Human Factor XIII Research Articles

Evaluation of Different Preparation Parameters for the Production and Cryopreservation of Seed Cultures with Recombinant Saccharomyces cerevisae

In this article we describe the intensive investigation of defined physicochemical parameters for their influence on the preparation, storage stability in liquid nitrogen, and the post-thaw productivity of a recombinant strain of Saccharomyces cerevisiae expressing the human factor XIIIa protein. Preparation of industrially sized seed cultures and their storage stability were monitored over more than a 1-year period. Major parameters recorded before and after thawing were number of colony-forming units on agar slant media, plasmid retention, and expression capacity of the recombinant protein. The viability of the cells after reconstitution of the frozen batches was found to improve significantly if a certain combination of prethaw growth conditions was applied and amino acids were included in the cryoprotectant media. In addition, the influence of different concentrations of glycerol as a cryoprotectant was investigated and the specific freezing conditions which improved the viability were identified. Even without complex freezing devices for the controlled freezing of cultures, viability rates of 85% and higher were obtained, enabling consistent production of the recombinant protein.

Structural evidence that the activation peptide is not released upon thrombin cleavage of factor XIII

The three-dimensional structure of the recombinant human factor XIII a 2 dimer after cleavage by thrombin has been determined by X-ray crystallography. Factor XIII zymogen was treated with bovine alpha-thrombin in the presence of 3 mM CaCl 2, and the cleaved protein was crystallized from Tris buffered at pH 6.5 using ethanol as the precipitating agent. Refinement of the molecular model of thrombin-cleaved factor XIII against diffraction data from 10.0 to 2.5 Å resolution has been carried out to give a crystallographic R factor of 18.2%. The structure of thrombin-cleaved factor XIII is remarkably similar to that of the zymogen: there are no large conformational changes in the protein and the 37 residue amino terminus activation peptide remains associated with the rest of the molecule. This work shows that the activation peptide, upon thrombin cleavage, has the same conformation and occupies the same position with respect to the rest of the molecule as it does in the zymogen structure.

Polymerization of Soybean Proteins and Spinach RuBisCO by FXIIIa with Respect to the Labeling of Reactive Glutaminyl Residues

Calcium-activated human placental factor XIII was assayed for polymerization on some food proteins. High molecular weight polymers were visualized on SDS-PAGE. Polymerization of soybean proteins, β-conglycin, a trimer [α,α',β], and glycinin, a hexamer of A-B dimer, depends on protein concentration, time, and the presence of DTT, although no disulfide bridge is known for β-conglycin. FXIIIa can also polymerize spinach RuBisCO, especially the L subunit. These proteins, β-conglycin, glycinin, and RuBisCO, were then assayed for incorporation of monodansylcadaverine to identify the subunit(s) containing reactive glutaminyl residues. Although all subunits can be polymerized, when purified, only the α of β-conglycin, A of glycinin, and S of RuBisCO subunits were labeled on the whole proteins. These findings could be explained by the better accessibility of the reactive glutaminyl residues from the isolated subunits to FXIIIa

Ｋａｓａｂａｃｈ‐Ｍｅｒｒｉｔｔ症候群患者の抜歯経験

Kasabach-Merritt syndrome is a coagulation disorder associated with giant hemangioma and consumption coagulopathy.Here we describe the procedure for hemostatic management for tooth extraction in a patient with Kasabach-Merritt syndrome.A 22-year-old woman was referred to our clinic to have a tooth extracted.Sixty mg/day of predonisolone p. o. and 10mg/h of nafamostat mesilate were given preoperatively, and 12ml (3V) of dried human antihemophilic factor XIII fraction was additionally administered just before extraction.We conclude that tooth extraction in a patient with consumption coagulopathy can be performed safety by carefully correcting for any coagulation disorders.

A Microtiter Assay for Factor XIII Using Fibrinogen and Biotinylcadaverine as Substrates

Efforts to develop an improved assay for plasma and tissue transglutaminase have led us to a convenient, sensitive microtiter plate assay for coagulation factor XIII using human fibrinogen as an immobilized substrate. Factor XIII was activated in the presence of calcium, thrombin, and immobilized fibrinogen and then assayed by adding biotinylcadaverine. The reaction was terminated by adding EDTA and the level of incorporated biotin was measured with streptavidin-β-galactosidase. In this assay, the analytical range for human platelet factor XIII was 0.01-100 ng and 1-100 ng for guinea pig liver transglutaminase. Fibrinogen-coated plates gave more than 100-fold increase in sensitivity compared with N, N-dimethylcasein-coated microtiter plates. The intraassay coefficient of variation was less than 5% ( n = 12) and interassay less than 6% ( n = 4). The sensitivity of this assay reduced the volumes of plasma samples required and consequently eliminated the need to remove fibrinogen from such test samples. As expected, factor XIII activity could be inhibited by putrescine and antibodies against factor XIII as well as by a monoclonal antibody that bound to the carboxyl terminus of human fibrin γ-chains. The assay provided a sensitive, simple, and rapid method for measuring factor XIII.

Analysis of the catalytic activity of human factor XIIIa by site-directed mutagenesis.

Factor XIIIa (FXIIIa) stabilizes fibrin clots by covalently cross-linking fibrin molecules. The purpose of this study was to determine the amino acid requirements at the active site of FXIIIa for catalysis. We selected amino acids 310-317 (Arg-Tyr-Gly-Gln-Cys-Trp-Val-Phe) in the human FXIII A-chain sequence for analysis based on the high degree of sequence homology among the different transglutaminases. We converted each amino acid in this region to Ala by site-directed mutagenesis. These recombinant FXIII A-chain mutants were expressed in Escherichia coli using the pTrc99A expression vector. FXIIIa activity was assessed by measuring the incorporation of 5-(biotinamido)pentylamine into N,N'-dimethylcasein in a solid-phase microtiter plate assay. The Cys-314-->Ala mutation yielded a recombinant protein with no FXIIIa activity. We also found that changing Gly-312 and Val-316 to Ala resulted in 22 and 65% decreases in activity, respectively. The other five mutations near the active-site Cys resulted in FXIIIa molecules in which the activity was reduced > 95%. The mechanism of SH protease catalysis is similar to transglutaminase catalysis in that both form thioester intermediates. His and Asp residues may stabilize this enzyme-substrate intermediate. Therefore, we performed site-directed mutagenesis on several His residues (His-342, His-373, and His-450) as well as Asp-396 in human FXIII. We found that changing His-342 to Ala reduced catalytic activity by 85%, while the His-373-->Ala mutant had no activity. In contrast, changing His-450 to Ala reduced FXIIIa activity by only 15%. We also examined the activity of all the mutants in a fibrin cross-linking assay. Four of the mutations (Phe-317-->Ala, Tyr-315-->Ala, Gln-313-->Ala, and Asp-396-->Ala), in which the activity toward the small primary amine was reduced by > 95%, were still capable of cross-linking the gamma-chain of fibrin. Even though these four mutants produced gamma-gamma dimers, they were not capable of forming higher molecular weight cross-linked products. Finally, we found that the binding of all the mutants to fibrin was similar to that of wild-type FXIIIa. In conclusion, we demonstrated that changing the specific amino acids Arg-310-Phe-317 to Ala substantially reduced FXIIIa activity. In addition, full catalytic activity was dependent on His-342, His-373, and Asp-396. These findings provide new insights into the catalytic mechanism of FXIIIa.

Three-dimensional structure of a transglutaminase: human blood coagulation factor XIII.

Mechanical stability in many biological materials is provided by the crosslinking of large structural proteins with gamma-glutamyl-epsilon-lysyl amide bonds. The three-dimensional structure of human recombinant factor XIII (EC 2.3.2.13 zymogen; protein-glutamine:amine gamma-glutamyltransferase a chain), a transglutaminase zymogen, has been solved at 2.8-A resolution by x-ray crystallography. This structure shows that each chain of the homodimeric protein is folded into four sequential domains. A catalytic triad reminiscent of that observed in cysteine proteases has been identified in the core domain. The amino-terminal activation peptide of each subunit crosses the dimer interface and partially occludes the opening of the catalytic cavity in the second subunit, preventing substrate binding to the zymogen. A proposal for the mechanism of activation by thrombin and calcium is made that details the structural events leading to active factor XIIIa'.

Purification and Characterization of Recombinant Human Coagulation Factor XIII A-Chains Expressed in E. coli

The purpose of this study was to develop an Escherichia coli expression system to facilitate study of the structure and function of blood coagulation factor XIII (FXIII) A-chains. We engineered an NcoI site into the full-length FXIII A-chain cDNA and subcloned it into pKK233-2 expression vector. A low level of full-length FXIII A-chain and a 30-kDa FXIII A-chain-related antigen were expressed in the JM 105 strain of E. coli. Protein sequencing of the 30-kDa protein demonstrated that it was synthesized by internal translation starting at either Met474 Or Met475. We mutated the internal ribosome-binding sequences from AGGA to TGGT (pKF13A2 construct) and found that it yielded a 30-fold increase in the production of full-length FXIII A-chains. JM105 harboring pKF 13A2 produced 20 mg of soluble FXIII A-chains antigen from 1 liter culture in TB medium. The recombinant FXIII A-chain was readily purified to homogeneity through PEG fractionation, Q-Sepharose, and mono-P column chromatography with a 2100-fold increase in specific activity and a yield of 150 to 200 μg of FXIII A-chains per liter of culture. The purified FXIII A-chains behaved as a dimer on gel filtration analysis, were thrombin- and calcium-activated, cross-linked fibrin, and bound to fibrin to the same extent as purified plasma FXIII A-chains and recombinant FXIII A-chains purified from yeast. These results document that FXIII A-chains can be readily expressed and purified from E. coli culture and that they retained properties similar to those of purified human factor XIII A-chains. The recombinant FXIII expressed in E. coli will be useful for studies on the structure and function of this important coagulation protein.

The human tissue transglutaminase gene maps on chromosome 20q12 by in situ fluorescence hybridization.

A cDNA encoding for the human tissue transglutaminase gene has been used to identify the chromosomal localization of the corresponding structural gene. The precise chromosomal and subregional localizations have been established by using in situ fluorescence mapping with a recombinant lambda-Zap phage containing the full cDNA coding sequence. The study showed that the human tissue transglutaminase gene is localized on chromosome 20 and, more precisely, within the band 20q12. To date, this is the third member of the transglutaminase gene family to be mapped. Human factor XIIIa (plasma transglutaminase), human keratinocyte transglutaminase (type I), and human tissue transglutaminase (type II) genes, although codifying for homologous enzymes, are localized on three different chromosomes.

Characterization of a Monoclonal Antibody Directed Against the Carboxyl-Terminus of Human Factor XIII

By deriving an anti-peptide monoclonal antibody, mAb 7A4, we characterized the relatively unstudied carboxyl-terminal end of the a-chain of human factor XIII, the plasma transglutaminase. MAb 7A4 was directed against the last eight amino acids (Gln-Ile-Gln-Arg-Arg-Pro-Ser-Met) and bound with a dissociation constant of 3.4 x 10(-8)M. In a solid assay format, mAb 7A4 bound equally well to factor XIII obtained from human plasma, platelets or placenta. However, in a solution-phase assay format, the epitope was largely unavailable but could be readily exposed by heat denaturation. Immunoblotting showed that this epitope is conserved among all species of plasma factor XIII tested except rabbit suggesting that the carboxyl-terminus might be an important structural element. Other competitive binding experiments with synthetic peptides as inhibitors pointed toward the final carboxyl-terminal amino acid, Met-731, as an immunochemically important determinant. This was used advantageously to confirm the finding that the carboxyl-terminal Met-731 is largely absent from placental factor XIII (1) as compared to platelet or plasma factor XIII.

Cross-linking of α2-antiplasmin to fibrin is a key factor in regulating blood clot lysis

The basal lysis rates ofex-vivoprepared blood clots from rats, mice, hamsters, dogs, and humans and levels of α2-antiplasmin (α2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an α2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIII) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma α2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited crosslinking by more than 80%. No significant cross-linking of α2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of α2-AP cross-linking was equal to that in human plasma indicating that rat α2-AP can be cross-linked to human fibrin. The same effect was obtained with human FXIII deficient plasma but not with human fibrinogen deficient plasma. These experiments indicate that rat fibrinogen is not a substrate for the cross-linking of rat α2-AP catalysed by rat FXIII, Addition of human plasma to hamster plasma did not result in an increased level of α2-AP cross-linking which indicates that other factors are involved.

Fibrin - recombinant human factor XIII a-subunit association

The association of factor XIII a-subunits with fibrin was characterized using recombinant human placental factor XIII (rFXIII) and native fully hydrated fibrin clots formed from purified fibrinogen and thrombin. Binding was assessed using small columns containing fibrin and perfusing them with radioiodinated rFXIII. Results show that thrombin activation of rFXIII led to fibrin binding. The association was partially reversible since much of the bound enzyme could be removed by percolating clots with more buffer. Binding was blocked by antibody directed against the COOH-terminal part of fibrinogen Aα-chain (Aα389–402) and also by the COOH-terminal Aα-chain peptide fragment Aα241–476 (Hi2-DSK).

2] Human plasma factor XIII: Subunit interactions and activation of zymogen

Publisher Summary This chapter focuses on the human plasma factor XIII that is the subunit interactions and activation of zymogen. Blood coagulation factor XIII, or fibrin-stabilizing factor, is the plasma zymogen of the enzyme responsible for strengthening the clot network and for endowing it with a high resistance to lysis. The chapter also provides a description for separating the A 2 and B 2 subunits of the purified human plasma factor XIII by using the Ca 2+ -specific, thrombin- independent pathway for directly dissociating the zymogen. Breaking the heterologous association of subunits seems to be the key for activating factor XIII. However, purified human plasma factor XIII can become activated to express full transamidating enzyme activity even without the prior removal of the N-terminal activation peptide (AP) segments from the A subunits by thrombin.

Limulus hemocyte transglutaminase. cDNA cloning, amino acid sequence, and tissue localization.

We have evidence that the limulus (Tachypleus tri-dentatus) hemocyte transglutaminase (TGase) has a molecular mass of 86 kDa and properties of the mammalian type II TGase-like enzyme (Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., and Davie, E.W. (1993) J. Biol. Chem. 268, 252-261). We present here the cDNA and amino acid sequences, and localization of the TGase in various tissues of limulus. The cloned cDNA for TGase consists of 2,884 base pairs. An open reading frame of 2,292 base pairs encodes a sequence comprising 764 residues of the mature protein with molecular masses of 87,021 and 87,110 Da, due to two different clones. The discrepancies of nucleotides in these two clones result in 3 amino acid exchanges at positions Gly452(GGT)-Arg(CGT), Ser477(AGT)-Cys(TGT), and Ile486(ATC)-Ser(AGC), respectively. Northern blot analysis on a total RNA extracted from various tissues of limulus revealed that TGase is expressed with 3.0 kilobases of a single type of mRNA, mainly in hemocytes, hepatopancreas, and gastric tissues. Limulus TGase shows significant sequence similarity with the mammalian TGase family, as follows: guinea pig liver TGase (32.7%), human factor XIIIa subunit (34.7%), human keratinocyte TGase (37.6%), and human erythrocyte band 4.2 (23.0%). Limulus TGase has a unique NH2-terminal cationic extension of 60 residues with no homology to the NH2 termini of mammalian TGases. Based on the alignment of the amino acid sequence of limulus TGase with those of the known TGase family, a phylogenetic tree representing an evolutionary relationship among the family members was inferred by the neighbor joining method.

Polymerization of meat and soybean proteins by human placental calcium-activated factor XIII

Placental FXIII a catalyzes the formation of e-(γ-glutamyl)-lysine bonds within and between protein molecules. Therefore, this enzyme, activated by calcium, was used to cross-link myosin, actin and 7S globulin. High-concentration myosin and globulin solutions formed gels when they were treated by FXIII a . The breaking strength of globulin gel is greater than that of myosin gel

Genomic structure of keratinocyte transglutaminase. Recruitment of new exon for modified function.

The gene for keratinocyte transglutaminase (TGK) spans 14 kilobase pairs and contains 15 exons. Many features of the TGK gene are very similar, if not identical, to those of the gene encoding the catalytic subunit of human clotting factor XIII: they have the same number of exons, corresponding introns always interrupt the coding region in the same phase of the codon, and most exons are of similar size (10 or 15 are exactly the same size). In these respects, the TGK and factor XIII catalytic subunit genes resemble each other more than either resembles the gene for erythrocyte band 4.2, a noncatalytic transglutaminase superfamily member. Exon II in both the TGK and factor XIII genes encodes an amino-terminal extension of nonhomologous sequence which in each protein confers a specialized function (membrane anchorage or activation of cross-linking, respectively). This suggests that the evolution of these genes included recruitment of a new exon to modify the enzyme action. Southern blots of genomic DNA reveal the presence of a TGK-like gene in birds, amphibians, and fish, but not in flies.

Isolation and characterization of cDNA clones to mouse macrophage and human endothelial cell tissue transglutaminases.

The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of both tissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophage tissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (greater than 80%). Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology (75% identity) was found in the active site region.

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.

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.

Nucleotide sequence of the gene for the b subunit of human factor XIII

Factor XIII (Mr 320,000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (Mr 75,000 each) and two b subunits (Mr 80,000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in lambda phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92% of the gene. The introns contained four Alu repetitive sequences, one each in introns A, E, I, and J. A fifth Alu repeat was present in the flanking 3' end of the gene. Two partial KpnI repeats were also found in the introns, including one in intron I and one in intron J. The KpnI repeat in intron J was 89% homologous to a sequence of approximately 2200 nucleotides flanking the gene coding for human beta globin and approximately 3800 nucleotides from the L1 insertion present in the gene for human factor VIII. Intron H also contained an "O" family repeat, while two potential regions for Z-DNA were identified within introns G and J. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.