Subunit Of Coagulation Factor XIII Research Articles

High prevalence of thrombophilic risk factors in patients with central retinal artery occlusion

IntroductionCentral retinal artery occlusion (CRAO) is a common cause of blindness and visual morbidity. In the majority of cases, it is related to thrombotic embolism. Nevertheless, the role of inherited or acquired thrombophilic risk factors in CRAO pathogenesis has not been comprehensively studied.MethodsIn 126 CRAO patients (66 [52.4%] men, median age 55 [range: 18–80] years) and 107 matched controls (56 [52.3%] men, median age 53 [range: 34–78] years) we evaluated classical atherosclerotic risk factors, including serum lipid profile and glucose level, analyzed intima-media complex thickness (IMT) of external carotid arteries, and performed transthoracic echocardiography. Furthermore, we established the prevalence of inherited and acquired thrombophilic risk factors, such as factor V Leiden (FVL) and prothrombin 20210 G/A genetic variants, plasma activity of factor (F) VIII, protein C and antithrombin activity, and free protein S levels. We also assessed the presence of antiphospholipid antibodies (APLA) and evaluated blood homocysteine in all enrolled subjects. Additionally, we estimated the occurrence of Val34Leu polymorphism of the A subunit of coagulation factor XIII (FXIII-A) in both groups as a potential thrombosis-protecting factor.ResultsAmong traditional atherosclerotic risk components, obesity/overweight and hypercholesterolemia were the most common in the CRAO group and occurred in 103 (81.7%) and 85 (67.5%) patients, respectively. CRAO patients also had elevated IMT and altered echocardiographic parameters, indicating diastolic cardiac dysfunction. In thrombophilia investigations, at least one laboratory risk factor occurred in 72.2% (n = 91) of CRAO patients, with APLA as the most frequent, detected in 38.1% (n = 48) of them (almost seven times more frequent than in controls, p < 0.001). Deficiencies in protein C activity and free protein S levels were also common in the CRAO group, reported in 17.5% (n = 22) and 19.8% (n = 25) of patients, respectively. Interestingly, among two analyzed prothrombotic genetic variants, only the FVL was related to CRAO, with the allelic frequency 2.4 times more prevalent than in controls (p = 0.044). Finally, the CRAO group was characterized by hyperhomocysteinemia, almost twice as common as in controls (p = 0.026). Antithrombin deficiency, elevated FVIII, and FXIII-A Val34Leu polymorphism were not associated with CRAO.ConclusionsOur findings suggest that thrombophilia plays a vital role in the pathogenesis of CRAO. Thus, proper laboratory screening should be considered in the primary and secondary prevention of those episodes, with implementing appropriate therapy as needed.

Cellular FXIII in Human Macrophage-Derived Foam Cells.

Macrophages express the A subunit of coagulation factor XIII (FXIII-A), a transglutaminase which cross-links proteins through Nε-(γ-L-glutamyl)-L-lysyl iso-peptide bonds. Macrophages are major cellular constituents of the atherosclerotic plaque; they may stabilize the plaque by cross-linking structural proteins and they may become transformed into foam cells by accumulating oxidized LDL (oxLDL). The combination of oxLDL staining by Oil Red O and immunofluorescent staining for FXIII-A demonstrated that FXIII-A is retained during the transformation of cultured human macrophages into foam cells. ELISA and Western blotting techniques revealed that the transformation of macrophages into foam cells elevated the intracellular FXIII-A content. This phenomenon seems specific for macrophage-derived foam cells; the transformation of vascular smooth muscle cells into foam cells fails to induce a similar effect. FXIII-A containing macrophages are abundant in the atherosclerotic plaque and FXIII-A is also present in the extracellular compartment. The protein cross-linking activity of FXIII-A in the plaque was demonstrated using an antibody labeling the iso-peptide bonds. Cells showing combined staining for FXIII-A and oxLDL in tissue sections demonstrated that FXIII-A-containing macrophages within the atherosclerotic plaque are also transformed into foam cells. Such cells may contribute to the formation of lipid core and the plaque structurization.

Coagulation Factor XIII Subunit A Is a Biomarker for Curative Effects and Prognosis in Malignant Solid Tumors, Especially Non-small Cell Lung Cancer.

BackgroundThe expression of coagulant factor XIII subunit A (FXIII-A) is significantly increased in some types of cancer cells and tumor-associated macrophages (TAMs). However, few studies on plasma FXIII-A in cancer patients have been conducted and have shown contradictory results, so the relationship of plasma FXIII-A with the progression and prognosis of malignant tumors is still unknown. This study explored the association of plasma FXIII-A with a curative effect and the prognosis of patients with malignant solid tumors.MethodsWe monitored plasma FXIII-A before and during systemic therapy and assessed its relationship with the curative effect and prognosis of malignant solid tumors, especially non-small cell lung carcinoma (NSCLC), by propensity-adjusted, multivariable logistic regression analysis and survival curve, in a prospective study of 1147 patients with different types of malignant solid tumors. The influencing factors of plasma FXIII-A were also analyzed.ResultsWe found that D-dimer (D2) = 1 mg/L was the inflection point for the association between FXIII-A and D2: FXIII-A was significantly negatively correlated with D2 (r = -0.39, p < 0.01) and FDP (r = -0.40, p < 0.01) in D2 > 1 mg/L but uncorrelated with D2 or FDP in D2 ≤ 1 mg/L, which provided a method to find a more realistic plasma FXIII-A level. Plasma FXIII-A was positively correlated with age, platelets, lymphocytes, monocytes and carcinoembryonic antigen (CEA). It was found for the first time that plasma FXIII-A was abnormally significantly increased (FXIII-A > 150%) in post-therapy patients, especially in NSCLC and lung metastasis patients, and the incidence of FXIII-A > 150% in lung adenocarcinoma was 16 times higher than that in lung squamous carcinoma. FXIII-A > 150% proved to be an independent risk factor for disease progression in NSCLC patients (OR=5.74, 95% CI: 1.20-27.60, p = 0.029), predicting poor efficacy. The marked decrease in plasma FXIII-A (FXIII-A < 40%) was related to coagulation disorders and poor prognosis with a short survival time (median survival time of 4 months).ConclusionsPlasma FXIII-A has the potential to be a real-time biomarker with bidirectional indicator effects to assess curative effects and prognosis in malignant solid tumors, especially NSCLC.

Expression Patterns of Coagulation Factor XIII Subunit A on Leukemic Lymphoblasts Correlate with Clinical Outcome and Genetic Subtypes in Childhood B-cell Progenitor Acute Lymphoblastic Leukemia.

Background: Based on previous retrospective results, we investigated the association of coagulation FXIII subunit A (FXIII-A) expression pattern on survival and correlations with known prognostic factors of B-cell progenitor (BCP) childhood acute lymphoblastic leukemia (ALL) as a pilot study of the prospective multi-center BFM ALL-IC 2009 clinical trial. Methods: The study included four national centers (n = 408). Immunophenotyping by flow cytometry and cytogenetic analysis were performed by standard methods. Copy number alteration was studied in a subset of patients (n = 59). Survival rates were estimated by Kaplan-Meier analysis. Correlations between FXIII-A expression patterns and risk factors were investigated with Cox and logistic regression models. Results: Three different patterns of FXIII-A expression were observed: negative (<20%), dim (20–79%), and bright (≥80%). The FXIII-A dim expression group had significantly higher 5-year event-free survival (EFS) (93%) than the FXIII-A negative (70%) and FXIII-A bright (61%) groups. Distribution of intermediate genetic risk categories and the “B-other” genetic subgroup differed significantly between the FXIII-A positive and negative groups. Multivariate logistic regression confirmed independent association between the FXIII-A negative expression characteristics and the prevalence of intermediate genetic risk group. Conclusions: FXIII-A negativity is associated with dismal survival in children with BCP-ALL and is an indicator for the presence of unfavorable genetic alterations.

N‐glycosylation of blood coagulation factor XIII subunit B and its functional consequence

BackgroundThe protective/inhibitory B subunits of coagulation factor XIII (FXIII‐B) is a ~80 kDa glycoprotein containing two N‐glycosylation sites. Neither the structure nor the functional role of the glycans on FXIII‐B has been explored. ObjectiveTo reveal the glycan structures linked to FXIII‐B, to design a method for deglycosylating the native protein, to find out if deglycosylation influences the dimeric structure of FXIII‐B and its clearance from the circulation. MethodsAsparagine‐linked carbohydrates were released from human FXIIII‐B by PNGase F digestion. The released N‐linked oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis. Structural identification utilized glycan database search and exoglycosidase digestion based sequencing. The structure of deglycosylated FXIII‐B was investigated by gel filtration. The clearance of deglycosylated and native FXIII‐B from plasma was compared in FXIII‐B knock out mice. ResultsPNGase F completely removed N‐glycans from the denatured protein. Deglycosylation of the native protein was achieved by repeated digestion at elevated PNGase F concentration. The total N‐glycan profile of FXIII‐B featured nine individual structures; three were fucosylated and each structure contained at least one sialic acid. Deglycosylation did not change the native dimeric structure of FXIII‐B, but accelerated its clearance from the circulation of FXIII‐B knock out mice. ConclusionCharacterization of the glycan moieties attached to FXIII‐B is reported for the first time. Complete deglycosylation of the native protein was achieved by a deglycosylation workflow. The associated glycan structure is not required for FXIII‐B dimer formation, but it very likely prolongs the half‐life of FXIII‐B in the plasma.

Coagulation FXIII-a Protein Expression Defines Three Novel Sub-Populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized By Distinct Gene Expression Signatures

Background Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). FXIII-A expression determined by flow cytometry (FC) exhibited characteristically distinct expression patterns in subgroups of lymphoblasts. The FXIII-A negative subgroup was significantly associated with the 'B-other' genetic category and had an unfavorable disease outcome. Methods RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology (GO) analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed (DE) genes were validated by RT-Q-PCR. Results We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the expression of FXIII-A protein, as determined by FC. We observed three well-defined categories of FXIII-A protein expression: FXIII-A negative (&lt;20% FXIII-A positive blasts), FXIII-A dim (20-80% FXIII-A positive blasts), and FXIII-A bright (&gt;80% FXIII-A positive blasts). The intensity of the FXIII-A expression increased continuously, which excluded the existence of a distinct FXIII-A negative and a FXIII-A bright subpopulation within the FXIII-A dim subgroup (Image 1/A). These three subgroups defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. There were 26 DE genes found when comparing the FXIII-A negative with the FXIII-A bright subgroup. The FXIII-A dim vs. bright comparison resulted in 155 DE genes and there were 88 DE genes identified between the FXIII-A negative and dim subgroups (Image 1/B). Expression of F13A1 gene was detected and readily validated by RT-Q-PCR in every sample. Intensity of gene expression; however, was characteristically different among samples of the three different FXIII-A protein expression subgroups with an increasing intensity in terms of relative fold-changes measured by RT-Q-PCR from the FXIII-A negative, through dim to bright subgroups. We selected 13/45 genes based on fold-change results detected by microarray, whereas an additional 32/45 genes were selected according to enriched functional categories of potential interest as defined by the GO analysis. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2, validated by RT-Q-PCR according to the FXIII-A status, followed the pattern of intensity of the expression of the F13A1 gene. Of these genes, ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 appear to have a role in leukemia and other forms of cancer. NUP43 has not yet been shown to be associated with any forms of human cancer in contrast to other members of the NUP gene family. PLAC8 which is a trophoblast lineage marker was most intensively expressed in the FXIII-A dim subgroup. This gene has been shown to be aberrantly activated in various types of cancer arising in mammals and mammalian cancer cell lines, but not in any subtype of human ALL (Image 1/C). Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of 'B-other' samples. We identified 14 DE genes by microarray overlapping with DE genes of the 'B-other' subgroup defined by the COALL Group. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the 'B-other' subgroup. Conclusions We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL. Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Validated genes proved biologically and clinically relevant. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment. Figure Disclosures No relevant conflicts of interest to declare.

Coagulation FXIII-A Protein Expression Defines Three Novel Sub-populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized by Distinct Gene Expression Signatures.

Background: Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). Flow cytometry (FC) revealed three distinct expression patterns, i.e., FXIII-A negative, FXIII-A dim, and FXIII-A bright subgroups. The FXIII-A negative subgroup was significantly associated with the “B-other” genetic category and had an unfavorable disease outcome.Methods: RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28,869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed genes were validated by RT-Q-PCR.Results: We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the FXIII-A protein expression subgroups which defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 followed the pattern of change in the intensity of the expression of the F13A1 gene. Common enhancer elements of these genes revealed by in silico analysis suggest that common transcription factors may regulate the expression of these genes in a similar fashion. PLAC8 was downregulated in the FXIII-A bright subgroup. Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of “B-other” samples. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the “B-other” subgroup. Validated genes proved biologically and clinically relevant. We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL.Conclusions: Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment.

First Step of the Transglutaminase Reaction Catalyzed by Activated Factor XIII Subunit A, Hybrid Quantum Chemistry/Molecular Mechanics Calculations.

The A subunit of blood coagulation factor XIII belongs to the family of transglutaminase enzymes. Its active form (FXIIIa) catalyzes isopeptide bond formation between Glu and Lys residues of specific substrates. Little data are available on the mechanism of this reaction. In this work, the first step of the proposed two-step process was investigated using two different protocols of hybrid QM/molecular mechanics (MM) calculations: an ONIOM-based model as well as QM/MM/molecular dynamics (MD) metadynamics simulations in explicit TIP3P solvent with Gromacs, PLUMED, and a DFTB3 package. Based on calculations involving a truncated system derived from docking of a peptide substrate, our study confirms the higher stability of a zwitterionic form of the catalytic Cys and His residues in the Michaelis complex as well as the "resting" state of the enzyme. Potential energy surfaces, obtained by geometry optimizations with Gaussian, show a two-step reaction mechanism with a zwitterionic tetrahedral intermediate formation in the first and NH3 dissociation in the second step in the case of our ONIOM system. In contrast, in QM/MM MD metadynamics simulations, all three steps occurred in a concerted manner. As a conclusion, our model is able to provide insights into the reaction mechanism of this enzyme.

Factor XIII Subunit A Immunohistochemical Expression is Associated With Inferior Outcomes in Acute Promyelocytic Leukemia.

Coagulation factor XIII subunit A (FXIIIa) intracellular expression has been described in platelets, megakaryocytes, monocytic cells, and leukemic blasts. Flow cytometric-based studies have suggested prognostic implications of FXIIIa expression, especially within the acute promyelocytic leukemia (APL) subgroup of acute myeloid leukemia (AML); however, its prognostic correlate by immunohistochemistry (IHC) is unknown. The aims of this study were to (1) define the clinicopathologic features of FXIIIa IHC-positive AML and (2) compare APL with other AML subtypes. Eighty-seven bone marrow biopsies or clot/particle preparations from our institution were evaluated with FXIIIa IHC. The study cohort consisted of bone marrow evaluations of 36 consecutive pretherapy APL, 42 selected pretherapy non-APL AML, and 9 negative staging cases. FXIIIa IHC expression was correlated with clinical and pathologic features and overall survival (OS). Leukemic blast FXIIIa cytoplasmic positivity was noted in 56% (20/36) APL and 74% (31/42) non-APL AML (P=0.10). FXIIIa IHC expression was associated with inferior OS within the APL cohort (P=0.04). No OS differences were noted in comparing FXIIIa IHC expression in all AML (P=0.17), or FXIIIa IHC expression within favorable, intermediate or adverse cytogenetic groups (P=0.14, 0.22 and 0.87, respectively). FXIIIa IHC expression is observed among a broad spectrum of AML subtypes and is not characterized by specific pathologic features. However, within the APL subgroup, FXIIIa IHC expression is associated with an inferior outcome and may be useful for additional prognostic risk stratification.

Expression of Coagulation Factor XIII Subunit A Correlates with Outcome in Childhood Acute Lymphoblastic Leukemia

Previously we identified B-cell lineage leukemic lymphoblasts as a new expression site for subunit A of blood coagulation factor XIII (FXIII-A). On the basis of FXIII-A expression, various subgroups of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be identified. Fifty-five children with BCP-ALL were included in the study. Bone marrow samples were obtained by aspiration and the presence of FXIII-A was detected by flow cytometry. G-banding and fluorescent in situ hybridization was performed according to standard procedures. The 10-year event-free survival (EFS) and overall survival (OS) rate of FXIII-A-positive and FXIII-A-negative patients showed significant differences (EFS: 84% vs. 61%, respectively; p=0.031; OS: 89% vs. 61%; p=0.008). Of all the parameters examined, there was correlation only between FXIII-A expression and 'B-other' genetic subgroup. Further multivariate Cox regression analysis of FXIII-subtype and genetic group or 'B-other' subgroup identified the FXIII-A negative characteristic as an independent factor associated with poor outcome in BCP-ALL. We found an excellent correlation between long-term survival and the FXIII-A-positive phenotype of BCP lymphoblasts at presentation. The results presented seem to be convincing enough to suggest a possible role for FXIII-A expression in the prognostic grouping of childhood BCP-ALL patients.

Expression of Blood Coagulation Factor Xiiia in Lymphoblasts Predicts Favorable Outcome in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction: Previously we identified B-cell lineage leukemic lymphoblasts as a new expression site for subunit A of blood coagulation factor XIII (FXIIIA)1. On the basis of FXIIIA expression, various subgroups of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be identified. Methods. Fifty-five children with BCP-ALL treated within the frame of BFM ALL-IC 2002 clinical trial were studied retrospectively. Bone marrow samples were obtained by aspiration and the expression of FXIIIA was detected by flow cytometry. G-banding and fluorescent in situ hybridization was performed according to standard procedures. Results. The 10-year event-free survival (EFS) and overall survival (OS) rate of FXIIIA-positive and FXIIIA-negative patients showed significant differences (EFS: 84% vs. 61%, respectively; p=0.031; OS: 89% vs. 61%; p=0.008). Of all the parameters examined, the only correlation was between the lack of FXIIIA expression and ’B-other’ genetic subgroup. Further multivariate Cox regression analysis of FXIII-subtype and genetic group or ’B-other’ subgroup identified FXIIIA-negative characteristics as an independent predictor for poor outcome in BCP-ALL. Conclusion. We found an excellent correlation between long-term survival and FXIIIA-positive phenotype of lymphoblasts in de novo childhood BCP-ALL. The results presented seem to be convincing enough to suggest a possible role for FXIIIA expression in the prognostic grouping of childhood BCP-ALL patients. In addition, lack of FXIIIA expression is associated with the ’B-other’ characteristics, therefore, FXIIIA can help to identify those cases that may require further detailed genetic examination by using expensive methods. Acknowledgment. The authors thank Dr. Erzsebet Balogh for performing the cytogenetic analyses and Csaba Antal for his administrative help. The authors are grateful to Dr. Kalman Nagy and their coworkers at the Borsod-Abaúj-Zemplén County Hospital and University Hospital for sending bone marrow samples for flow cytometry. This study was supported by grant OTKA K-108885 (CK). Authors declare no conflict of interest. References. 1. Kiss F, Hevessy Z, Veszpremi A, Katona E, Kiss C, Vereb G, Muszbek L, Kappelmayer JN. Leukemic lymphoblasts, a novel expression site of coagulation factor XIII subunit A. Thromb Haemost. 2006; 96: 176-82. Legend to figures. Figure 1. Prognostic value of FXIII-A expression of lymphoblasts in children with B-cell precursor ALLKaplan Meier plots of event-free (A) and overall survival (B) showed significant difference between the FXIIIA-positive and FXIII-A-negative groups (p=0.031 and p=0.008). Figure 2. Relationship between FXIIIA expression profile and genetic classification The distribution of patients in the various genetic groups differed significantly in terms of FXIII-A profile using Chi square test. Recurrent genetic abnormalities: BCR-ABL1, KMT2A (MLL) gene rearrangements, ETV6-RUNX1 (TEL-AML1), E2A/PBX1 and high hyperdiploidy. DisclosuresNo relevant conflicts of interest to declare.

Coagulation Factor XIIIA Subunit Missense Mutations Affect Structure and Function at the Various Steps of Factor XIII Action

Inherited defects of coagulation Factor XIII (FXIII) can be categorized into severe and mild forms based on their genotype and phenotype. Heterozygous mutations occurring in F13A1 and F13B genes causing mild FXIII deficiency have been reported only in the last few years primarily because the mild FXIII deficiency patients are often asymptomatic unless exposed to some kind of a physical trauma. However, unlike mutations causing severe FXIII deficiency, many of these mutations have not been comprehensively characterized based on expression studies. In our current article, we have transiently expressed 16 previously reported missense mutations detected in the F13A1 gene of patients with mild FXIII deficiency and analyzed their respective expression phenotype. Complimentary to expression analysis, we have used in silico analysis to understand and explain some of the in vitro findings. The expression phenotype has been evaluated with a number of expression phenotype determining assays. We observe that the mutations influence different aspects of FXIII function and can be functionally categorized on the basis of their expression phenotype. We identified mutations which even in heterozygous form would have strong impact on the functional status of the protein (namely mutations p.Arg716Gly, p.Arg704Gln, p.Gln602Lys, p.Leu530Pro, p.His343Tyr, p.Pro290Arg, and p.Arg172Gln).

Blood coagulation factor XIII-A subunit Val34Leu polymorphisms and intracerebral hemorrhage risk: A meta-analysis of case-control studies

Background. Previous studies investigating the association between factor XIII-A subunit (FXIII-A) Val34Leu polymorphisms and intracerebral hemorrhage (ICH) had provided inconsistent results and no large systematic review or meta-analysis had been conducted regarding this issue. Methods. We conducted a meta-analysis to confirm whether the FXIII-A Val34Leu polymorphisms increased the risk of ICH. Relevant studies were identified from the Pubmed, Medline, Embase, China National Knowledge Infrastructure, and Chinese Biomedicine Databases published up to September 2013. Summary odds ratios (ORs) and 95% confidence intervals (CIs) for FXIII-A Val34Leu polymorphisms and ICH were calculated in a fixed-effects model or a random-effects model when appropriate. We also carried out the stratified analyses and sensitivity analyses by region, source of control group, and sample size. Results. Eight eligible studies were reviewed. As FXIII Val34Leu was absent or had a very low prevalence among East Asians, only six studies in Caucasians were analyzed, involving 564 cases and 1276 controls. Overall, the Leu allele of FXIII gene had a trend to slightly increased odds of having ICH, but there is no statistic significance (OR1.23, 95% CI 0.94–1.61, P = 0.13). The OR of genotypes Leu+(Leu/Leu or Leu/Val) for the risk of ICH was 1.21, 95% CI 0.98–1.50, P = 0.08. And the OR of recessive model genotypes was 1.53, 95% CI 0.81–2.88, P = 0.19. There was no difference of the association between the Leu allele of FXIII gene and risk of ICH in stratified analysis. Conclusions. Our meta-analysis suggests that there is no evidence for strong association between FXIII Val34Leu polymorphisms and ICH, but Leu allele of FXIII gene might slightly increase the risk of ICH in Caucasians. Since limited studies and subjects were included, larger scale association studies exploring the gene–gene interactions and gene–environment interactions are necessary to further validate the association.

Structural and functional influences of coagulation factor XIII subunit B heterozygous missense mutants.

The coagulation factor XIII(FXIII) is a plasma circulating heterotetrameric protransglutaminase that acts at the end of the coagulation cascade by covalently cross-linking preformed fibrin clots (to themselves and to fibrinolytic inhibitors) in order to stabilize them against fibrinolysis. It circulates in the plasma as a heterotetramer composed of two homomeric catalytic Factor XIIIA2 (FXIIIA2) and two homomeric protective/carrier Factor XIIIB2 subunit (FXIIIB2). Congenital deficiency of FXIII is of two types: severe homozygous/compound heterozygous FXIII deficiency which results in severe bleeding symptoms and mild heterozygous FXIII deficiency which is associated with mild bleeding (only upon trauma) or an asymptomatic phenotype. Defects in the F13B gene (Factor XIIIB subunit) occur more frequently in mild FXIII deficiency patients than in severe FXIII deficiency. We had recently reported secretion-related defects for seven previously reported F13B missense mutations. In the present study we further analyze the underlying molecular pathological mechanisms as well as the heterozygous expression phenotype for these mutations using a combination of in vitro heterologous expression (in HEK293T cells) and confocal microscopy. In combination with the in vitro work we have also performed an in silico solvated molecular dynamic simulation study on previously reported FXIIIB subunit sushi domain homology models in order to predict the putative structure-functional impact of these mutations. We were able to categorize the mutations into the following functional groups that: (1) affect antigenic stability as well as binding to FXIIIA subunit, that is, Cys5Arg, Cys316Phe, and Pro428Ser (2) affect binding to FXIIIA subunit with little or no influence on antigenic stability, that is, Ile81Asn and Val401Gln c) influence neither aspects and are most likely causality linked polymorphisms or functional polymorphisms, that is, Leu116Phe and Val217Ile. The Cys5Arg mutation was the only mutation to show a direct secretion-based defect since the mutated protein was observed to accumulate in the endoplasmic reticulum.

Subunit a of Coagulation Factor XIII As a New Biomarker in Childhood Acute Lymphoblastic Leukemia?

Background: Using multiparameter flow cytometry (FC), Western blot, ELISA and laser scanning microscopy, leukemic B-cell progenitor (BCP) lymphoblasts were identified as a novel expression site of coagulation factor XIII subunit A (FXIIIA; Kiss F. et al. Thomb Hemost, 2006.).Objectives: The significance of FXIIIA expression, defined by FC, on the clinical outcome of children with BCP acute lymphoblastic leukemia (ALL) was studied. We used gene expression profile (GEP) analysis and pathway analysis to identify genes which may contribute to the phenotype of the F13a1 low expression patient subgroup. These genes will be tested for the presence of mutations by next generation sequencing in the next phase of the study.Method: ALL immunophenotyping, including FXIIIA expression and detection of minimal residual disease (MRD) in Day 15 bone marrow was determined by four color FC analysis. Clinical course of FXIIIA-positive and –negative children with ALL was retrospectively studied and 3-yr overall survival (OS) data were calculated and compared by Kaplan-Mayer plot analysis. We analyzed 3 public datasets in the GEO Database (GSE47051, GSE13351 and GSE13425), including the dataset of the University of Padova (GSE47051) containing data on GEPs of children with ALL using GeneSpring and Ingenuity Pathway Analysis softwares.Results: Sixty-three per cent of BCP ALL cases, investigated by FC at the Department of Laboratory Medicine, University of Debrecen expressed FXIIIA. Three-year overall survival (OS) of children with FXIIIA-positive ALL was significantly higher (87%) than 3-yr OS of patients with FXIIIA-negative ALL (65%). As expected, Day 15 FC-MRD successfully separated patients into three well-defined groups with different OS rates: 92%, 72% and 43% in FC-MRD SR, IR and HR groups, respectively. FXIIIA expression has merged FC-MRD SR and IR groups with a significant difference between FXIIIA-positive vs. -negative patients.In the GeneSpring analysis, patients were separated into two groups: F13a1 low expression, and F13a1 high expression group, with at least 2-fold difference in their F13a1 expression level. F13a1 low and high expression groups exhibited a characteristically different GEP (at least 2-fold difference, p≤0.05, t-test, Benjamini-Hochberg correction for FDR). Low F13a1 expression level was prevalent among the genetic subgroup of “B-other” samples, high F13a1 expression level was associated with the t(1;19) genetic subgroup of childhood ALL. We found 8 genes that were significantly down-regulated, and one, that was upregulated in the F13a1 low expression group in all three datasets (Table 1), and 28 similarly deregulated genes in at least two of the three datasets. We identified two chromosomal loci, 19p13.3 and 16q22, with 11 and 2 deregulated genes, respectively, within the F13a1 low expression group (Table 1). Using Ingenuity Pathway Analysis, we identified a network of genes participating in B cell development that were deregulated in the F13a1 low expression group. Using the Ingenuity upstream regulator analysis, deregulation of NUPR1, TCF3 and IKZF1 were predicted in the F13a1 low expression group.Conclusion: FXIIIA expression by FC may define a new subgroup of childhood ALL, with a partial overlap in the GEPs of the F13a1 low expression group and the recently defined BCR-ABL1-like group (Table 1). FXIIIA expression by FC may help in selecting those cases which require a more sophisticated – and more expensive - genetic diagnosis to define the optimal risk-tailored therapy. In addition, FXIIIA may become a useful marker for Day 15 MRD detection.Grant sponsor TÁMOP 4.2.2.A-11/1/KONV-2012-0025 - the project is co-financed by the European Union and the European Social Fund; The AIRC (Associazione Italiana Ricerca su Cancro) project; OTKA K108885. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Catridecacog: First drug to treat rare genetic blood clotting disorder

Catridecacog, or coagulation factor XIII A-subunit, recombinant (Tretten—Novo Nordisk), a minimally invasive option for treating rare congenital factor XIII A-subunit deficiency, has received FDA approval. According to Novo Nordisk, catridecacog helps to prevent bleeding in children and adults who have the coagulation factor XIII A-subunit deficiency, an extremely rare genetic disorder. Catridecacog is the first drug to receive FDA approval for this indication. FDA also granted catridecacog orphan drug status because it treats a rare disease. Factor XIII is a terminal enzyme in the blood coagulation cascade that stimulates cross-linking of fibrin and other proteins in the fibrin clot to promote hemostasis. In human plasma, factor XIII circulates as a heterotetramer of two functional subunit proteins—two factor XIII A-subunits and two factor XIII B-subunits. The factor XIII A-subunit acts as the enzyme, while factor XIII B-subunit acts as a carrier for the factor XIII A-subunit. During coagulation, the factor XIII is activated by thrombin and calcium; the factor XIII A-subunit dissociates from the factor XIII B-subunit to become active and help cross-link fibrin to enhance platelet and clot adhesion to injured tissue. Patients with the factor XIII A-subunit deficiency have a significantly lower level of the active subunit, thus resulting in less stable clotting for injured tissue. Catridecacog binds to the free human factor XIII B-subunit, as factor XIII A-subunit, to strengthen fibrin clots, impede fibinolysis, and enhance platelet adhesion to the injury site. Give patients the Médication Guide and review it with them carefully. Verify the diagnosis, since catridecacog should not be used for prophylactic treatment of bleeding in patients with congenital factor XIII B-subunit deficiency. Advise patients that breakthrough bleeding may be a sign and symptom of inhibitor formation, and they may have to see their health care provider to measure antibody concentrations. Tell patients to inform their health care provider of all the medications they may be taking. Patients with the factor XIII A-subunit deficiency have a significantly lower level of the active subunit, thus resulting in less stable clotting for injured tissue. During clinical development, researchers studied the effectiveness of catridecacog in 77 patients with coagulation factor XIII A-subunit deficiency. Catridecacog was effective in preventing bleeding in 90% of the patients when given the recommended dose of 35 IU/ kg. The mean age of study participants was approximately 26 years (63.4% > 18 years), and the male to female ratio was 3:2. The most common related adverse events (occurring in >1% of patients treated with catridecacog) were headache, pain in the extremities, injection site pain, and an increase in nonneutralizing antibodies. Use of catridecacog is contraindicated in patients who have known hypersensitivity to the active substance or to any of the excipients. Because increased levels of rFXIII may increase the risk of thrombosis, health professionals should exercise caution in patients with a predisposition to conditions of thrombosis. Incorrect storage of the product after reconstitution may result in loss of sterility and increased levels of activated rFXIII. Catridecacog, coagulation factor XIII A-subunit, recombinant (Tretten)Manufacturer: Novo NordiskDrug class: Blood coagulation factorsIndication: Treatment of congenital factor XIII A-subunit deficiencyDosage: 35 IU/kg once monthly■To administer, reconstitute with sterile water for injection with a 10-mL syringe.■A dose recommendation cannot be made for older adults, pregnant patients, or children younger than age 6 years due to lack of data.Of note: Treatment should be initiated under the supervision of a physician experienced in the treatment of rare bleeding disorders. Do not use vial if solid matter and discoloration are present in the reconstituted product. Administer at a rate not exceeding 1–2 mL per minute. Do not administer as a drip or with other infusion solutions. Manufacturer: Novo Nordisk Drug class: Blood coagulation factors Indication: Treatment of congenital factor XIII A-subunit deficiency Dosage: 35 IU/kg once monthly■To administer, reconstitute with sterile water for injection with a 10-mL syringe.■A dose recommendation cannot be made for older adults, pregnant patients, or children younger than age 6 years due to lack of data. Of note: Treatment should be initiated under the supervision of a physician experienced in the treatment of rare bleeding disorders. Do not use vial if solid matter and discoloration are present in the reconstituted product. Administer at a rate not exceeding 1–2 mL per minute. Do not administer as a drip or with other infusion solutions.

Expression Level Of A Coagulation Factor Gene, F13A1 Defines Characteristic Subpopulations Of Childhood Acute Lymphoblastic Leukemia

Using multiparameter flow cytometry, Western blot, ELISA and laser scanning microscopy, leukemic B-cell progenitor lymphoblasts were identified as a novel expression site of coagulation factor XIII subunit A (FXIIIA) (Kiss et al Thromb Haemost 2006). Three-year overall survival (OS) of children with FXIIIA-positive acute lymphoblastic leukemia (ALL) was significantly higher (87%) than 3-yr OS of patients with FXIIIA-negative ALL (65%). Here we report on gene expression profiles (GEP) associated with FXIIIA-positive vs. FXIIIA–negative childhood (ch)ALL. Finnally, the expression levels of F13A1 gene in cytogenetic subgroups were investigated.GEP of 11 FXIIIA-positive and 9 FXIIIA-negative samples of patients treated at the Department of Hematology-Oncology, University of Debrecen was investigated using HG-U 133 plus 2.0 Affymetrix Platform Array. Expression sequence tags (EST) characterized by expression levels with at least 2 logs difference among FXIIIA-positive vs. FXIIIA-negative samples were selected and validated with real-time quantitative PCR. Extending the analysis, we have searched the database containing GEPs of 317 children with ALL treated at the Division of Hemato-Oncology, Department of Women's and Children's Health, University of Padova, using R package and Partek Genomic Suite softwares.FXIIIA-positive and FXIIIA-negative samples exhibited a characteristically different GEP. In the FXIIIA-positive samples one of the overexpressed genes was F13A1. FXIIIA-negative samples contained two characteristic groups of overexpressed genes. One of these consisted of genes participating in B-cell development: EBF1, IKZF1 and PAX5. The second set consisted of genes encoding for tyrosine kinases: JAK1, JAK3, NC07523, NC08002, NC06523, NC08345 and for serine-threonine kinase NC00027. In contrast, FXIIIA-positive samples contained only two overexpressed tyrosine kinases: C-KIT and JAK2.A wide variation of F13A1 expression levels of the 317 Padova patients was observed. Since expression level of FXIIIA protein of these patients has not been determined, we have arbitrarily defined F13A1 gene expression levels below 106 as “low” and gene expression levels exceeding 109 as “high”. Considering these two groups, we have investigated the distribution of F13A1 expression among the known cytogenetic subgroups of ALL. Low F13A1 expression was prevalent among “B-other” samples, high F13A1 expression was associated with t(1;19).The pattern of overexpressed ESTs, accumulation of low F13A1 expressing samples in the “B-other” cytogenetic group of ALL and the unfavorable disease outcome of FXIIIA-negative cases may suggest a possible overlap between FXIIIA-negative ALL and BCR-ABL1-like ALL identified recently in the “B-other” group. The nature and extent of this overlap will be investigated prospectively in the BFM ALL-IC 2009 clinical trial. In contrast, high expression levels of F13A1 accumulated preferentially within the t(1;19) genetic group, associated with a good prognosis. Detection of FXIIIA expression by flow cytometry may offer an easy and non-expensive tool for defining new prognostic subpopulations of ALL. Grant supportTÁMOP 4.2.2.A-11/1/KONV-2012-0025 project (CK, KG, HZ, IG, IS and JK). The project is co-financed by the European Union and the European Social Fund and the AIRC (Associazione Italiana Ricerca su Cancro; SB) project. Disclosures:No relevant conflicts of interest to declare.

Expression of coagulation factor XIII subunit A in acute promyelocytic leukemia

Leukemic cells often express markers, which are not characteristic of their particular cell lineage. In this study, we identified the "A" subunit of coagulation factor XIII (FXIII-A) in leukemic promyelocytes in de novo AML M3 cases. The cytoplasmic presence of factor XIII-A has previously been shown only in platelets/megakaryocytes and monocytes/macrophages. Furthermore, more recently we described the presence of FXIII-A in leukemic lymphoblasts. We studied 14 patients with this rare type of acute leukemia in a period of 4 years and investigated their bone marrow samples by 3-color flow cytometry upon diagnosis, mainly focusing on FXIII-A expression of leukemic cells. We detected FXIII-A also by ELISA, Western-blot, and confocal laser scanning microscopy. This was a homogenous group of AML M3 patients with translocation t(15;17)(q22;q21) detected by fluorescence in situ hybridization (FISH). In 10 out of 14 samples, FXIII-A was detectable by flow cytometry and was coexpressed with markers characteristic for leukemic promyleocytes (CD45dim/CD13+/CD33+/CD117+/cyMPO+ and HLA-DR-/CD34-/CD14-/CD15-). Staining for the markers GPIIb and GPIX were negative, and FXIII-A was identified in the cytoplasm of the cells by confocal microscopy in a relatively high quantity, as measured by ELISA. By Western blot analysis we could identify FXIII-A in the native 82 kDa form and in cleaved forms corresponding to cleavage products observed when purified FXIII-A was treated by human neutrophil elastase. This novel expression site of FXIII-A in AML M3 can be considered as a leukemia associated immunophenotype and may have pathophysiological significance.

Exploring the interaction between SNP genotype and postmenopausal hormone therapy effects on stroke risk.

BackgroundGenome-wide association studies have identified several genomic regions that are associated with stroke risk, but these provide an explanation for only a small fraction of familial stroke aggregation. Genotype by environment interactions may contribute further to such an explanation. The Women's Health Initiative (WHI) clinical trial found increased stroke risk with postmenopausal hormone therapy (HT) and provides an efficient setting for evaluating genotype-HT interaction on stroke risk.MethodsWe examined HT by genotype interactions for 392 SNPs selected from candidate gene studies, and 2,371 SNPs associated with changes in blood protein concentrations after hormone therapy, in analyses that included 2,045 postmenopausal women who developed stroke during WHI clinical trial and observational study follow-up and one-to-one matched controls. A two-stage procedure was implemented where SNPs passing the first stage screening based on marginal association with stroke risk were tested in the second stage for interaction with HT using case-only analysis.ResultsThe two-stage procedure identified two SNPs, rs2154299 and rs12194855, in the coagulation factor XIII subunit A (F13A1) region and two SNPs, rs630431 and rs560892, in the proprotein convertase subtilisin kexin 9 (PCSK9) region, with an estimated false discovery rate <0.05 based on interaction tests. Further analyses showed significant stroke risk interaction between these F13A1 SNPs and estrogen plus progestin (E+P) treatment for ischemic stroke and for ischemic and hemorrhagic stroke combined, and suggested interactions between PCSK9 SNPs with either E+P or estrogen-alone treatment.ConclusionsGenotype by environment interaction information may help to define genomic regions relevant to stroke risk. Two-stage analysis among postmenopausal women generates novel hypotheses concerning the F13A1 and PCSK9 genomic regions and the effects of hormonal exposures on postmenopausal stroke risk for subsequent independent validation.

Prothrombotic Gene Variants and Mortality after Cerebral Ischemia of Arterial Origin

Background: Several functional prothrombotic gene variants have been associated with cerebral ischemia and myocardial infarction. We hypothesized that such gene variants may also be associated with mortality after cerebral ischemia of arterial origin because of an increased risk of fatal vascular events. Methods: We performed a case-control study in 316 long-term survivors and 887 patients with recent cerebral ischemia of arterial origin. False discovery rate q values were calculated to account for multiple testing. The mean duration between occurrence of cerebral ischemia and DNA collection was 16.8 years in long-term survivors and 3.2 months in recent patients. Results: Two of 23 variants were associated with mortality: the 95Arg allele of the coagulation factor XIII subunit B (F13B) His95Arg variant (OR, 1.5 for Arg/Arg and His/Arg vs. His/His genotype; 95% CI, 1.1–2.2, q = 0.29) and the 4G allele of the plasminogen activator inhibitor-1 (PAI-1) 4G/5G variant (OR, 1.5 for 4G/4G and 5G/4G vs. 5G/5G genotype; 95% CI, 1.1–2.0, q = 0.29). Both associations disappeared after accounting for multiple testing. Data analysis restricted to recently deceased patients (n = 133) yielded similar results. Conclusions: In this hospital-based study none of 23 prothrombotic gene variants were associated with long-term mortality after cerebral ischemia of arterial origin. Prothrombotic gene variants do not appear to play an important role in long-term mortality after cerebral ischemia.