Heterozygosity in factor XIII genes and the manifestation of mild inherited factor XIII deficiency.

BACKGROUND: The characterization of inherited mild factor XIII deficiency is more imprecise than its rare, inherited severe forms. It is known that heterozygosity at FXIII genetic loci results in mild FXIII deficiency, characterized by circulating FXIII activity levels ranging from 20% to 60%. There exists a gap in information on 1) how genetic heterozygosity renders clinical bleeding manifestations among these individuals and 2) the reversal of unexplained bleeding upon FXIII administration in mild FXIII-deficient individuals. OBJECTIVES: To assess the prevalence and burden of mild FXIII deficiency among the apparently healthy German-Caucasian population and correlate it with genetic heterozygosity at FXIII and fibrinogen gene loci. METHODS: Peripheral blood was collected from 752 donors selected from the general population with essentially no bleeding complications to ensure asymptomatic predisposition. These were assessed for FXIII and fibrinogen activity, and FXIII and fibrinogen genes were resequenced using next-generation sequencing. For comparison, a retrospective analysis was performed on a cohort of mild inherited FXIII deficiency patients referred to us. RESULTS: The prevalence of mild FXIII deficiency was high (∼0.8%) among the screened German-Caucasian population compared with its rare-severe forms. Although no new heterozygous missense variants were found, certain combinations were relatively dominant/prevalent among the mild FXIII-deficient individuals. CONCLUSION: This extensive, population-based quasi-experimental approach revealed that the burden of heterozygosity in FXIII and fibrinogen gene loci causes the clinical manifestation of inherited mild FXIII deficiency, resulting in ''unexplained bleeding'' upon provocation.

Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products.

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

Structure functional insights into calcium binding during the activation of coagulation factor XIII A.

The dimeric FXIII-A2, a pro-transglutaminase is the catalytic part of the heterotetrameric coagulation FXIII-A2B2 complex that upon activation by calcium binding/thrombin cleavage covalently cross-links preformed fibrin clots protecting them from premature fibrinolysis. Our study characterizes the recently disclosed three calcium binding sites of FXIII-A concerning evolution, mutual crosstalk, thermodynamic activation profile, substrate binding, and interaction with other similarly charged ions. We demonstrate unique structural aspects within FXIII-A calcium binding sites that give rise to functional differences making FXIII unique from other transglutaminases. The first calcium binding site showed an antagonistic relationship towards the other two. The thermodynamic profile of calcium/thrombin-induced FXIII-A activation explains the role of bulk solvent in transitioning its zymogenic dimeric form to an activated monomeric form. We also explain the indirect effect of solvent ion concentration on FXIII-A activation. Our study suggests FXIII-A calcium binding sites could be putative pharmacologically targetable regions.

Identification of Potential Novel Interacting Partners for Coagulation Factor XIII B (FXIII-B) Subunit, a Protein Associated with a Rare Bleeding Disorder.

Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.

Disruption of Structural Disulfides of Coagulation FXIII-B Subunit; Functional Implications for a Rare Bleeding Disorder.

Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine's, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds.

Revisiting the mechanism of coagulation factor XIII activation and regulation from a structure/functional perspective.

The activation and regulation of coagulation Factor XIII (FXIII) protein has been the subject of active research for the past three decades. Although discrete evidence exists on various aspects of FXIII activation and regulation a combinatorial structure/functional view in this regard is lacking. In this study, we present results of a structure/function study of the functional chain of events for FXIII. Our study shows how subtle chronological submolecular changes within calcium binding sites can bring about the detailed transformation of the zymogenic FXIII to its activated form especially in the context of FXIIIA and FXIIIB subunit interactions. We demonstrate what aspects of FXIII are important for the stabilization (first calcium binding site) of its zymogenic form and the possible modes of deactivation (thrombin mediated secondary cleavage) of the activated form. Our study for the first time provides a structural outlook of the FXIIIA2B2 heterotetramer assembly, its association and dissociation. The FXIIIB subunits regulatory role in the overall process has also been elaborated upon. In summary, this study provides detailed structural insight into the mechanisms of FXIII activation and regulation that can be used as a template for the development of future highly specific therapeutic inhibitors targeting FXIII in pathological conditions like thrombosis.

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).

Free factor XIII activation peptide (fAP-FXIII) is a regulator of factor XIII activity via factor XIII-B.

In a factor XIIIa (FXIIIa) generation assay with recombinant FXIII-A2 (rFXIII-A2 ) free FXIII activation peptide (fAP-FXII) prolonged the time to peak (TTP) but did not affect the area under the curve (AUC) or concentration at peak (CP). Addition of recombinant factorXIII-B2 (rFXIII-B2 ) restored the characteristics of the FXIIIa generation parameters (AUC, TTP and CP) to those observed for plasma FXIII (FXIII-A2 B2 ). FXIII-A2 B2 reconstituted from rFXIII-A2 and rFXIII-B2 showed a similar effect on AUC, TTP and CP in the presence of fAP-FXII as observed for plasma FXIII-A2 B2 , indicating a role for FXIII-B in this observation. An effect of fAP-FXIII on thrombin, the proteolytic activator of FXIII, was excluded by thrombin generation assays and clotting experiments. In a purified system, fAP-FXIII did not interfere with the FXIIIa activity development of thrombin-cleaved rFXIII-A2 (rFXIII-A2 ') also excluding direct inhibition of FXIIIa. However, FXIIIa activity development of FXIII-A2 'B2 was inhibited in a concentration-dependent manner by fAP-FXIII, indicating that an interaction between AP-FXIII and FXIII-B2 contributes to the overall stability of FXIII-A2 'B2 . In addition to its well-known role, FXIII-B also contributes to FXIII-A2 B2 stability or dissociation depending on fAP-FXIII and calcium concentrations.

Factor XIIIa generation assay: a tool for studying factor XIII function in plasma.

Triggering the extrinsic coagulation pathway in plasma and using a fluorogenic factor XIIIa (FXIIIa) substrate for continuously monitoring FXIIIa activity, an FXIIIa generation curve is obtained. The parameters area under the curve (AUC), time to peak (TTP), and concentration at peak (CP) were calculated. In dilutions of normal plasma in FXIII-deficient plasma, AUC and CP showed linear dose-response relationships, whereas TTP increased from 9.9 min for 25% FXIII to 11.6 min for 100% FXIII. Three FXIII-A preparations (rFXIII, rFXIII(V34L), and cellular FXIII [cFXIII]) showed a linear dose response for AUC and CP. The TTP increased slightly for rFXIII from 13.5 to 15.0 min, but surprisingly for cFXIII TTP increased concentration dependently from 13.5 to 28.7 min. Adding 5 µg/ml FXIII-B at a concentration of 1U of FXIII-A increased the AUC for rFXIII(V34L) and cFXIII by approximately 20% and accelerated TTP from 27.3 to 20.8 min for cFVIII, indicating a supportive function of FXIII-B in orientating cFXIII-A for thrombin cleavage. A commercial assay quantifying FXIII after complete activation in a restricted time window did not reveal differences in the cFXIII preparation with or without FXIII-B. The FXIIIa generation assay provides additional information about activation and function of FXIII. This advantage was underlined in experiments with an irreversible FXIIIa inhibitor.

The hyaluronic acid-binding protease: a novel vascular and inflammatory mediator?

A serine protease in human plasma termed hyaluronan-binding protease HABP is structurally related to plasminogen-activators, coagulation FXII and hepathocyte growth factor activator. This protease has coagulation and fibrinolysis-related activities, although a physiologic role in haemostasis still requires confirmation. In more recent years accumulating information became available supporting the hypothesis that HABP plays also a significant role in the regulation of cells in the vasculature and in the perivascular environment. On the one hand HABP generates bradykinin or bFGF on the surface of human umbilical vein endothelial cells (HUVECs), triggering intracellular signalling via the bradykinin receptor 2 and FGFR-1. Other data indicate that beside endothelial cells also vascular smooth muscle cells are a target for HABP. As major mechanism of cell regulation a high affinity of HABP to growth factors with the subsequent proteolytic cleavage and inactivation has been identified. The current knowledge of the physiologic and clinical relevance of HABP as a vascular and possibly inflammatory mediator is summarized in this review.

A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma.

Based on the iso-peptidase activity of human plasma FXIII, a novel fluorometric assay that determines FXIII concentrations in human plasma below 0.05 IU/ml is introduced. We considered a peptide sequence derived from alpha(2)-antiplasmin (n =12) to yield high sensitivity. Peptide Abz-NE(Cad-Dnp)EQVSPLTLLK exhibits a K(m) value of 19.8+/-2.8 microM and is used in a concentration of 50 microM. The assay design is suitable for measurements in cuvettes (1 ml volume) as well as for the microtiter plate (MTP) format (0.2 ml volume). It provides linear dose-response curves over a wide range of FXIII concentrations (0.05-8.8 IU/ml). The assay was validated with respect to precision, detection and quantitation limits, accuracy/specificity, linearity, and range. A comparison of the fluorometric assay with the photometric assay for FXIII determinations in plasma pools as well as single donor plasma revealed suitability of the fluorometric assay for FXIII determination in plasma of healthy individuals. FXIII concentrations in plasma samples of patients with severe FXIII deficiency are discussed in the context of FXIII antigen levels. These assays correlate well in the critical range below 0.1 IU/ml, whereas the photometric assay may overestimate residual FXIII activity in severe FXIII-deficient patients.

Induction of intracellular signalling in human endothelial cells by the hyaluronan-binding protease involves two distinct pathways.

Recently a novel plasma serine protease with high affinity to hyaluronic acid and glycosaminoglycans, such as heparin and heparan sulfate, has been described and termed hyaluronan-binding protease (HABP). HABP cleaves kininogen in vitro, releasing the vasoactive peptide bradykinin, and activates plasminogen activators, suggesting a vascular cell-directed physiological function of this novel plasma protease. Here we show that HABP stimulates human umbilical vein endothelial cells (HUVECs) by activating two distinct cell-surface receptors. On the one hand, HABP releases bradykinin from cell surface-bound or soluble kininogen and triggers a bradykinin B2-receptor-dependent mobilisation of intracellular Ca2+. On the other hand, HABP activates the p44/42-dependent MAPK (ERK1/2) signalling cascade independent of the B2-receptor, but involving the fibroblast growth factor receptor-1 and basic fibroblast growth factor. This signalling pathway leads to phosphorylation of the kinases Raf, MEK1/2 and ERK1/2. The extracellular activity of HABP also affects the gene expression level through phosphorylation of two transcription factors, the cAMP-responsive element binding protein CREB and the proto-oncogene c-Myc. Our results indicate a proangiogenic potential of HABP, which, in combination with a profibrinolytic activity, directs the physiological function of this plasma protease to processes in which clot lysis, cell motility and neovascularisation are pivotal processes, e.g., in wound healing, tissue repair and tumour progression.

A secreted metallo protease from Aeromonas hydrophila exhibits prothrombin activator activity.

Detection, purification, and partial characterization of a protease from Aeromonas hydrophila capable of cleaving prothrombin into active thrombin is described. The protease has been characterized with respect to enzymatic characteristics such as optimum reaction conditions for prothrombin activation, usage of additional substrates, as well as sensitivity against inhibitors. The protease activity can reversibly be inhibited by Me2+ chelating agents like ethylenediamine tetraacetic acid. The enzyme exhibits a pI value of 4.4 and can withstand temperatures up to 55 degrees C without loss of activity. With respect to prothrombin the enzyme exhibits a K(M) value of 1.47 micromol/l and a vmax value of 1.66 mol/min per mol enzyme. Amino terminal sequence analysis as well as mass spectrometry of fragments obtained by trypsin digest showed identity to a recently described elastase type protease from the same organism and homology to known proteases from other procaryotes (e.g. Aeromonas caviae, Vibrio proteolytica, Pseudomonas aeruginosa).

Inhibition of bFGF/EGF-dependent endothelial cell proliferation by the hyaluronan-binding protease from human plasma.

Recently we identified a plasma serine protease with a high affinity to glycosaminoglycans like heparin or hyaluronic acid, termed hyaluronan-binding protease (HABP). Since glycosaminoglycans are found on cell surfaces and in the extracellular matrix a physiological role of this plasma protease in a pericellular environment was postulated. Here we studied the influence of HABP on the regulation of endothelial cell growth. We found that HABP efficiently prevented the basic fibroblast growth factor/epidermal growth factor (bFGF/EGF)-dependent proliferation of human umbilical vein endothelial cells. Proteolytic cleavage of adhesion molecules was found to be involved, but was not solely responsible for the anti-proliferative activity. Pre-treatment of growth factor-supplemented cell culture medium with HABP indicated that no direct contact between the active protease and cells was required for growth inhibition. In vitro studies revealed a growth factor-directed activity of HABP, resulting in complexation and partial hydrolysis and, thus, inactivation of basic fibroblast growth factor, a potent mitogen for endothelial cells. Heparin and heparan sulfate fully protected bFGF from complexation and cleavage by HABP, although these glycosaminoglycans are known to enhance the proteolytic activity of HABP. This finding suggested that free circulating bFGF rather than bFGF bound to heparan sulfate proteoglycans would be a physiologic substrate. In conclusion, down-regulation of bFGF-dependent endothelial cell growth represents an important mechanism through which HABP could control cell growth in physiologic or pathologic processes like angiogenesis, wound healing or tumor development.

Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase.

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.