Fbg αC 389-402 Enhances Factor XIII Cross-Linking in the Fibrinogen αC Region Via Electrostatic and Hydrophobic Interactions.

Coagulation Factor XIII (FXIII) stabilizes blood clots by cross-linking glutamines and lysines in fibrin and other proteins. FXIII activity in the fibrinogen αC region (Fbg αC 221-610) is critical for clot stability and growth. Fbg αC 389-402 is a binding site for thrombin-activated FXIII, (FXIII-A*), with αC E396 promoting FXIII-A* binding and activity in αC. The current study aimed to discover additional residues within Fbg αC 389-402 that accelerate transglutaminase activity toward αC. Electrostatic αC residues (E395, E396, and D390), hydrophobic αC residues (W391 and F394), and residues αC 328-425 were studied by mutations to recombinant Fbg αC 233-425. FXIII activity was monitored through MS-based glycine ethyl ester (GEE) cross-linking and gel-based fluorescence monodansylcadaverine (MDC) cross-linking assays. Truncation mutations 403 Stop (Fbg αC 233-402), 389 Stop (Fbg αC 233-388), and 328 Stop (Fbg αC 233-327) reduced Q237-GEE and MDC cross-linking compared to wild-type (WT). Comparable cross-linking between 389 Stop and 328 Stop showed that FXIII is mainly affected by the loss of Fbg αC 389-402. Substitution mutations E396A, D390A, W391A, and F394A decreased cross-linking relative to WT, whereas E395A, E395S, E395K, and E396D had no effect. Similar FXIII-A* activities were observed for double mutants (D390A, E396A) and (W391A, E396A), relative to D390A and W391A, respectively. In contrast, cross-linking was reduced in (F394A, E396A), relative to F394A. In conclusion, Fbg αC 389-402 boosts FXIII activity in Fbg αC, with D390, W391, and F394 identified as key contributors in enhancing αC cross-linking.

Cloning of human anti-factor XIII monoclonal antibody dissects mechanisms of polyclonal antibodies in a single patient.

BACKGROUND: Coagulation factor XIII (FXIII) consists of 2 A (FXIII-A) and 2 B (FXIII-B) subunits that cross-link and strengthen the hemostatic fibrin thrombus; thus, abnormal bleeding occurs when FXIII is significantly reduced. Autoimmune-acquired FXIII deficiency (AiF13D) is characterized by lethal bleeding secondary to the development of autoantibodies against FXIII. However, since anti-FXIII autoantibodies are polyclonal, the mechanism underlying FXIII dysfunction is unclear. OBJECTIVES: The objective of this study was to dissect the inhibitory mechanisms of polyclonal anti-FXIII autoantibodies. METHODS: In this study, we prepared the human monoclonal antibodies (hmAbs) from the peripheral blood of an 86-year-old man with AiF13D by using a new complementary DNA cloning method and analyzed the properties of each autoantibody. RESULTS: Seventeen clones obtained from hmAbs were divided into the following 3 groups: dissociation inhibitors of FXIII-A2B2 (6 clones), assembly inhibitors of FXIII-A2B2 (3 clones), and nonneutralizing/inhibitory hmAbs (8 clones). Dissociation inhibitors strongly inhibited fibrin cross-linking and amine incorporation. Assembly inhibitors extracted FXIII-A from FXIII-A2B2, strongly inhibited binding of FXIII-A to FXIII-B, and activation peptide cleavage. However, the patient's plasma presented a strong inhibition of A2B2 heterodimer assembly but only a slight inhibition of thrombin-Ca2+-dependent dissociation, suggesting that the assembly inhibitors concealed the effect of dissociation inhibitors in plasma. By contrast, nonneutralizing antibodies had little effect on the function of FXIII, suggesting that nonneutralizing hmAbs (and/or dissociation inhibitors and/or assembly inhibitors) promoted the clearance of FXIII-A from the blood. CONCLUSION: Cloning of anti-FXIII autoantibodies enabled us to not only elucidate the mechanism and pathophysiology of AiF13D but also develop a completely new type of anticoagulant.

Biochemical characterization of medaka (Oryzias latipes) fibrinogen gamma and its gene disruption resulting in anemia as a model fish.

At the final stages of blood coagulation, fibrinogen is processed into insoluble fibrin by thrombin resulting in fibril-like structure formation. Via further cross-linking reactions between the fibrin gamma subunit by the catalytic action of blood transglutaminase (Factor XIII), this molecule gains further physical stability. Meanwhile, since fibrinogen is expressed in various cells and tissues, this molecule can exhibit other functions apart from its role in blood coagulation. To create a system studying on aberrant coagulation and investigate the physiological functions, using a model fish medaka (Oryzias latipes), we established gene-deficient mutants of fibrinogen gamma subunit protein in parallel with its biochemical analysis, such as tissue distribution pattern and substrate properties. By genetic deletion via genome editing, two distinct mutants displayed retardation of blood coagulation. The mutants showed lower hematocrit with aberrant erythrocyte maturation, which indicates that fibrin deficiency caused severe anemia, and also appeared as a model for investigation of the fibrin function.

Factor XIII and Fibrin Clot Properties in Acute Venous Thromboembolism.

Coagulation factor XIII (FXIII) is converted by thrombin into its active form, FXIIIa, which crosslinks fibrin fibers, rendering clots more stable and resistant to degradation. FXIII affects fibrin clot structure and function leading to a more prothrombotic phenotype with denser networks, characterizing patients at risk of venous thromboembolism (VTE). Mechanisms regulating FXIII activation and its impact on fibrin structure in patients with acute VTE encompassing pulmonary embolism (PE) or deep vein thrombosis (DVT) are poorly elucidated. Reduced circulating FXIII levels in acute PE were reported over 20 years ago. Similar observations indicating decreased FXIII plasma activity and antigen levels have been made in acute PE and DVT with their subsequent increase after several weeks since the index event. Plasma fibrin clot proteome analysis confirms that clot-bound FXIII amounts associated with plasma FXIII activity are decreased in acute VTE. Reduced FXIII activity has been associated with impaired clot permeability and hypofibrinolysis in acute PE. The current review presents available studies on the role of FXIII in the modulation of fibrin clot properties during acute PE or DVT and following these events. Better understanding of FXIII's involvement in the pathophysiology of acute VTE might help to improve current therapeutic strategies in patients with acute VTE.

The Nature of Resistance of the Coagulation Factor XIII Structure to Hypochlorite-Induced Oxidation.

The damage to blood coagulation factor XIII (FXIII) at different stages of its enzymatic activation under the action of various physiological amounts of hypochlorite ion was studied. The results obtained by HPLC-MS/MS, SDS-PAGE, and colorimetry showed that, during the conversion of FXIII to FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased. FXIII oxidized with 150 µM hypochlorite completely retained its enzymatic activity inherent to the intact protein, whereas FXIIIa treated with 50 µM hypochlorite showed sharply reduced enzymatic activity. It was shown that a number of methionine and cysteine residues on the catalytic subunit can perform antioxidant function; additionally, the regulatory subunits of FXIII-B contribute to the antioxidant protection of the catalytic center of the FXIII-A subunit, which, together with the tight packing of the tetrameric structure of the FXIII proenzyme, are the three factors that provide high protein resistance to the oxidizing agent.

The Structure of Blood Coagulation Factor XIII Is Adapted to Oxidation.

The blood coagulation factor XIII (FXIII) plays a critical role in supporting coagulation and fibrinolysis due to both the covalent crosslinking of fibrin polymers, rendering them resistant to plasmin lysis, and the crosslinking of fibrin to proteins of the fibrinolytic system. The hypochlorite-mediated oxidation of the blood coagulation factor XIII (FXIII) at the different stages of its enzymatic activation is studied for the first time in this paper. The consolidated results obtained with the aid of MS/MS, electrophoresis, and colorimetry demonstrate that in the process of FXIII's conversion into FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased as follows: native FXIII < FXIII + Ca2+ << FXIII + Ca2+/thrombin. The modification sites were detected among all the structural regions of the catalytic FXIII-A subunit, except for the activation peptide, and embraced several sushi domains of the FXIII-B subunit. Oxidized amino acid residues belonging to FXIII-A are surface-exposed residues and can perform an antioxidant role. The regulatory FXIII-B subunits additionally contribute to the antioxidant defense of the catalytic center of the FXIII-A subunits. Taken together, the present data along with the data from previous studies demonstrate that the FXIII proenzyme structure is adapted to oxidation.

The Plasma Factor XIII Heterotetrameric Complex Structure: Unexpected Unequal Pairing within a Symmetric Complex.

Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with two catalytic A subunits and two carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B subunits, activated FXIII (FXIIIa) covalently cross links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamics simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly or activity to reduce bleeding and thrombotic complications.

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.

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.

Exploring the structural similarity yet functional distinction between coagulation factor XIII-B and complement factor H sushi domains.

Coagulation factor XIII (FXIII) covalently crosslinks pre-formed fibrin clots preventing their premature fibrinolysis. In plasma, FXIII circulates as a zymogenic heterotetramer composed of catalytic FXIII-A subunits, and carrier/regulatory FXIII-B subunits. FXIII-A is a well characterized component of this complex, and has been associated with several pleiotropic roles outside coagulation as well. In comparison only protective/regulatory roles towards the FXIII-A subunit have been identified for FXIII-B. Strong homology between FXIII-B and complement regulator Complement factor H suggests a putative role of FXIII-B in complement activation. In the current study we have analyzed the similarities and yet functional divergence of these two proteins using in silico sequence alignment and structural analysis. We have evaluated complement activation post reconstitution of FXIII components into FXIII deficient and CFH deficient plasma. We have also transiently expressed FXIII-B in SH-SY5Y cell lines and evaluated its effect on the endogenous complement activation. Our investigations show no effect of FXIII-B subunit on the rate of complement activation. Therefore we conclude that at a physiological level, FXIII-B subunit plays no role in the complement system, although a vestigial function in altered pathological states might still exist.

Factor XIII topology: organization of B subunits and changes with activation studied with single-molecule atomic force microscopy.

Essentials Factor XIII is a heterotetramer with 2 catalytic A subunits and 2 non-catalytic B subunits. Structure of active and inactive factor XIII was studied with atomic force microscopy. Inactive factor XIII is made of an A2 globule and 2 flexible B subunits extending from it. Activated factor XIII separates into a B2 homodimer and 2 monomeric active A subunits. SUMMARY: Background Factor XIII (FXIII) is a precursor of the blood plasma transglutaminase (FXIIIa) that is generated by thrombin and Ca2+ and covalently crosslinks fibrin to strengthen blood clots. Inactive plasma FXIII is a heterotetramer with two catalytic A subunits and two non-catalytic B subunits. Inactive A subunits have been characterized crystallographically, whereas the atomic structure of the entire FXIII and B subunits is unknown and the oligomerization state of activated A subunits remains controversial. Objectives Our goal was to characterize the (sub)molecular structure of inactive FXIII and changes upon activation. Methods Plasma FXIII, non-activated or activated with thrombin and Ca2+ , was studied by single-molecule atomic force microscopy. Additionally, recombinant separate A and B subunits were visualized and compared with their conformations and dimensions in FXIII and FXIIIa. Results and Conclusions We showed that heterotetrameric FXIII forms a globule composed of two catalytic A subunits with two flexible strands comprising individual non-catalytic B subunits that protrude on one side of the globule. Each strand corresponds to seven to eight out of 10 tandem repeats building each B subunit, called sushi domains. The remainder were not seen, presumably because they were tightly bound to the globular A2 dimer. Some FXIII molecules had one or no visible strands, suggesting dissociation of the B subunits from the globular core. After activation of FXIII with thrombin and Ca2+ , B subunits dissociated and formed B2 homodimers, whereas the activated globular A subunits dissociated into monomers. These results characterize the molecular organization of FXIII and changes with activation.

Live Confocal Visualization of FXIII Activity.

In addition to its important functions in angiogenesis, wound healing, bone biology, immunology, pregnancy, and adipogenesis, transglutaminase factor XIII is an outstanding determinant of clot characteristics. Factor XIII mediates clot stability by covalent cross-linking of fibrin-fibrils and inhibition of fibrinolysis. For decades it was assumed that factor XIII mediates the formation of a 3-dimensional net structure of the clot. It was the aim of our experiment to prove this thesis.

Sortase A as a cross-linking enzyme in tissue engineering.

The bacterial ligase Sortase A (SA) and its mutated variants have become increasingly popular over the last years for post-translational protein modifications due to their unparalleled specificity and efficiency. The aim of this work was to study SA as a cross-linking enzyme for hydrogel-based tissue engineering. For this, we optimized SA pentamutant production and purification from E. coli to achieve high yields and purity. Then using hyaluronan (HA) as a model biopolymer and modifying it with SA-substrate peptides, we studied the cross-linking kinetics obtained with SA, the enzyme stability, cytocompatibility, and immunogenicity, and compared those to state-of-the-art standards. The transglutaminase activated factor XIII (FXIIIa) was used as the reference cross-linking enzyme, and the clinical collagen scaffold Chondro-Gide (CG) was used as a reference biocompatible material for in vivo studies. We found SA could be produced in large amounts in the lab without special equipment, whereas the only viable source of FXIIIa is currently a prescription medicine purified from donated blood. SA was also remarkably more stable in solution than FXIIIa, and it could provide even much faster gelation, making it possible to achieve nearly-instantaneous gel formation upon delivery with a double-barrel syringe. This is an interesting improvement for in vivo work, to allow in situ gel formation in a wet environment, and could also be useful for applications like bioprinting where very fast gelation is needed. The cytocompatibility and lack of immunogenicity were still uncompromised. These results support the use of SA as a versatile enzymatic cross-linking strategy for 3D culture and tissue engineering applications. STATEMENT OF SIGNIFICANCE: Enzymatic crosslinking has immense appeal for tissue engineers as one of the most biocompatible methods of hydrogel crosslinking. Sortase A has a number of unique advantages over previous systems. We show an impressive and tunable range of crosslinking kinetics, from almost instantaneous gelation to several minutes. We also demonstrate that Sortase A crosslinked hydrogels have good cytocompatibility and cause no immune reaction when implanted in vivo. With its additional benefits of excellent stability in solution and easy large-scale synthesis available to any lab, we believe this novel crosslinking modality will find multiple applications in high throughput screening, tissue engineering, and biofabrication.

The role of ß-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit.

Essentials The roles of ß-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking ß-barrel 2, both ß-barrels, or full length FXIII, were made. Removing ß-barrel 2 caused total loss of activity, removing both ß-barrels returned 30% activity. ß-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII is composed of an activation peptide segment, a ß-sandwich domain, a catalytic core, and, finally, ß-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of ß-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to ß-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to ß-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of ß-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of ß-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the ß-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the ß-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual ß-barrel domains in modulating access to the FXIII active site region.

Activation of factor XIII is accompanied by a change in oligomerization state.

Factor XIII A (FXIIIA) is a member of the transglutaminase enzyme family that cross-links both intra- and extracellular protein substrates. To prevent undesired cross-linking, FXIIIA is expressed as an inactive zymogen and exists intracellularly as an A2 homodimer. In plasma, FXIII A2 is complexed with two protective factor XIII B subunits (A2 B2 ) that dissociate upon activation of the zymogen. Based on limited experimental data, activated FXIII was considered a dimer of two catalytically active A subunits. However, accumulating but indirect evidence has suggested activation may lead to a monomeric state instead. In the present study, we employed analytical ultracentrifugation (AUC) to directly explore the oligomerization state of zymogen as well as active FXIIIA in solution. We first confirmed that the zymogen was a FXIIIA2 dimer. When we activated FXIIIA nonproteolytically (by high mm Ca2+ ), the protein dissociated to monomers. More importantly, FXIIIA incubation with its physiological partner, the protease thrombin, led to a monomeric state as well. AUC studies of partially cleaved FXIIIA further suggested that thrombin cleavage of a single activation peptide in a zymogen dimer is sufficient to weaken intersubunit interactions, initiating the transition to monomer. The enzymatic activity of the thrombin-cleaved species was higher than nonproteolytically activated enzyme, suggesting that displacement of the activation peptide renders the FXIIIA more accessible to substrates. Thus, results provide evidence that FXIII undergoes a change in oligomerization state as part of the activation process, and emphasize the role of the activation peptide in preventing FXIIIA catalytic activity. ENZYMES: Factor XIIIA (EC2.3.2.13).

Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation.

Essentials Corn Trypsin Inhibitor (CTI) is a selective inhibitor of coagulation Factor XII (FXII). Molecular modelling of the CTI-FXIIa complex suggested a canonical inhibitor binding mode. Mutagenesis revealed the CTI inhibitory loop and helices α1 and α2 mediate the interaction. This confirms that CTI inhibits FXII in canonical fashion and validates the molecular model. SUMMARY: Background Corn trypsin inhibitor (CTI) has selectivity for the serine proteases coagulation factor XII and trypsin. CTI is in widespread use as a reagent that specifically inhibits the intrinsic pathway of blood coagulation but not the extrinsic pathway. Objectives To investigate the molecular basis of FXII inhibition by CTI. Methods We performed molecular docking of CTI, using its known crystal structure, with a model of the activated FXII (FXIIa) protease domain. The interaction model was verified by use of a panel of recombinant CTI variants tested for their ability to inhibit FXIIa enzymatic activity in a substrate cleavage assay. Results The docking predicted that: (i) the CTI central inhibitory loop P1 Arg34 side chain forms a salt bridge with the FXIIa S1 pocket Asp189 side chain; (ii) Trp22 from CTI helix α1 interacts with the FXIIa S3 pocket; and (iii) Arg43 from CTI helix α2 forms a salt bridge with FXIIa H1 pocket Asp60A. CTI amino acid substitution R34A negated all inhibitory activity, whereas the G32W, L35A, W22A and R42A/R43A substitutions reduced activity by large degrees of 108-fold, 41-fold, 158-fold, and 100-fold, respectively; the R27A, W37A, W39A and R42A substitutions had no effect. Synthetic peptides spanning CTI residues 20-44 had inhibitory activity that was three-fold to 4000-fold less than that of full-length CTI. Conclusions The data confirm the validity of a canonical model of the FXIIa-CTI interaction, with helix α1 (Trp22), central inhibitory loop (Arg34) and helix α2 (Arg43) of CTI being required for effective binding by contacting the S1, S3 and H1 pockets of FXIIa, respectively.

Biochemical characterization of a medaka (Oryzias latipes) orthologue for mammalian Factor XIII and establishment of a gene-edited mutant.

In the final process of blood coagulation, fibrin molecules are stabilized via a catalytic reaction by Factor XIIIA (FXIIIA), a member of the transglutaminase (TGase) family that catalyzes protein cross-linking reactions. In this study, we characterized the orthologue of this enzyme in medaka (Oryzias latipes), an established model fish in which a coagulation system is also preserved. The recombinant protein of this orthologue enzyme was produced in baculovirus-infected insect cells and used for analysis of its biochemical properties including activation by thrombin proteolysis and calcium dependence of the TGase enzymatic activity. Immunostaining and immunoblotting revealed that medaka FXIIIA is expressed in the kidney, bone, and esophagus in addition to blood cells. Furthermore, a gene-mutant fish was established using the CRISPR/Cas9 system. The loss of FXIIIA expression was validated in the mutants, and phenotypes, such as absence of fibrin cross-linking, were investigated in the established mutant fish.

Modification of the catalytic subunit of plasma fibrin-stabilizing factor under induced oxidation.

For the first time, by using mass-spectrometry method, the oxidation-mediated modification of the catalytic FXIII-A subunit of plasma fibrin-stabilizing factor, pFXIII, has been studied. The oxidative sites were identified to belong to all structural elements of the catalytic subunit: the ß-sandwich (Tyr104, Tyr117, and Cys153), the catalytic core domain (Met160, Trp165, Met266, Cys328, Asp352, Pro387, Arg409, Cys410, Tyr442, Met475, Met476, Tyr482, and Met500), the ß-barrel 1 (Met596), and the ß-barrel 2 (Met647, Pro676, Trp692, Cys696, and Met710), which correspond to 3.9%, 1.11%, 0.7%, and 3.2%, respectively, of oxidative modifications as compared to the detectable amounts of amino acid residues in each of the structural domains. Lack of information on some parts of the molecule may be associated with the spatial unavailability of residues, complicating analysis of the molecule. The absence of oxidative sites localized within crucial areas of the structural domains may be brought about by both the spatial inaccessibility of the oxidant to amino acid residues in the zymogen and the screening effect of the regulatory FXIII-B subunit.

Fibrinogen splice variation and cross-linking: Effects on fibrin structure/function and role of fibrinogen γ' as thrombomobulin II.

Fibrin is an important matrix protein that provides the backbone to the blood clot, promoting tissue repair and wound healing. Its precursor fibrinogen is one of the most heterogeneous proteins, with an estimated 1 million different forms due to alterations in glycosylation, oxidation, single nucleotide polymorphisms, splice variation and other variations. Furthermore, ligation by transglutaminase factor XIII (cross-linking) adds to the complexity of the fibrin network. The structure and function of the fibrin network is in part determined by this natural variation in the fibrinogen molecule, with major effects from splice variation and cross-linking. This mini-review will discuss the direct effects of fibrinogen αEC and fibrinogen γ' splice variation on clot structure and function and also discuss the additional role of fibrinogen γ' as thrombomodulin II. Furthermore, the effects of cross-linking on clot function will be described. Splice variation and cross-linking are major determinants of the structure and function of fibrin and may therefore impact on diseases affecting bleeding, thrombosis and tissue repair.

The conversion of fibrinogen to fibrin: A brief history of some key events.

The conversion of fibrinogen to fibrin is a process that has long fascinated an army of researchers. In this brief review some early break-through observations are noted and a few later unexpected results described.