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.

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.

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.

Why fibrin biomechanical properties matter for hemostasis and thrombosis.

Polymeric fibrin displays unique structural and biomechanical properties that contribute to its essential role of generating blood clots that stem bleeds. The aim of this review is to discuss how the fibrin clot is formed, how protofibrils make up individual fibrin fibers, what the relationship is between the molecular structure and fibrin biomechanical properties, and how fibrin biomechanical properties relate to the risk of thromboembolic disease. Fibrin polymerization is driven by different types of bonds, including knob-hole interactions displaying catch-slip characteristics, and covalent crosslinking of fibrin polypeptides by activated factor XIII. Key biophysical properties of fibrin polymer are its visco-elasticity, extensibility and resistance to rupture. The internal packing of protofibrils within fibers changes fibrin biomechanical behavior. There are several methods to analyze fibrin biomechanical properties at different scales, including AFM force spectroscopy, magnetic or optical tweezers and rheometry, amongst others. Clinically, fibrin biomechanical characteristics are key for the prevention of thromboembolic disorders such as pulmonary embolism. Future studies are needed to address unanswered questions regarding internal molecular structure of the fibrin polymer, the structural and molecular basis of its remarkable mechanical properties and the relationship of fibrin biomechanical characteristics with thromboembolism in patients with deep vein thrombosis and ischemic stroke.

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.

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.

Expression, Purification and Characterization of Recombinant Human Coagulation Factor XIIIa in Pichia Pastoris.

BACKGROUND: Coagulation factor XIIIa(FXIIIa) plays a critical role in the final stage of blood coagulation. It is extremely important in wound healing, tissue repairing and promoting cell adhesion. The deficiency of the coagulation factor can cause hemorrhage and slow wound healing. OBJECTIVE: In this study, recombinant pPICZαC-FXIIIa was expressed in Pichia pastoris, purified as well as its biological activity was determined. METHODS: The FXIIIa fragment obtained from the human placenta was inserted into pPICZαC to obtain pPICZαC-FXIIIa, which was transformed into X33 after linearization, and FXIIIa inserted into Pichia pastoris X33 was screened for methanol induction. The expressed product was identified by western blotting, then the supernatant was purified by affinity chromatography, and the purified product was determined by plasma coagulation experiment. RESULTS: Polymerase Chain Reaction(PCR) showed that the FXIIIa fragment of 2250 bp was inserted successfully into pPICZαC. The expression and purification products of the same molecular weight as target protein(about 83 kDa) were obtained, which solidified significantly when reacted with plasma. CONCLUSION: The expression and purification products were successful, with sufficient biological activity, which can be used as a candidate FXIIIa hemostatic agent in genetic engineering.

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.

On the early events of the calcium-induced activation of coagulation factor XIII-A2 and tissue transglutaminase: an in silico study.

Both coagulation factor XIII-A2 (FXIII-A2) and tissue transglutaminase (TG2) play distinctive and important roles in homeostasis by crosslinking proteins or peptides via isopeptide bonds. In this present study, a series of microsecond-long all-atom molecular dynamics (MD) simulations were carried out in order to reveal the dynamic, atomic-level events which may contribute to the activation of these proteins via the binding of calcium ions. In addition to previously conducted in vitro and crystallographic studies, further suggestions have been made concerning the calcium binding features of these enzymes. The different systems used for running the simulations were based on the zymogen, computationally cleaved and even the activation peptide (AP-FXIII) free FXIII-A2' homodimer form. The effects of various ionic environments have also been explored in the simulations of FXIII-A2. Our results suggest that the presence of calcium ions can cause increased AP fluctuations, which ultimately could lead to their relocation on the homodimer surface. The release of these APs seems to be crucial for rotation of the A subunits based on equilibrium MD simulations. The primary evidence for this assumption comes from the predicted principal component eigenvector which is considered as the first, large-scale event of the overall activation process. To get a more accurate atomic-level description, the calcium binding sites of TG2 have also been investigated in our extensive in silico experiments, which suggests the presence of previously unidentified binding sites as well.Communicated by Ramaswamy H. Sarma.

Proteolytic and nonproteolytic activation mechanisms result in conformationally and functionally different forms of coagulation factor XIII A.

Factor XIIIA (FXIIIA) is a transglutaminase that cross-links intra- and extracellular protein substrates. FXIIIA is expressed as an inactive zymogen, and during blood coagulation, it is activated by removal of an activation peptide by the protease thrombin. No such proteolytic FXIIIA activation is known to occur in other tissues or the intracellular form of FXIIIA. For those locations, FXIIIA is assumed instead to undergo activation by Ca2+ ions. Previously, we demonstrated a monomeric state for active FXIIIA. Current analytical ultracentrifugation and kinetic experiments revealed that thrombin-activated FXIIIA has a higher conformational flexibility and a stronger affinity toward glutamine substrate than does nonproteolytically activated FXIIIA. The proteolytic activation of FXIIIA was further investigated in a context of fibrin clotting. In a series of fibrin cross-linking assays and scanning electron microscopy studies of plasma clots, the activation rates of FXIIIA V34X variants were correlated with the extent of fibrin cross-linking and incorporation of nonfibrous protein into the clot. Overall, the results suggest conformational and functional differences between active FXIIIA forms, thus expanding the understanding of FXIIIA function. Those differences may serve as a basis for developing therapeutic strategies to target FXIIIA in different physiological environments. ENZYMES: Factor XIIIA ( EC 2.3.2.13).

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.

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.

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.

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.

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.

Biophysical Mechanisms Mediating Fibrin Fiber Lysis.

The formation and dissolution of blood clots is both a biochemical and a biomechanical process. While much of the chemistry has been worked out for both processes, the influence of biophysical properties is less well understood. This review considers the impact of several structural and mechanical parameters on lytic rates of fibrin fibers. The influences of fiber and network architecture, fiber strain, FXIIIa cross-linking, and particle transport phenomena will be assessed. The importance of the mechanical aspects of fibrinolysis is emphasized, and future research avenues are discussed.

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 .

Genomic variants reveal differential evolutionary constraints on human transglutaminases and point towards unrecognized significance of transglutaminase 2.

Transglutaminases (TGMs) catalyze Ca2+-dependent transamidation of proteins with specified roles in blood clotting (F13a) and in cornification (TGM1, TGM3). The ubiquitous TGM2 has well described enzymatic and non-enzymatic functions but in-spite of numerous studies its physiological function in humans has not been defined. We compared data on non-synonymous single nucleotide variations (nsSNVs) and loss-of-function variants on TGM1-7 and F13a from the Exome aggregation consortium dataset, and used computational and biochemical analysis to reveal the roles of damaging nsSNVs of TGM2. TGM2 and F13a display rarer damaging nsSNV sites than other TGMs and sequence of TGM2, F13a and TGM1 are evolutionary constrained. TGM2 nsSNVs are predicted to destabilize protein structure, influence Ca2+ and GTP regulation, and non-enzymatic interactions, but none coincide with conserved functional sites. We have experimentally characterized six TGM2 allelic variants detected so far in homozygous form, out of which only one, p.Arg222Gln, has decreased activities. Published exome sequencing data from various populations have not uncovered individuals with homozygous loss-of-function variants for TGM2, TGM3 and TGM7. Thus it can be concluded that human transglutaminases differ in harboring damaging variants and TGM2 is under purifying selection suggesting that it may have so far not revealed physiological functions.

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).

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII.

UNLABELLED: Essentials Factor XIIIa inhibits fibrinolysis by forming fibrin-fibrin and fibrin-inhibitor cross-links. Conflicting studies about magnitude and mechanisms of inhibition have been reported. Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots. Cross-links of α2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot. SUMMARY: Background Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links α2 -antiplasmin to fibrin, the topic remains a matter of debate. Objective To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of α2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of α2 -antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of α2 -antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction.