Peptidic Inhibitors and a Fluorescent Probe for the Selective Inhibition and Labelling of Factor XIIIa Transglutaminase.

Factor XIIIa (FXIIIa) is a transglutaminase of major therapeutic interest for the development of anticoagulants due to its essential role in the blood coagulation cascade. While numerous FXIIIa inhibitors have been reported, they failed to reach clinical evaluation due to their lack of metabolic stability and low selectivity over transglutaminase 2 (TG2). Furthermore, the chemical tools available for the study of FXIIIa activity and localization are extremely limited. To combat these shortcomings, we designed, synthesised, and evaluated a library of 21 novel FXIIIa inhibitors. Electrophilic warheads, linker lengths, and hydrophobic units were varied on small molecule and peptidic scaffolds to optimize isozyme selectivity and potency. A previously reported FXIIIa inhibitor was then adapted for the design of a probe bearing a rhodamine B moiety, producing the innovative KM93 as the first known fluorescent probe designed to selectively label active FXIIIa with high efficiency (kinact/KI = 127,300 M-1 min-1) and 6.5-fold selectivity over TG2. The probe KM93 facilitated fluorescent microscopy studies within bone marrow macrophages, labelling FXIIIa with high efficiency and selectivity in cell culture. The structure-activity trends with these novel inhibitors and probes will help in the future study of the activity, inhibition, and localization of FXIIIa.

Identification of amino acid residues that are crucial for FXIII-A intersubunit interactions and stability.

Coagulation factor XIII (FXIII) is the main stabilizer of the fibrin clot. It circulates in plasma as a tetramer of two A-subunits and two B-subunits. Under physiological conditions, FXIII-A exists as a dimer (FXIII-A2). The interactions between the FXIII-A-subunits that stabilize the FXIII-A2 dimer are not fully understood. We therefore designed a systematic approach to identify amino acid residues crucial for the expression and stability of FXIII-A2. Based on the available FXIII-A2 crystal structure, we identified 12 amino acid residues forming intersubunit salt bridges and 21 amino acid residues forming hydrogen bonds between the two A-subunits. We chose 10 amino acid residues that form 5 particularly strong interactions, performed site-directed mutagenesis, and expressed the mutants in CHO cells. Disruption of these interactions by single mutation of Lys257, Lys113, Asp343, Glu401, or Asp404 abolished the expression of properly folded, soluble, and functional FXIII-A in CHO cells. On the contrary, mutation of Glu111, Arg100, or Asn112 had no significant effect on FXIII-A expression. Our results suggest that 4 intersubunit interactions (Arg11-Asp343, Lys113-Asp367, Lys257-Glu401, and Arg260-Asp404) are essential for the stability of FXIII-A2. Our findings are supported by reported mutations at Lys257, Arg260, and Asp404 found in patients with congenital FXIII-A deficiency.

Factor XIII-A: An Indispensable "Factor" in Haemostasis and Wound Healing.

Factor XIII (FXIII) is a transglutaminase enzyme that catalyses the formation of ε-(γ-glutamyl)lysyl isopeptide bonds into protein substrates. The plasma form, FXIIIA2B2, has an established function in haemostasis, with fibrin being its principal substrate. A deficiency in FXIII manifests as a severe bleeding diathesis emphasising its crucial role in this pathway. The FXIII-A gene (F13A1) is expressed in cells of bone marrow and mesenchymal lineage. The cellular form, a homodimer of the A subunits denoted FXIII-A, was perceived to remain intracellular, due to the lack of a classical signal peptide for its release. It is now apparent that FXIII-A can be externalised from cells, by an as yet unknown mechanism. Thus, three pools of FXIII-A exist within the circulation: plasma where it circulates in complex with the inhibitory FXIII-B subunits, and the cellular form encased within platelets and monocytes/macrophages. The abundance of this transglutaminase in different forms and locations in the vasculature reflect the complex and crucial roles of this enzyme in physiological processes. Herein, we examine the significance of these pools of FXIII-A in different settings and the evidence to date to support their function in haemostasis and wound healing.

Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia.

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients' plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and "D:D" interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

Mass spectrometry-based molecular mapping of native FXIIIa cross-links in insoluble fibrin clots.

The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the αC region of fibrinogen α. Our findings expand the known native cross-linked species from one to over 100 and suggest distinct antiparallel registries for interprotofibril association and covalent attachment of serpins that regulate clot dissolution.

Protein-peptide based assay for the characterization of human blood coagulation factor XIII-A isopeptidase activity.

Blood coagulation factor XIII-A (FXIII-A), a member of the transglutaminase enzyme family, is best known for its fibrin clot stabilizing function during blood coagulation. It possesses amine incorporating and protein crosslinking transamidase activities, but it is also able to cleave the previously formed isopeptide bond by its isopeptidase activity. Our aim was to develop a protein-based assay for better characterization of FXIII-A isopeptidase activity. The first attempt applying the crosslinked D-dimer of fibrin as a substrate was not successful because of poor reproducibility. Then, the principle of an earlier published anisotropy based activity assay was adapted for the measurement of FXIII-A isopeptidase activity. After crosslinking the fluorescently labelled α2-antiplasmin derived peptide and S100A4(GST) lysine donor protein, this protease-resistant γ-glutamyl-ε-lysine isopeptide bond containing protein-peptide product was applied as a substrate for FXIII-A. Using this substrate and detecting decreasing anisotropy, kinetic measurement of FXIII-A isopeptidase activity was achieved at high sensitivity even in a complex biological sample and in the presence of inhibitor.

Rapid paper diagnostic for plasma fibrinogen concentration.

Fibrinogen is a blood protein that is essential for clotting. It is converted into the polymer fibrin by the blood enzymes thrombin and factor XIIIa. Fibrinogen is one of the first proteins to be depleted in heavily bleeding patients. Patients with early hypofibrinogenemia need urgent fibrinogen replenishment to prevent the onset of haemorrhage and death. However, currently there is no rapid, sensitive, cheap and easy-to-use fibrinogen assay that can detect fibrinogen concentrations. In this study, we have developed a new paper-based diagnostic to quantify the fibrinogen concentration in blood at room temperature. This diagnostic is a 2-step process: first, plasma is added onto thrombin-treated paper strips where fibrinogen is converted to fibrin; then the strips are placed into an aqueous dye bath where elution occurs. The test operates by measuring the change in hydrophobicity, which increases with fibrinogen concentration under otherwise constant conditions. The diagnostic can precisely measure fibrinogen concentration within the range of 0-2 g L-1, which is ideal for the clinical diagnosis of hypofibrinogenemia. Furthermore, testing needs only 12 µL of plasma, 60 mU of thrombin and 7.5 minutes of testing. This diagnostic has the potential to revolutionise point of care testing and save many lives.

Comparison of F13A1 gene mutations in 73 patients treated with recombinant FXIII-A2.

INTRODUCTION: Congenital factor XIII (FXIII) deficiency is a rare, autosomal recessive bleeding disorder usually caused by mutations in the F13A1 gene that produce a severe quantitative (type I) deficiency of the FXIII-A subunit. AIM: To determine the genotypes of patients with severe FXIII-A deficiency treated with recombinant FXIII-A subunit (rFXIII-A2 ) participating in three international efficacy and safety trials. METHODS: We determined the genotypes of 73 patients in total; 32 had already undergone genotype analysis and were known to carry F13A1 mutations that have been previously reported in the literature. Mutation screening was performed in 41 patients with unknown genetic status using direct sequencing. RESULTS: In total, 51 distinct mutations in 73 patients were identified. Two patients showed a phenotype of severe FXIII-A deficiency, despite having heterozygous missense mutations. Two siblings carried a missense mutation in the F13A1 gene (p.Ser296Arg) in combination with a novel, probably polymorphic variant of the F13B gene (p.Ser654Phe). Molecular modelling of five F13A1 novel missense mutations (p.Leu171Phe, p.Glu204Lys, p.Leu276Phe, p.Asp405His and p.Gly411Cys) predicted a damaging effect of these mutations on protein structure. Although five patients treated with rFXIII-A2 had transient, low-titre, non-neutralizing anti-rFXIII antibodies, no patients developed FXIII-neutralizing antibodies (inhibitors). CONCLUSION: The identified mutations are causally implicated in severe FXIII deficiency; however, they do not appear to increase the risk of neutralizing antibody development against rFXIII-A2 .

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).

Longitudinal orientation of cross-linked polypeptide γ chains in fibrin fibrils.

The crosslinking of fibrin γ-polypeptide chains under the influence of the plasma fibrin-stabilizing factor (FXIIIa), which causes their conversion to γ-γ dimers, is the major enzyme reaction of covalent fibrin stabilization. We studied the self-assembly of soluble cross-linked fibrin oligomers. The results of analytical ultracentrifugation as well as elastic and dynamic light scattering showed that the double-stranded fibrin oligomers formed under the influence of moderate concentrations of urea are cross-linked only due to formation of γ-γ dimers, which can dissociate into single-stranded structure when the concentration of urea increases. This fact proves that γ-γ dimers are formed in the end-to-end manner.

Ozone-induced oxidative modification of plasma fibrin-stabilizing factor.

The plasma fibrin-stabilizing factor (pFXIII) function is to maintain a hemostasis by the fibrin clot stabilization. The conversion of pFXIII to the active form of the enzyme (FXIIIа) is a multistage process. Ozone-induced oxidation of pFXIII has been investigated at different stages of its enzyme activation. The biochemical results point to a decrease of an enzymatic activity of FXIIIа depending largely on the stage of the pFXIII conversion into FXIIIа at which oxidation was carried out. UV-, FTIR- and Raman spectroscopy demonstrated that chemical transformation of cyclic, NH, SH and S-S groups mainly determines the oxidation of amino acid residues of pFXIII polypeptide chains. Conversion of pFXIII to FXIIIa proved to increase protein sensitivity to oxidation in the order: pFXIII

Hybrid photopatterned enzymatic reaction (HyPER) for in situ cell manipulation.

The ability to design artificial extracellular matrices as cell-instructive scaffolds has opened the door to technologies capable of studying the fate of cells in vitro and to guiding tissue repair in vivo. One main component of the design of artificial extracellular matrices is the incorporation of biochemical cues to guide cell phenotype and multicellular organization. The extracellular matrix (ECM) is composed of a heterogeneous mixture of proteins that present a variety of spatially discrete signals to residing cell populations. In contrast, most engineered ECMs do not mimic this heterogeneity. In recent years, photo-deprotection has been used to spatially immobilize signals. However, this approach has been limited mostly to small peptides. Here we combine photo-deprotection with enzymatic reaction to achieve spatially controlled immobilization of active bioactive signals that range from small molecules to large proteins. A peptide substrate for transglutaminase factor XIII (FXIIIa) was caged with a photo-deprotectable group, which was then immobilized to the bulk of a cell-compatible hydrogel. With focused light, the substrate can be deprotected and used to immobilize patterned bioactive signals. This approach offers an innovative strategy to immobilize delicate bioactive signals, such as growth factors, without loss of activity and enables in situ cell manipulation of encapsulated cells.

In situ cell manipulation through enzymatic hydrogel photopatterning.

The physicochemical properties of hydrogels can be manipulated in both space and time through the controlled application of a light beam. However, methods for hydrogel photopatterning either fail to maintain the bioactivity of fragile proteins and are thus limited to short peptides, or have been used in hydrogels that often do not support three-dimensional (3D) cell growth. Here, we show that the 3D invasion of primary human mesenchymal stem cells can be spatiotemporally controlled by micropatterning the hydrogel with desired extracellular matrix (ECM) proteins and growth factors. A peptide substrate of activated transglutaminase factor XIII (FXIIIa)--a key ECM crosslinking enzyme--is rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of the caged FXIIIa substrate into poly(ethylene glycol) hydrogels and subsequent laser-scanning lithography affords highly localized biomolecule tethering. This approach for the 3D manipulation of cells within gels should open up avenues for the study and manipulation of cell signalling.

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay.

Activated factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetic assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting only the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, Staphylococcus aureus fibronectin binding protein A, and thrombin-activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character, and the P-2 and P-3 substrate positions are sensitive to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.

Interplay of fibrinogen αEC globular domains and factor XIIIa cross-linking dictates the extensibility and strain stiffening of fibrin networks.

BACKGROUND: Fibrinogen is a plasma protein forming the fibrin scaffold of blood clots. Its mechanical properties therefore affect the risk of bleeding as well as thrombosis. There has been much recent interest in the biophysical mechanisms controlling fibrin mechanics; however, the role of molecular heterogeneity of the circulating fibrinogen in determining clot mechanical function remains poorly characterized. OBJECTIVES: By comparing 2 fibrinogen variants where the only difference is the Aα-chain length, with one variant having a globular domain at its C-terminus, this study aimed to reveal how the molecular structure impacts the structure and mechanics of fibrin networks. METHODS: We characterized the mechanical response to large shear for networks formed from 2 recombinant fibrinogen variants: the most prevalent variant in circulation with a molecular weight of 340 kDa (recombinant human fibrinogen [rFib] 340) and a minor variant with a molecular weight of 420 kDa (rFib420). RESULTS: We show that the elastic properties of the 2 variants are identical when fibrin is cross-linked with factor XIIIa but differ strongly in its absence. Uncross-linked rFib420 networks are softer and up to 3-fold more extensible than rFib340 networks. Electron microscopy imaging showed that the 2 variants formed networks with a comparable structure, except at 4 mg/mL, where rFib420 formed denser networks. CONCLUSION: We propose that the αEC domains of rFib420 increase the extensibility of uncross-linked fibrin networks by promoting protofibril sliding, which is blocked by FXIIIa cross-linking. Our findings can help explain the functional role of different circulating fibrinogen variants in blood clot mechanics and tissue repair.

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.

Identification of a novel mutation in the factor XIII A subunit in a patient with inherited factor XIII deficiency.

Inherited factor XIII (FXIII) deficiency is an extremely rare and under-diagnosed autosomal recessive inherited coagulopathy, which is caused by genetic defects in the F13A1 or F13B gene. More than 200 genetic mutations have been identified since the first case of inherited FXIII deficiency was reported. This study aimed to identify underlying gene mutations in a patient with inherited FXIII deficiency who presented with recurrent intracerebral hemorrhage. Levels of plasma FXIII-A antigen were measured, F13A1 and F13B genes were sequenced, mutation information was analyzed, and the mutated protein structure was predicted using bioinformatics methods. Molecular genetic analysis identified four mutations of FXIII-related genes in the proband, including three previously reported mutations inherited from his parents (c.631G>A, p.Gly210Arg and c.1687G>A, p.Gly562Arg of F13A1 gene and c.344G>A, p.Arg115His of F13B gene) and a novel spontaneous mutation of F13A1 gene (c.2063C>G, p.Ser687Cys). Molecular structural modeling demonstrated that the novel Ser687Cys mutation may cause changes in the spatial structure of FXIII-A and increase its instability. In conclusion, we identified a novel and likely pathogenic mutation of the F13A1 gene, which enriched the gene mutation spectrum of inherited FXIII deficiency. The findings may provide promising targets for diagnosis and treatment of inherited FXIII deficiency.

Identification of respective lysine donor and glutamine acceptor sites involved in factor XIIIa-catalyzed fibrin α chain cross-linking.

Factor XIIIa-catalyzed ε-(γ-glutamyl)-lysyl bonds between glutamine and lysine residues on fibrin α and γ chains stabilize the fibrin clot and protect it from mechanical and proteolytic damage. The cross-linking of γ chains is known to involve the reciprocal linkages between Gln(398) and Lys(406). In α chains, however, the respective lysine and glutamine partners remain largely unknown. Traditional biochemical approaches have only identified the possible lysine donor and glutamine acceptor sites but have failed to define the respective relationships between them. Here, a differential mass spectrometry method was implemented to characterize cross-linked α chain peptides originating from native fibrin. Tryptic digests of fibrin that underwent differential cross-linking conditions were analyzed by high resolution Fourier transform mass spectrometry. Differential intensities associated with monoisotopic masses of cross-linked peptides were selected for further characterization. A fit-for-purpose algorithm was developed to assign cross-linked peptide pairs of fibrin α chains to the monoisotopic masses relying on accurate mass measurement as the primary criterion for identification. Equipped with hypothesized sequences, tandem mass spectrometry was then used to confirm the identities of the cross-linked peptides. In addition to the reciprocal cross-links between Gln(398) and Lys(406) on the γ chains of fibrin (the positive control of the study), nine specific cross-links (Gln(223)-Lys(508), Gln(223)-Lys(539), Gln(237)-Lys(418), Gln(237)-Lys(508), Gln(237)-Lys(539), Gln(237)-Lys(556), Gln(366)-Lys(539), Gln(563)-Lys(539), and Gln(563)-Lys(601)) on the α chains of fibrin were newly identified. These findings provide novel structural details with respect to the α chain cross-linking compared with earlier efforts.

Reactivity of the N-terminal region of fibronectin protein to transglutaminase 2 and factor XIIIA.

Transglutaminase 2 (TG2) is secreted by a non-classical pathway into the extracellular space, where it has several activities pertinent to fibronectin (FN), including binding to the gelatin-binding domain of FN and acting as an integrin co-receptor. Glutamines in the N-terminal tail of FN are known to be susceptible to transamidation by both TG2 and activated blood coagulation factor XIII (FXIIIa). We used immunoblotting, limited proteolysis, and mass spectrometry to localize glutamines within FN that are subject to TG2-catalyzed incorporation of dansylcadaverine in comparison to residues modified by FXIIIa. Such analysis of plasma FN indicated that Gln-3, Gln-7, and Gln-9 in the N-terminal tail and Gln-246 of the linker between fifth and sixth type I modules ((5)F1 and (6)F1) are transamidated by both enzymes. Only minor incorporation of dansylcadaverine was detected elsewhere. Labeling of C-terminally truncated FN constructs revealed efficient TG2- or FXIIIa-catalyzed dansylcadaverine incorporation into the N-terminal residues of constructs as small as the 29-kDa fragment that includes (1-5)F1 and lacks modules from the adjacent gelatin-binding domain. However, when only (1-3)F1 were present, dansylcadaverine incorporation into the N-terminal residues of FN was lost and instead was in the enzymes, near the active site of TG2 and terminal domains of FXIIIa. Thus, these results demonstrate that FXIIIa and TG2 act similarly on glutamines at either end of (1-5)F1 and transamidation specificity of both enzymes is achieved through interactions with the intact 29K fragment.

Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase.

In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin-pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction may influence the TSG-6/heavy Chain 2-mediated transfer of heavy chains observed during inflammation.