Subunit Of Coagulation Factor XIII Research Articles

Mutations in coagulation factor XIII subunit A in severe factor XIII deficiency patients: five novel mutations detected

Mutations in coagulation factor XIII subunit A in severe factor XIII deficiency patients: five novel mutations detected

The Val34Leu genetic variation in the A Subunit of Coagulation Factor XIII in recurrent spontaneous abortion

The present study was carried out to assess the frequencies of factor XIII (FXIII) Val34Leu genetic variation in the patients with recurrent spontaneous abortion and healthy fertile women of Azeri Turkish origin. A total of 54 patients with recurrent spontaneous abortion and 46 healthy fertile women as controls were studied for the FXIII Val34Leu genetic variation by a RFLP-PCR method. Statistical analysis showed that patients (χ2 = 2.4, p value = 0.292) and controls (χ2 = 1.035, p value = 0.596) were in agreement with Hardy-Weinberg equilibrium. The Leu allele frequency was 0.18 and 0.13 in patients and controls, respectively. FXIII Leu34Leu (homozygous for 34Leu) genotype was not found in patients and controls. FXIII Val/Leu and Val/Val genotype frequencies were 19 (35.19%) and 31 (64.81%) in patients and 12 (26.09%) and 34 (73.91%) in controls, respectively. FXIII 34Leu allele and Val34Leu genotypes were more common in the case group containing individuals with unexplained RSA but the differences of FXIII Val34Leu (G/T genotype) (odds ratio = 0.65 (0.25 < OR < 1.67) corresponding to 95% CI, χ2 = 0.96, p value = 0.32) and FXIII 34Leu (T allele) (odds ratio = 0.70 (0.30 < OR < 1.64) corresponding to 95% CI, χ2 = 0.78, p value = 0.37) was not statistically significant. These results suggest that factor XIII Val34Leu genetic variation is not associated with recurrent spontaneous abortion.

Coagulation factor XIII activation peptide and subunit levels in patients with acute ischaemic stroke: A pilot study

IntroductionWe have recently shown that FXIII activation peptide (AP-FXIII) can be measured in plasma. The objective of this pilot study was to investigate for the first time if AP-FXIII can be detected in plasma from patients with acute ischaemic stroke. Materials and methodsWe included 66 patients with acute ischaemic stroke admitted between 1 and 7hours after the onset of clinical symptoms. We collected plasma samples upon admission and on the two following days and measured AP-FXIII and subunit levels by ELISA. Clinical stroke severity was assessed by NIHSS stroke score. ResultsAP-FXIII could be detected in 34 patients upon admission (range 0.2-26.3ng/ml), on day 1 in 15 patients (0.2-10.4ng/ml), and on day 2 in 11 patients (0.1-15.1ng/ml. AP-FXIII was higher in patients with severe stroke. Lower AP-FXIII levels upon admission were associated with clinical improvement. FXIII-A and FXIII-B subunit levels decreased significantly from day 0 to day 1. ConclusionsFor the first time, we detected AP-FXIII in patients upon an acute thrombotic event. The decrease in FXIII subunit levels during acute ischaemic stroke is evidence for ongoing coagulation activity and FXIII consumption. Our results suggest that FXIII activation and concomitant AP-FXIII release might be associated with an unfavourable short-term clinical outcome. Larger studies are needed to further investigate whether AP-FXIII might serve as a diagnostic and/or prognostic marker for acute thrombotic diseases.

Identification of eight novel coagulation factor XIII subunit A mutations: implied consequences for structure and function

Severe hereditary coagulation factor XIII deficiency is a rare homozygous bleeding disorder affecting one person in every two million individuals. In contrast, heterozygous factor XIII deficiency is more common, but usually not associated with severe hemorrhage such as intracranial bleeding or hemarthrosis. In most cases, the disease is caused by F13A gene mutations. Causative mutations associated with the F13B gene are rarer. We analyzed ten index patients and three relatives for factor XIII activity using a photometric assay and sequenced their F13A and F13B genes. Additionally, structural analysis of the wild-type protein structure from a previously reported X-ray crystallographic model identified potential structural and functional effects of the missense mutations. All individuals except one were heterozygous for factor XIIIA mutations (average factor XIII activity 51%), while the remaining homozygous individual was found to have severe factor XIII deficiency (<5% of normal factor XIII activity). Eight of the 12 heterozygous patients exhibited a bleeding tendency upon provocation. The identified missense (Pro289Arg, Arg611His, Asp668Gly) and nonsense (Gly390X, Trp664X) mutations are causative for factor XIII deficiency. A Gly592Ser variant identified in three unrelated index patients, as well as in 200 healthy controls (minor allele frequency 0.005), and two further Tyr167Cys and Arg540Gln variants, represent possible candidates for rare F13A gene polymorphisms since they apparently do not have a significant influence on the structure of the factor XIIIA protein. Future in vitro expression studies of the factor XIII mutations are required to confirm their pathological mechanisms.

Distinct C-terminus of the B subunit of factor XIII in a population-associated major phenotype: the first case of complete allele-specific alternative splicing products in the coagulation and fibrinolytic systems.

The purpose of this study was to elucidate the molecular bases of the heterogeneity of the B subunit of coagulation factor XIII (FXIII-B), classified by isoelectric focusing into its three population-associated major phenotypes. By genetic sequencing and polymerase chain reaction (PCR)-restriction fragment length polymorphism analyses, a C-to-G change was identified in intron K for the Asian-associated major phenotype FXIII-B*3. A transcript containing the novel exon XII' was detected by reverse transcription PCR using hepatocyte cell lines with this allele. The exclusive existence of a novel C-terminal peptide in a homozygote of FXIII-B*3 was also detected by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The FXIII-B*3 isoform had a C-terminus 15 residues longer than the other isoforms, containing two additional basic amino acids and one extra acidic amino acid. Accordingly, the C-to-G nucleotide substitution created an efficient splice acceptor AG dinucleotide, which resulted in allele-specific alternative splicing in intron K. When compared with FXIII-B*1, the third major phenotype, FXIII-B*2, had an A-to-G change in exon III, converting His95 to Arg, and a rare phenotype, FXIII-B*4, had an A-to-T change in exon VII, converting Glu368 to Val. We found an extremely rare event of complete allele-specific alternative splicing for FXIII-B. The FXIII-B*3 isoform had a distinct C-terminal peptide, while the FXIII-B*2 and FXIII-B*4 isoforms had His95 to Arg and Glu368 to Val substitutions, respectively, which led to differential isoelectric points of these isoforms. Such variations in the amino acid sequence of FXIII-B may have profound effects on its structure-function relationship, plasma FXIII levels, and disease susceptibility.

Prothrombotic Gene Variation in Patients with Large and Small Vessel Disease

The classification of ischemic stroke into different subtypes is supported in genetic association studies. While several gene variants have been identified as being associated with ischemia subtypes, most studies have not been powered to study subtypes separately. We investigated 887 patients with nondisabling cerebral ischemia of arterial origin and classified ischemia as being caused by large or small vessel disease (LVD or SVD), primarily based on neuroimaging findings. In total, 621 patients had LVD and 266 SVD. The coagulation factor XIII subunit B gene (F13B) His95Arg variant was more common in patients with LVD (mean prevalence difference 9.9%; 95% CI 4.7–15). None of 21 other prothrombotic gene variants was associated with LVD or SVD. Many gene variants may not be specific for one subtype. We could not replicate the genetic associations identified in previous smaller previous studies. Thus claims about genetic associations with specific subtypes of ischemia should be interpreted with caution. The association between F13B His95Arg and LVD requires replication.

Coagulation factor XIII gene variation, oral contraceptives, and risk of ischemic stroke

AbstractProthrombotic conditions are associated with ischemic stroke in young women. In particular, the combination of oral contraceptive use and prothrombotic genetic variants appears to increase the risk of ischemic stroke. We performed a population-based case-control study in 190 women aged 20 to 49 years with ischemic stroke and 767 women without cardiovascular disease stratified for age, calendar year of the index event, and residence. A total of 4 variants of coagulation factor XIII subunit A and B genes (F13A1 and F13B) were investigated. The Phe allele of the F13A1 Tyr204Phe variant was present in 59 (31%) patients and 43 (6%) controls; the odds ratio for ischemic stroke was 9.1 for Phe/Phe and Phe/Tyr versus Tyr/Tyr genotype; the 95% confidence interval was 5.5 to 15. Homozygous genotypes (Phe/Phe) conferred a higher risk (odds ratio, 77; 95% confidence interval, 7.0-848) than heterozygous (Tyr/Phe) genotypes (odds ratio, 8.2; 95% confidence interval, 4.9-14). The risk of ischemic stroke was further increased in carriers of the 204Phe allele using oral contraceptives (odds ratio, 20; 95% confidence interval, 9-46) compared with nonusers with Tyr/Tyr genotype. In conclusion, the F13A1 204Phe allele was strongly associated with ischemic stroke in young women. Oral contraceptive use further increased the risk of ischemic stroke.

Further Investigation of the Role of Factor XIII in Priapism Associated with SCD.

Genetic risk factors may contribute to the incidence of certain sickle cell disease (SCD) complications. Priapism, a painful, unwanted and sustained erection, is caused by decreased venous drainage in the penile vessels. Our previous work suggested an association between multiple non-coding polymorphisms in the A1 subunit of coagulation Factor XIII (F13A1) gene and a history of priapism (Elliott et al., 2007). The purpose of this study was to further investigate the possible association of polymorphisms in the A1 and B (F13B) subunits of the Factor XIII gene with priapism in order to better understand how differences in the coagulation pathway might affect the risk for priapism. The larger data set from which this study was derived included 524 unrelated patients with any form of SCD. These individuals had been recruited at three southeastern US medical centers. A subset of 186 males from this study population was used in the current analysis. These patients all had a diagnosis of Hb SS, had provided a DNA sample for genetic analysis and had responded to a standardized questionnaire regarding the history of priapism. The patients had a mean age of 32.4 years, and 78 (42%) reported a positive history of priapism. Four coding single nucleotides polymorphism (SNPs) in the F13A1 gene and 10 SNPs (2 coding, 8 non-coding) in the F13B gene were genotyped in our dataset. Genotyping was performed using Applied Biosystems Taqman Allelic Discrimination Assays. For quality control, blinded duplicate and CEPH samples were included on all gels and required to match 100%. Further, the genotypes of at least 95% of the samples had to be called with certainty to be considered for statistical analysis. Deviations from Hardy-Weinberg equilibrium (HWE) were identified using exact tests implemented by the Genetic Data Analysis program; no markers in F13A1 or F13B significantly deviated from HWE. For each SNP, exact tests of association for the genotypes by occurrence of priapism were performed using SAS. No SNPs in F13B were associated with priapism, and two F13A polymorphisms previously associated with risk for thrombosis (rs5985 and rs6003) were also not associated with risk for priapism in our data set. However, a single coding SNP in F13A1 (rs5988) was strongly associated with priapism (global p-value of 0.03). The odds that males with the C/C genotype suffered from priapism were 2.43 times higher than for those with the C/G genotype. This SNP resides in the C- terminus of FXIIIA. This area of the protein functions to prevent substrates from gaining access to the Factor XIII active site. While there is no evidence that this area directly regulates catalysis, it may play a role in aligning the substrate with the active site. Factor XIII could modify fibrin formation and alter red cell interaction with the vasculature. We plan to express the molecule encoded by the allele associated with risk to see if it has altered activity. In conclusion, identification of risk groups for priapism by genetic studies may lead to investigation of newly identified pathophysiologic mechanisms for this complication of SCD.Priapism Genotype Frequencies for rs5988Negative HistoryPositive HistoryTotalC/C6861129C/G381452G/G235Total10878186

Genetic polymorphisms associated with priapism in sickle cell disease

Priapism occurs in 30-45% of male patients with sickle cell disease (SCD), but the possible influence of genetic risk factors on the incidence of priapism is not well understood. We examined genetic polymorphisms in 199 unrelated, adult (>18 years), male patients with Hb SS and Hb Sbeta(0)-thalassaemia, 83 (42%) of whom reported a history of priapism. Candidate genes for association with priapism were identified based on their involvement in adhesion, coagulation, inflammation and cell signalling. Additionally, we examined genes involved in nitric oxide biology (NOS2, NOS3, SLC4A1), as well as polymorphisms in the klotho (KL) gene, which has previously been associated with priapism. Strong evidence of association was found for single nucleotide polymorphisms in transforming growth factor-beta receptor, type III (TGFBR3) (rs7526590; P = 0.00058), aquaporin (AQP1) (rs10244884; P = 0.00068), integrin alphav (ITGAV) (rs3768780; P = 0.00090), and the A1 subunit of coagulation factor XIII (F13A1) (hcv1860621; P = 0.00156). Associations with TGFBR3, AQP1, and ITGAV remained significant after adjusting for multiple testing, using the Benjamini-Hochberg procedure. Our data suggest that genes involved in the TGFbeta pathway, coagulation, cell adhesion and cell hydration pathways may be important in risk for priapism.

Genetic Polymorphisms Associated with Priapism in Sickle Cell Disease.

Priapism, a painful and prolonged erection, has been reported to occur in 30–45% of male patients with sickle cell disease (SCD). However, little is known about the pathological processes and genetic risk factors that contribute to the occurrence of priapism. The identification of genetic variables that are associated with priapism may therefore help define both critical pathophysiologic mechanisms not otherwise apparent, as well as patients at increased risk. We examined genetic variation in our sample of 199 unrelated, adult (>18 years), male patients with Hb SS and Hb Sβ0-thalassemia, 83 (42%) of whom reported a history of priapism. Candidate genes for association with priapism were identified based on their involvement in adhesion, coagulation, inflammation, and cell signaling. Additionally, we examined genes involved in NO biology (NOS2, NOS3, SOD1, SLC4A1). Finally, we also examined polymorphisms in the KLOTHO gene, which has previously been associated with priapism. We examined a total of 389 SNPs in 48 candidate genes. Except for the gene encoding the β2 adrenergic receptor, SNP genotyping was performed by TaqMan, using Assays-on-Demand or Assays-by-Design genotyping products (Applied Biosystems). Allele tests were used to detect genetic associations with priapism. Strong evidence of association was found for SNP rs7526590 in the transforming growth factor-β receptor, type III (TGFBR3) gene (p=.00058), SNP rs10244884 in the aquaporin (AQP1) gene (p=.00068), and SNP rs3768780 in the integrin αV (ITGAV) gene (p=0.00090). A second ITGAV SNP (rs3768778), in linkage disequilibrium (r2=.59) with the first, also showed association with priapism (p=.00888). The A1 subunit of coagulation factor XIII (F13A1) had four SNPs (hcv1860621, rs1032045, rs1674074, rs381061) with p-values less than 0.010 (p-values = 0.00156, 0.00415, 0.00648, and 0.00712, respectively). The linkage disequilibrium among these F13A1 SNPs is negligible (r2 <.15). We also adjusted for multiple testing using the Benjamini-Hochberg procedure (significance threshold <.10). SNP rs7526590 in TGFBR3, SNP rs10244884 in AQP1 and SNP rs3768780 in ITGAV each had a false discovery rate (FDR) p-value of .09834. SNP rs1674074 in F13A1 had an FDR p-value of .12733. The other SNPs in F13A1 had large FDR p-values, close to .30. We did not detect an association between priapism and genetic variation in the Klotho gene, as was previously reported by Nolan et al. (2005). Specifically, SNPs rs2249358, rs211234 and rs211239 showed a virtually identical distribution of genotypes for individuals with and without a history of priapism. However, our population is not identical to the previous study, which included patients as young as 10 years old. In conclusion, our data support the hypothesis that genetic variation is associated with risk for priapism among males with SCD and suggest that genes involved in the TGFβ pathway, coagulation, cell adhesion and cell hydration pathways may be important. [Display omitted]

Transglutaminases in mineralized tissues

Bone development and formation during embryogenesis as well as postnatally during bone remodeling is a complex process controlled systemically and locally by hormones, growth factors and matrix molecules. Transglutaminases (TGases) are the protein cross-linking enzymes, which have long been implicated in bone development and formation. Two members of TGase family, TG2 (also called tissue transglutaminase) and FXIIIA (the enzymatic A subunit of coagulation factor XIII), are expressed in chondrocytes and osteoblasts. The results of analyses in vivo and in vitro accumulated to date indicate an important role of these enzymes in promoting chondrocyte and osteoblast differentiation and matrix mineralization. These effects could be mediated by protein cross-linking activity of TGases, by GTPase activity of TG2 or via non-catalytic signaling effects. The aim of this review is to summarize the available data regarding the expression, localization and activity of TG2 and FXIIIA in mineralizing tissues and to discuss a number of mechanisms by which TGases could exert their promineralizing effects.

Leukemic lymphoblasts, a novel expression site of coagulation factor XIII subunit A

Blood coagulation factor XIII (FXIII) is a protransglutaminase circulating as a tetramer formed by two types of subunits (A2B2). The intracellular dimeric form of FXIII (A2) is present in platelets, megakaryocytes, monocytes and macrophages and has been detected in mono- and megakaryocytic leukemias. The aim of our study was to investigate FXIII-A expression in newly diagnosed B cell acute lymphoblastic leukemia (ALL) samples. We examined 47 de novo ALL cases of B cell origin by triple color labeling with flow cytometry. FXIII-A was detected by a FITC conjugated monoclonal antibody combined with CD34 and CD45 staining. In selected cases FXIII-A was investigated on slides prepared from blasts and visualized with a fluorescent microscope. In addition, blasts were studied by Western blot analysis and FXIII-A was measured by a highly sensitive ELISA method. By flow cytometry 19 samples of the 47 cases were found to be FXIII-A positive. Antigen concentration was 3.11 +/- 1.19 fg/blast, while normal lymphoid precursors and mature lymphocytes from B-CLL did not contain FXIII-A. In the lysate of lymphoblasts that were positive by flow cytometry, a single band (82 kDa) corresponding to FXIII-A was detected on Western blots. Confocal laser scanning microscopic examination revealed the presence of FXIII-A in the cytoplasm of these lymphoblasts. This novel expression site of FXIII-A in leukemic lymphoblasts can be utilized as a diagnostic tool and may also gain functional significance in B-lineage ALL.

Rapid detection of the factor XIII Val34Leu (163 G-->T) polymorphism by real-time PCR using fluorescence resonance energy transfer detection and melting curve analysis.

The Val34Leu polymorphism in the A subunit of blood coagulation factor XIII (FXIII-A) is located in the activation peptide, just three amino acids upstream of the thrombin cleavage site. The Val-->Leu replacement accelerates the rate of the proteolytic activation of FXIII and it seems to provide protection against myocardial infarction. Methods available for the assessment of the FXIII-A Val34Leu polymorphism are rather time-consuming, laborious and not easily applicable for large-scale studies. In this study a new method based on real-time PCR with fluorescence resonance energy transfer (FRET) detection and melting curve analysis was developed. The rapid, simple method was adapted to the widely used real-time PCR instrument, LightCycler (Roche Diagnostics). The results showed 100% coincidence with those obtained by the traditional PCR-restriction fragment length polymorphism (RFLP) assay and fluorescent DNA sequencing. Using this method, an allele frequency of 24.2% was obtained (n=113), which well agrees with the allele frequency obtained by PCR-RFLP on a different group of the same ethnic Hungarian population (25.9%).

Genetic and phenotypic polymorphisms of the A subunit of Coagulation factor XIII in Japanese population.

To clarify whether genetic polymorphisms in exon 14 of Coagulation factor XIII A-subunit gene (FXIIIA) affect phenotype expressions, we studied genetic polymorphisms in exon 14 of FXIIIA in a Japanese population and the relationship between the genetic polymorphisms and phenotype expression. Genetic polymorphisms in exon 14 of FXIIIA of 144 unrelated Japanese were analyzed by single-strand conformation polymorphism. Plasma FXIIIA antigen concentrations, FXIII activities, and phenotype were also determined by two-dimensional electrophoresis. The frequencies of the three genotypes, the homozygote (AD), the homozygote (BC) and the heterozygote (AD/BC), were 77.1, 0.7, and 22.2%, respectively. The gene frequencies of AD and BC were 0.88 and 0.12. We detected AD (GTT x GAG) and BC (ATT x CAG) at codon 650 and 651 of exon 14. There were no significant differences of FXIIIA antigen concentrations and FXIII activities between these genotypes. We detected three pl differences among them as being pls of 5.3, 5.6, 5.8 in the homozygote (AD) and the heterozygote (AD/BC), and a pl of 5.8 in the homozygote (BC). These polymorphisms affected isoelectric mobility, but did not affect protein levels, enzyme activities, or the molecular weight of FXIII.

Enzyme-linked immunosorbent assay for the determination of blood coagulation factor XIII A-subunit in plasma and in cell lysates

A new one-step ELISA was developed for the determination of the concentration of blood coagulation factor XIII subunit A (FXIII-A) in plasma and in cell lysates. Monoclonal antibodies directed against different epitopes on FXIII-A were used for the assay. The capture antibody was biotinylated on its carbohydrate moiety and the detection antibody was labelled with horseradish peroxidase. The antigen–antibody reaction was carried out in the well of a streptavidin-coated microplate. Complex formation with FXIII subunit B (FXIII-B) and association to fibrinogen did not influence the accessibility of the antibodies to FXIII-A. The method could be performed within 2 h and demonstrated good reproducibility, recovery and sensitivity. Plasma samples could be assayed after storage at −20 °C for at least 6 months. However, in the case of platelet lysates freezing and rethawing resulted in a significant loss of FXIII-A. FXIII-A concentrations measured in the plasma samples of healthy individuals and patients correlated well with the concentrations of complexed plasma FXIII (A 2B 2) and with the results of FXIII activity measurements. A reference range of 46–82 fg/platelet was established for platelet FXIII-A.

Val34Leu polymorphism of plasma factor XIII: biochemistry and epidemiology in familial thrombophilia

AbstractVal34Leu polymorphism of the A subunit of coagulation factor XIII (FXIII-A) is located in the activation peptide (AP) just 3 amino acids away from the thrombin cleavage site. This mutation has been associated with a protective effect against occlusive arterial diseases and venous thrombosis; however, its biochemical consequences have not been explored. In the current study it was demonstrated that the intracellular stability and the plasma concentration of FXIII of different Val34Leu genotypes are identical, which suggests that there is no difference in the rate of synthesis and externalization of wild-type and mutant FXIII-A. In contrast, the release of AP by thrombin from the Leu34 allele proceeded significantly faster than from its wild-type Val34 counterpart. By molecular modeling larger interaction energy was calculated between the Leu34 variant and the respective domains of thrombin than between the Val34 variant and thrombin. In agreement with these findings, the activation of mutant plasma FXIII by thrombin was faster and required less thrombin than that of the wild-type variant. Full thrombin activation of purified plasma FXIII of different genotypes, however, resulted in identical specific transglutaminase activities. Similarly, the mean specific FXIII activity in the plasma was the same in the groups with wild-type, heterozygous, and homozygous variants. Faster activation of the Leu34 allele hardly could be associated with its presumed protective effect against venous thrombosis. No such protective effect was observed in a large group of patients with familial thrombophilia.

Val34Leu polymorphism of plasma factor XIII: biochemistry and epidemiology in familial thrombophilia

Val34Leu polymorphism of the A subunit of coagulation factor XIII (FXIII-A) is located in the activation peptide (AP) just 3 amino acids away from the thrombin cleavage site. This mutation has been associated with a protective effect against occlusive arterial diseases and venous thrombosis; however, its biochemical consequences have not been explored. In the current study it was demonstrated that the intracellular stability and the plasma concentration of FXIII of different Val34Leu genotypes are identical, which suggests that there is no difference in the rate of synthesis and externalization of wild-type and mutant FXIII-A. In contrast, the release of AP by thrombin from the Leu34 allele proceeded significantly faster than from its wild-type Val34 counterpart. By molecular modeling larger interaction energy was calculated between the Leu34 variant and the respective domains of thrombin than between the Val34 variant and thrombin. In agreement with these findings, the activation of mutant plasma FXIII by thrombin was faster and required less thrombin than that of the wild-type variant. Full thrombin activation of purified plasma FXIII of different genotypes, however, resulted in identical specific transglutaminase activities. Similarly, the mean specific FXIII activity in the plasma was the same in the groups with wild-type, heterozygous, and homozygous variants. Faster activation of the Leu34 allele hardly could be associated with its presumed protective effect against venous thrombosis. No such protective effect was observed in a large group of patients with familial thrombophilia.

A study of human coagulation factor XIII A-subunit by electrospray ionisation mass spectrometry

A study of factor XIII A-subunit has been made by electrospray ionisation mass spectrometry. Factor XIII has been isolated and rigorously purified, and the intact A-subunit protein has been analysed to confirm its molecular weight accurately for the first time. Thrombin proteolysis of the A-subunit has been monitored and the resulting cleavage products, the activated A-subunit protein and the Activation Peptide, have been analysed by electrospray ionisation mass spectrometry to confirm the site of proteolysis. Facile and rapid separation of the Activation Peptide from the activated A-subunit protein has been achieved using simple on-line separation techniques. The molecular weight measurements have been repeated numerous times to provide an indication of the precision and variability of the analyses.

Alternative method of subtyping the A subunit of coagulation factor XIII (FXIIIA).

An alternative isoelectric focusing method for the A subunit of coagulation factor XIII (FXIIIA) subtyping is described which employs three different carrier ampholytes, pH 4-6.5, pH 5-6 and pH 5-8 (2:2:1), and sample application at the anode. With this technique ten common subtypes, including a variant, were clearly and reliably identified in Japanese and Chinese population samples.

Expression and release of the a and b subunits for human coagulation factor XIII in baby hamster kidney (BHK) cells.

The a subunit of coagulation factor XIII lacks a hydrophobic signal sequence for secretion from cells, while the b subunit has a typical signal sequence. To determine whether the a subunit can be synthesized and released, expression vectors containing the cDNA for either subunit were transfected into baby hamster kidney (BHK) cells. Western blotting analysis and gel filtration chromatography demonstrated that the recombinant a and b subunits (rXIIIa and rXIIIb) had the same molecular weights and subunit structures (a2, b2, and a2b2) as the native molecules. rXIIIa was enzymatically active when activated by thrombin. Most rXIIIb was secreted as measured by ELISA, while most rXIIIa was detected in the cytosol by subcellular fractionation. Co-expression with rXIIIb in the same cells did not promote the release of rXIIIa. Treatment of the cells with brefeldin A, a potent inhibitor of protein transportation, blocked the secretion of rXIIIb, although it had no effect on the release of rXIIIa. Several drugs and heat stress induced the release of rXIIIa, which correlated directly with that of cytoplasmic lactate dehydrogenase. These results suggest that the a subunit is released from cells as a consequence of cell injury, which is independent of the classical secretory pathway.