrombin can be measured by 14C- putrescine incorporation into casein. Modification of this assay be substituting 3H-putrescine of high specific activity as the donor amine permits measurement of amine incorporation by plasma even in the absence of added thrombin. Incorporation is calcium dependent, inhibited by iodoacetamide, and absent from congenital factor XIII- deficient plasma and from normal platelets. The transamidating activity detected by radioenzymatic assay catalyzed the formation of gamma-gamma dimers and alpha polymers of fibrin and was thus biologically functional. This fibrin cross-linking activity was absent from factor XIII-deficient plasma. These experiments show (1) some factor XIII is present in plasma as factor XIIIa; (2) this factor XIIIa can cross-link fibrin and thus has biologic activity as well; and (3) this activity is not present in factor XIII-deficient plasma. Factor XIIIa in normal plasma is possibly activated in vivo, perhaps by circulating thrombin, factor Xa, or other proteolytic enzymes.     

Fibrinogen Chapel Hill II: Defective in reactions with thrombin, factor XIIIa and plasmin

Fibrinogen Chapel Hill II is a hereditary, abnormal fibrinogen which is characterized by poor substrate reactivity toward thrombin, factor XIIIa and plasmin. The patient has a low plasma level of clottable protein with normal antigen concentration, high amounts of fibrinogen related material in serum, and prolonged thrombin and reptilase clotting times. Fibrinopeptide release was decreased with both thrombin and ancrod, indicating that release of fibrinopeptide A from the abnormal fibrinogen was impaired. Sequence analysis indicated that the A peptide was normal. Light scattering indicated that the fibrils formed by thrombin were unusually short and thick. When clotted under crosslinking conditions gamma dimers formed normally but alpha polymer formation was defective. Under conditions which yielded complete plasmin digestion of normal fibrinogen only half of the patient fibrinogen was degraded beyond the fragment X stage. The rate of fibrinopeptide release from patient fragment X and NH2-terminal disulphide knot (N-DSK) was similar to that from the fibrinogen, indicating that the defect was contained within the N-DSK. A simple amino acid substitution could result in a conformational defect in the N-DSK sufficient to perturb the reactions involving thrombin, factor XIIIa and plasmin and also polymerization.     

Deficiency of Blood Coagulation Factor XIII in Crohn's Disease

Objective : Refractory fistula formation is one of most intractable complications in Crohn's disease. Recently, the role of blood coagulation Factor XIII has been recognized as an important wound-healing factor. We investigated the plasma concentration and functional activity of blood coagulation Factor XIIIa, active subunit of the Factor XIII, in patients with Crohn's disease and in healthy volunteers. Methods : Peripheral blood was obtained from 24 patients with Crohn's disease and from 10 healthy volunteers. The functional activity of Factor XIIIa was measured by its transglutaminase activity, and plasma concentration was measured by the immunoelectrophoresis method. Results : The differences in Crohn's disease patients and healthy volunteers were not significant. However, Crohn's disease patients with fistula had significantly lower functional activity than Crohn's disease patients without fistula. Conclusions : The deficiency in this wound-healing factor might be one reason for refractory fistulas in Crohn's disease, and supplementation of the factor might be useful therapeutically.     

2''-Phosphoadenylylation of eukaryotic proteins: a type of covalent modification.

An enzymatic system in rat liver microsomal preparations has been detected that catalyzes the transfer of the 2'-phospho-AMP moiety from NADP to endogenous polypeptides; the major acceptor is a polypeptide of about 40 kDa (p40). Modification of the acceptor by 2'-phospho-AMP residues was deduced from the simultaneous transfer of 2'-[33P]phosphate and [3H]adenine residues from double-labeled NADP, while no incorporation of radioactivity into p40 was seen with NADP species labeled in the NMN moiety. The true substrate of this phosphoadenylylation reaction was 2'-phospho-ADP-ribose rather than NADP, because labeled phospho-ADP-ribose was as efficient as or more efficient than NADP in forming modified p40. Also, NADP was rapidly converted to phospho-ADP-ribose during incubation with microsomes. Furthermore, isonicotinic acid hydrazide, an inhibitor of NADP glycohydrolase, prevented phosphoadenylylation from NADP, but not from phospho-ADP-ribose, and glycohydrolase-resistant NADPH could not substitute for NADP. Transferase activity was found in liver and brain microsomes and, to a smaller extent, in the cytosol fractions. In Ehrlich ascites tumor cells, most of the activity resided in the cytosol, from which it could be partially purified. The apparent Km for phospho-ADP-ribose was about 2 X 10(-4) M, and the pH optimum was around 7. Divalent cations like Mg2+ and Mn2+ inhibited the reaction. In all compartmental preparations, activity was eliminated by heating or short treatment with alkali or acid. In submitochondrial particles from rat liver, a system with different characteristics led to the phosphoadenylylation of several endogenous polypeptides.     

Affinity of human erythrocyte transglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin.

Complex formation between the human erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. A 42-kDa gelatin-binding structure, obtained from human plasma fibronectin by thermolytic digestion, showed as high an affinity for the cytosolic enzyme as the parent fibronectin chains themselves. A 21-kDa fragment comprising type I modules 8 and 9, the last two modules in the 42-kDa fragment, bound with an affinity 100-fold less than the 42-kDa fragment. Binding was remarkably specific and could be exploited for the affinity purification of transglutaminase directly from the hemoglobin-depleted erythrocyte lysate. In spite of the high affinity, it was possible to elute active enzyme from the 42-kDa fragment column with 0.25% monochloroacetic acid. This solvent might have general applicability in other systems involving separation of tightly bound ligands.     

The Effect of Acetaldehyde on Human Plasma Factor XIII Function

The effect of acetaldehyde on the transglutaminase activity in pooled normal human plasma has been investigated. In this study, 0.05, 0.2, and 0.7 ml of pooled human plasma were preincubated for 30 min. at room temperature with buffer or acetaldehyde at final concentrations of 40.6, 22.4, and 11.2 mM before being utilized for Factor XIIIa assay with fibrinogen and thrombin which had been preheated at 40°C to destroy endogenous Factor XIII/XIIIa activity. At all concentrations of acetaldehyde and all concentrations of plasma-containing Factor XIII which were employed, prolongation of both clotting time and stabilization time was observed. The 11.2 mM acetaldehyde is within the range of daily acetaldehyde production to be predicted in severe alcoholics as a consequence of imbibing alcohol. The stabilization times measured for Factor XIIIa activity appear to be the most sensitive to acetaldehyde compared to acetaldehyde effects on thrombin, Factor Xa, and fibrinogen studied earlier in this laboratory, as well as Factors II, VII, and X.     

Human erythrocyte protein 4.2: isoform expression, differential splicing, and chromosomal assignment.

Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).