Human Coagulation Factor XIII Research Articles

Suppressive effect of human blood coagulation factor XIII on the vascular permeability induced by anti-guinea pig endothelial cell antiserum in guinea pigs

We investigated the effect of blood coagulation factor XIII(FXIII) on enhanced permeability induced by anti-endothelial cell antiserum, that was produced by the immunization of guinea pig endothelial cells with adjuvant into rabbits repeatedly. We have found that this antiserum reacts to human and guinea pig endothelial cells but not guinea pig fibroblast cells. The permeability was enhanced by intradermal injection of 400-fold dilution of this antiserum into dorsal skin of guinea pigs. The mixture of equal volume of antiserum and FXIII was intradermally injected into dorsal skin of guinea pig after Evans blue injection, and 15 minutes later the quantity of Evans blue at the each injection site was determined. We recognized the suppressive effect of FXIII on the dye leakage. We also studied the suppressive effect on swelling induced by the antiserum. After the subcutaneous injection of the mixture of antiserum and FXIII into the back of guinea pigs, we measured the thickness of skins at the injection site after day 1, 2 and 3. As a result, FXIII significantly suppressed the swelling. We found that FXIII suppresses the acute and subacute permeability enhancement. These results suggest that FXIII plays an important role on an inflammatory site and that it may exert as an anti-inflammatory protein.

Molecular cloning of the b subunit of mouse coagulation factor XIII and assignment of the gene to chromosome 1: close evolutionary relationship to complement factor H.

The b subunit of human coagulation factor XIII (FXIII-b) is composed of 10 short consensus repeats (SCRs) characteristic of the regulatory proteins of complement activation system. A full-length cDNA clone of mouse FXIII-b was isolated and the entire sequence was determined. The predicted amino acid sequence showed 77.5% homology with human FXIII-b, although mouse FXIII-b contained seven extra amino acid residues at the carboxyl terminal. The strong reactivity of the translation product of this clone with rabbit anti-human FXIII-b antiserum confirmed that it encodes a mouse counterpart of the human FXIII-b. By in situ hybridization and mapping studies using 66 interspecific backcross mice, the mouse FXIII-b gene (designated F13b) was shown to be located on distal chromosome 1 closely linked to Cfh, extending a conserved linkage group between human and mouse chromosome 1. In addition, a significant structural similarity between FXIII-b and complement factor H is described.

3] Molecular approach to structure-function relationship of human coagulation factor XIII

This chapter focuses on the molecular approach to structure–function relationship of human coagulation factor XIII. Genomic DNAs from patients with a or b subunit deficiencies have been analyzed by Southern blotting, polymerase chain reaction (PCR), and nucleotide sequencing, employing amplification primers that are designed from the genomic nucleotide sequence of each subunit. These experiments are performed to find possible defects in the sites and amino acids essential for the functions of factor XIII and to understand genetic regulatory mechanisms for its biosynthesis. The complementary DNAs (cDNA) are also utilized for the expression and characterization of recombinant a and b subunits in yeast and mammalian cell systems. A number of deletion mutants for the a subunit lacking each exon have been made by a site-directed mutagenesis method to search for the functional regions in the molecule.

Variation of short tandem repeats within and between populations.

Using the polymerase chain reaction (PCR) the frequency distributions of three short tandem repeats (STR) were investigated in five populations: North European, Cypriot, Pakistani, Gujarati and Vietnamese. Each STR is situated within an intron; the markers are in the genes for human coagulation factor XIII (4bp repeat), lipoprotein lipase (4bp repeat) and CD4 (5bp repeat). Population data were generated for each STR and allele frequencies calculated. A calculation of the level of population substructuring for the three systems was also made. The lipoprotein lipase STR data showed no evidence for population substructuring, but there was a significant level of substructuring in the other two systems. This initial pilot study demonstrates the need to validate each marker used for DNA profiling in different human populations, and that some markers (such as LPL) can be used with confidence in widely differing ethnic groups, while others (such as CD4 and F13A) may be of value in distinguishing sub-groups.

Secretion of human blood coagulation factor XIIIa by the yeast Yarrowia lipolytica

The industrial yeast, Yarrowia lipolytica, secretes high yields of an alkaline extracellular protease (AEP), which is synthesized as a preproprotein encoded by the XPR2 gene. We investigated the possibility of using this system for the secretion of human coagulation factor XIII subunit a (FXIIIa). This protein is naturally secreted in the plasma by an unknown, signal peptide-independent mechanism and has so far been found to be nonsecretable in yeast. We have designed six hybrid genes encoding fusion proteins between increasing portions of the AEP preprodomain and the precursor or mature forms of FXIIIa. All constructs directed translocation of the FXIIIa precursor into the endoplasmic reticulum. Transport of the translocated and core-glycosylated hybrid precursor to the Golgi apparatus appeared to be strongly rate limiting, and most of the precursors appeared to be partially proteolysed. One of these constructs directed the extracellular secretion of a low amount of hyperglycosylated FXIIIa. These results indicate that fusion to the yeast AEP signal peptide and dipeptide stretch allows FXIIIa to be translocated, albeit inefficiently, through the endoplasmic reticulum and to follow a classical secretory transit.

Identification of a Point Mutation in Factor XIII A Subunit Deficiency

Oligonucleotide primers have been designed for the amplification of all 15 exons of the human coagulation factor XIII A subunit gene. Each exon and its intron flanking regions has been amplified and sequenced from a patient with severe A subunit deficiency. A single G to A transition in the last base of exon 14 has been identified in the homozygous proband and in his heterozygous parents. The mutation would result in the substitution 681 Arg to His in the mature protein product. However, because the mutation is at a splice junction, the deficiency may result from a defect in pre-messenger RNA splicing.

Identification of a point mutation in factor XIII A subunit deficiency

Oligonucleotide primers have been designed for the amplification of all 15 exons of the human coagulation factor XIII A subunit gene. Each exon and its intron flanking regions has been amplified and sequenced from a patient with severe A subunit deficiency. A single G to A transition in the last base of exon 14 has been identified in the homozygous proband and in his heterozygous parents. The mutation would result in the substitution 681 Arg to His in the mature protein product. However, because the mutation is at a splice junction, the deficiency may result from a defect in pre-messenger RNA splicing.

Organization of the gene for human erythrocyte membrane protein 4.2: structural similarities with the gene for the a subunit of factor XIII.

Human erythrocyte band 4.2 is a major membrane-associated protein with an important, but still undefined, role in erythrocyte survival. We previously sequenced the complete cDNA for band 4.2 and showed that the protein has a strong sequence identity with the transglutaminase family of proteins but lacks transglutaminase activity. Here we have analyzed the genomic organization of band 4.2. The band 4.2 gene is approximately 20 kilobases, consisting of 13 exons and 12 introns. Reticulocytes contain two different sized messages for band 4.2, and our results show that the major, smaller, message is produced by alternative splicing within band 4.2 exon I. The upstream region of the gene has several prospective promoter elements arranged in a pattern similar to that of two other erythroid genes, beta-globin and porphobilinogen deaminase. Alignment of the band 4.2 amino acid sequence with that of the a subunit of human coagulation factor XIII and division of the sequences into exons reveal a remarkable correspondence, and in most cases identity, in the sizes of the paired exons. Moreover, each corresponding intron of the two genes is of an identical splice junction class. These and other similarities suggest that the gene for band 4.2 is closely related to and possibly derived from that for the a subunit of factor XIII and that the proteins may share common structural and functional properties.

Tetranucleotide repeat polymorphism at the human coagulation factor XIII A subunit gene (F13A1)

Tetranucleotide repeat polymorphism at the human coagulation factor XIII A subunit gene (F13A1)

Genetic polymorphisms of the A and B subunits of human coagulation factor XIII in mainland Italy and Sardinia: Description of a new FXIIIA variant allele

The distribution of the two alleles of FXIIIA and the three alleles of FXIIIB were studied in populations from mainland Italy and from Sardinia. The frequencies of the FXIIIA*2 allele were 0.266 and 0.265. The frequencies of FXIIIB*1 were 0.787 and 0.765; of B*2, 0.070 and 0.094; of B*3, 0.143 and 0.141. A new cathodal FXIIIA allele (A*7) was described in the Rome sample. No significant difference in the distribution of allele frequencies for either system was found between the two populations studied. For typing both markers, good results were also obtained by using whole blood instead of plasma.

Effect of human coagulation factor XIII on wound healing of guinea-pigs

Effect of human coagulation factor XIII on wound healing of guinea-pigs

Synthesis of human coagulation factor XIII in yeast

The active form of coagulation factor XIII, factor XIIIa, is a transglutaminase that covalently cross-links fibrin molecules by joining γ-glutamyl and ϵ-lysyl primary amino groups. In this paper we report the design of a yeast expression vector called pPH3 which includes the GAL1-GAL10 promoter, multiple cloning sites, the URA3 selectable marker, the 2μ sequences and the ampicillin-resistance gene. We have placed full-length factor XIII cDNA into the PstI site of this vector, transformed the yeast cells and expressed human coagulation factor XIII which was found to be enzymatically active based on observations that this recombinant factor XIII: (a) showed immunological identity with placental factor XIII; (b) was demonstrated to have transglutaminase activity, and (c) cross-linked fibrin in a manner identical to human placental factor XIIIa-catalyzed polymerization (γ-γ dimerization and α polymerization).

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.

The subunit composition of factor XIII proteins in normal and factor XIII deficient plasma and serum analysed by line immunoelectrophoresis

Line immunoelectrophoresis was used for the characterization of human coagulation factor XIII in normal and factor XIII-deficient plasma and serum. In normal plasma, two immunoreactive factor XIII proteins termed XIII-p ( M r 330 000 Da) and XIII-s ( M r 170 000 Da) were identified within the beta-globulin fraction. Their reactions with monospecific antisera against known factor XIII subunits, suggests that the factor XIII-p and -s proteins recognized represent the factor XIII a 2b 2 tetramer and -b 2 dimer, respectively. The concentration of the XIII-p protein was, in normal plasma, approximately 6 mg/l, in plasma from a case of congenital factor XIII, approximately 1 mg/l and in normal serum approximately 3 mg/l. The concentration of the XIII-s protein (approximately 18 mg/l) was unaffected by the coagulation process and normal in plasma and serum from the case of congenital factor XIII deficiency. Thus, the present investigation indicates the presence of an excess of the anticatalytic, DNA-binding factor XIII carrier protein in normal as well as in factor XIII-deficient plasma and serum.

A new method for FXIIIA genetic variants determination using isoelectric focusing in 1 M urea. Evidence for three structural gene loci, FXIIIA1, FXIIIA2, and FXIIIA3.

A new method for separating genetic variants of the A subunit of human coagulation factor XIII using ultrathin layer polyacrylamide gel isoelectric focusing in 1 M urea followed by immunoblotting is described. The pattern obtained by this method differs from that reported previously: Three sets of unrelated band patterns are observed and can be explained by the existence of two additional gene loci, designated FXIIIA2 and FXIIIA3, besides the previously reported FXIIIA locus, now renamed FXIIIA1. The FXIIIA2 locus is polymorphic and shows three commonly occurring phenotypes, FXIIIA2 1, FXIIIA2 2-I, and FXIIIA2 2. These are determined by two common alleles, FXIIIA2*1 and FXIIIA2*2, with respective frequencies of 0.7965 and 0.2035 in the Japanese population. The studied population conforms to a Hardy-Weinberg equilibrium, and family data confirmed autosomal codominant transmission. The FXIIIA3 locus is monomorphic.

The genetic relationships between the kringle domains of human plasminogen, prothrombin, tissue plasminogen activator, urokinase, and coagulation factor XII

A computer-based statistical evaluation of the optimal alignments of the kringle domains of human plasminogen, human prothrombin, human tissue plasminogen activator, human urokinase, and human coagulation Factor XIIa, as well as the putative kringle of human haptoglobin, has been performed. A variety of different alignments has been examined and scores calculated in terms of the number of standard deviations (SD) of a given match from randomness. With the exception of human haptoglobin, it was found that very high alignment scores (8.9-23.0 SD from randomness) were obtained between each of the kringles, with the kringle 1 and kringle 5 regions of human plasminogen displaying the highest similarity, and the S kringle of human prothrombin and the human Factor XII kringle showing the least similarity. The relationships obtained were employed to construct an evolutionary tree for the kringles. The predicted alignments have also allowed nucleotide mutations in these regions to be evaluated more accurately. For those regions for which nucleotide sequences are known, we have employed the maximal alignments from the protein sequences to assess nucleotide sequence similarities. It was found that a range of approximately 40-55% of the nucleotide bases were placed at identical positions in the kringles, with the highest number found in the alignment of the two kringles of human tissue plasminogen activator and the lowest number in the alignment of the S kringle of prothrombin with the second kringle of tissue plasminogen activator. From both protein and nucleotide alignments, we conclude that haptoglobin is not statistically homologous to any other kringle. Secondary structural comparisons of the kringle regions have been predicted by a combination of the Burgess and Chou-Fasman methods. In general, the kringles display a very high number of beta-turns, and very low alpha-helical contents. From analysis of the predicted structures in relationship to the functional properties of these domains, it appears as though many of their functional differences can be related to possible conformational alterations resulting from amino acid substitutions in the kringles.

0.9. FXIIIB ∗7, a new allele in the human coagulation factor XIII subunit B system, detected by immunofixation agarose gel isoelectric focusing (IAGIF)

0.9. FXIIIB ∗7, a new allele in the human coagulation factor XIII subunit B system, detected by immunofixation agarose gel isoelectric focusing (IAGIF)

Improved coagulation factor XIII B (FXIIIB) phenotyping after neuraminidase treatment of plasma and first description of the FXIIIB 2 phenotype.

The genetically determined polymorphism of the B subunit of the human coagulation factor XIII (FXIIIB) was investigated by an improved technique of immunofixation agarose gel electrophoresis (IAGE). Employing neuraminidase (CPN) treatment of fivefold-concentrated fresh plasma and monospecific anti-human FXIIIB antiserum an excellent resolution of phenotypes was possible. Six phenotypes, FXIIIB 1,2-1,2,3-1, 3-2 and 3 were detected among 178 unrelated blood donors from Hessen, Germany, the FXIIIB 2 homozygote for the first time. Our findings confirm the 'reduced' two-allele-model of Kera et al. [10]. For the first time the allele frequencies were determined in a European population: FXIIIB*1 = 0.708, FXIIIB*2 = 0.109, FXIIIB3* = 0.183. As in family studies no unexpected phenotypes were observed in the offspring, FXIIIB might become a useful genetic marker in paternity testing, the single exclusion chance for non-fathers being 23.7%.

Genetic polymorphism of the B subunit of human coagulation factor XIII in Japanese.

Polymorphism of the B subunit of human coagulation factor XIII was investigated in a total of 119 plasma samples from unrelated adult Japanese using agarose gel electrophoresis followed by immunofixation. Three common phenotypes were observed, which corresponded to the types 1, 1–3 and 3 reported by Board (1980) in Australians. The estimated allele frequencies ofF13B*1 andF13B*3 were 0.303 and 0.697, respectively. The observed numbers of the phenotypes were in good agreement with those expected on the basis of Hardy-Weinberg's law. These results are not inconsistent with the hypothesis that these phenotypes are controlled by a pair of autosomal, codominant alleles.

Human coagulation factor XIII A (F XIII A) phenotyping by immunofixation agarose gel electrophoresis (IAGE).

The genetic polymorphism of the A subunit of human coagulation factor XIII (FXIIIA) was investigated in platelet lysates from 239 blood donors from Hessen, Germany, and thereby for the first time in an European population. FXIIIA activity was detected by using a monospecific rabbit anti-human factor XIII subunit A antiserum for immunofixation after high voltage thin-layer agarose gel electrophoresis (IAGE). Three common phenotypes were observed (FXIII A 1, 2-1, and 2). The calculated allele frequencies were FXIIIA*1=0.797 and FXIIIA*2=0.203. The application of the FXIIIA polymorphism in blood group genetics is discussed.