The antifibrinolytic function of factor XIII is exclusively expressed through α₂-antiplasmin cross-linking

Factor XIII (FXIII) is activated by thrombin to form an active transglutaminase, FXIIIa. FXIIIa significantly alters the rheologic properties of fibrin by introducing intramolecular cross-links between fibrin strands.^1,2  A deficiency in FXIII results in bleeding, delayed wound healing and spontaneous abortion in humans and mice.^3,4  Initially FXIIIa forms a γ-γ dimer between Gln388/389 on one γ-chain of fibrin and Lys406 on another.^5,6  High molecular mass polymers of the α-chain follow⁶  with hybrid γ-α cross-links generated over prolonged periods.⁷ 

FXIIIa cross-links inhibitors of fibrinolysis to fibrin, dramatically altering its susceptibility to lysis.⁸  The most extensively characterized is α₂-antiplasmin (α₂AP), which cross-links to the Aα chain of fibrin(ogen)⁹  at Lys303 via Gln2.¹⁰  Plasminogen activator inhibitor 2 (PAI-2)¹¹  and thrombin activatable fibrinolysis inhibitor (TAFI)¹²  are also substrates for FXIIIa. Despite evidence of inhibitor cross-linking, it has been challenging to observe the role of FXIII in modulating fibrinolysis. We recently showed that thrombi formed under flow, even in the absence of cells, allows the impact of FXIII on fibrinolysis to be visualized and quantified.¹³  The thrombus model has also proved invaluable in determining the role of different inhibitors in regulating fibrinolysis.¹⁴  This study examines the contribution of fibrin-fibrin cross-links and fibrin-inhibitor cross-links in conferring resistance to fibrinolysis. We show for the first time that the role of FXIII in protecting fibrin against fibrinolytic degradation is fully explained by its ability to cross-link α₂AP into the fibrin network.

Plasma thrombi were formed in a Chandler loop as described.¹³  Briefly, FITC-labeled fibrinogen was added to pooled normal plasma (PNP) or plasma depleted of FXIII, α₂AP, TAFI or PAI (Affinity Biologicals Inc). Plasma was recalcified with 10.9mM CaCl₂ in a total volume of 0.575 mL. A nonreversible transglutaminase inhibitor, 1,3-dimethyl-2-[(2-oxopropyl) thio] imidazolium chloride (1mM)^13,15  (TG inhibitor), FXIII (0.1,0.3 or 1 U/mL Fibrogammin P; Aventis) or neutralizing antibody to α₂AP¹⁴  (150 μg/mL; Technoclone) were added in some experiments before thrombus formation. Thrombi were incubated in 10mM Tris (pH 7.5); 0.01% Tween-20 containing tissue plasminogen activator (tPA; 1 μg/mL) at 37°C. Samples (10 μL) were diluted 1/25 in 10mM phosphate (pH 7.4), 150mM NaCl, and fluorescence measured (excitation 485 nm: emission 530 nm) in a Biotek Instruments Fluorometer.

Plasma thrombi, formed as described in the preceding paragraph, were washed 3 times in 0.9% (wt/vol) NaCl before dissolving in 8M urea, 0.2M Tris (pH 8), 40mM dithiothreitol and 4% SDS at 72°C for approximately 1 hour. Samples were diluted in 0.9% NaCl and separated on 7.5% acrylamide gels before transferring to nitrocellulose and immunoblotting for fibrinogen α-chain, γ-chain (Santa Cruz Biotechnology Inc) or α₂AP (Affinity Biologicals).

Quantitative data are expressed as mean ± SEM. Data were analyzed in GraphPad Prism 5 (GraphPad Software) and shown as fluorescence units (FU) released or rates of lysis (FU/minutes), as determined by linear regression. Statistical analysis was performed by t test and Western blots were analyzed using Image J software (Version 1.44).

We examined lysis of thrombi prepared from PNP and from plasma immunodepleted of FXIII and the inhibitors, α₂AP, TAFI and PAI-1. FXIIIa can cross-link α₂AP⁸  and TAFI¹²  to fibrin, whereas PAI-1 is not a substrate.¹¹  Depletion of FXIII or α₂AP resulted in a 9-fold increase in lysis rate over PNP thrombi (Figure 1A; P < .005). Depletion of TAFI or PAI-1 did not significantly alter thrombus lysis (Figure 1A; P = .133 and P = .285, respectively). These data clearly confirm the major role of cross-linked α₂AP in down-regulating fibrinolysis. Consistent with this, addition of a TG inhibitor¹³  to thrombi formed from α₂AP depleted plasma had no effect on lysis (Figure 1B; P = .472), while in PNP a 9-fold (P < .005) increase in lysis was observed, as in Mutch et al.¹³ 

The absence of α₂AP and FXIII had strikingly similar effects on fibrinolysis. Addition of increasing concentrations of PNP normalized thrombus lysis (Figure 1C), but different concentrations were required to stabilize lysis in FXIII and α₂AP depleted plasma. The α₂AP data are consistent with previous studies on α₂AP deficiency.^16,17  Protection against premature fibrinolysis was achieved at approximately 50% plasma FXIII, as we have previously shown.¹³  We then defined the proportion of α₂AP (70 kDa) cross-linked to fibrin by Western blotting of plasma thrombi (Figure 1D). Total cross-linked α₂AP (140 kDa band and those of higher mass) was analyzed by densitometry and plotted against the mean lysis rate for each of the mixed plasma thrombi (Figure 1E). The lysis rate inversely correlated with total cross-linked α₂AP, whether thrombi were formed from FXIII or α₂AP depleted plasma (r² = 0.998; P = .383).

We then assessed the ability of FXIII to stabilize plasma thrombi in the absence of functional α₂AP by incorporating a neutralizing antibody to the inhibitor¹⁴  before thrombus formation; it should be noted that the antibody inhibits α₂AP function but not cross-linking of the inhibitor to fibrin. Reconstitution of FXIII into thrombi prepared from FXIII depleted plasma dramatically decreased the mean lysis rate, with complete stabilization of thrombi at 0.5 U/mL FXIII (Figure 2A closed circles). In contrast, when a neutralizing antibody to α₂AP was also added, no change in lysis was observed (Figure 2A open circles). We verified that the addition of FXIII had no effect on lysis of thrombi formed from α₂AP depleted plasma (Figure 2A closed triangles). Cross-linking patterns were examined by Western blot at 0 and 1 U/mL FXIII using antibodies to fibrin(ogen) and α₂AP. We confirmed that α₂AP was still cross-linked to fibrin in the presence of antibody (Figure 2B left panel). Normal fibrin-fibrin cross-links were formed when FXIII was present (Figure 2B). The key finding is that, despite the formation of normal fibrin-fibrin cross-links, the stability of the thrombi to lysis (Figure 2A) depends on FXIIIa cross-linking functional α₂AP into the thrombus.

Our data show that the ability of FXIII to modulate fibrinolysis is dependent on its action in cross-linking active α₂AP to fibrin during thrombus formation. We therefore infer that the introduction of fibrin-fibrin cross-links alone has a negligible impact on fibrinolysis. γ-dimers and α-polymers are reported to play a limited role in stabilizing clots against fibrinolytic degradation; with protection achieved only after prolonged incubation because of γ-multimer formation.⁷  These studies were performed with purified proteins in the absence of α₂AP.⁷  Our results indicate that α₂AP cross-linking determines thrombus stability in a plasma milieu.

Fibrin-fibrin cross-links, particularly α-polymers, γ-multimers and α-γ-hybrids enhance the ability of clots to resist mechanical stresses.^18,19  Together with our cross-linking data, these observations indicate that FXIII plays a dual role during clot formation (1) to strengthen individual fibrin fibers against mechanical stress and (2) to incorporate inhibitors into a forming clot to protect against premature degradation by fibrinolytic proteases.

Two forms of α₂AP exist in plasma, Met-α₂AP and Asn-α₂AP. Asn-α₂AP is produced on cleavage of Met-α₂AP by antiplasmin cleaving enzyme, and is more rapidly cross-linked to fibrin.¹⁷  We quantified α₂AP by ELISA in plasma and in the serum postthrombus formation. By subtraction, approximately 30%-50% of plasma α₂AP was bound into thrombi, consistent with other studies.^17,20  Inhibition of lysis occurs with incorporation of increasing amounts of Asn-α₂AP, demonstrating the effectiveness of cross-linked α₂AP in down-regulating fibrinolysis.¹⁷ 

This study addresses the complex question of whether fibrin-fibrin cross-links or fibrin-inhibitor cross-links regulate fibrinolysis of thrombi. We clearly show that, despite their importance in clot strength,^18,19  fibrin-fibrin cross-links do not protect against degradation by fibrinolytic proteases. Instead it is inhibitor cross-linking, specifically cross-linking of α₂AP to fibrin, that is the key determinant of fibrinolytic resistance.

This work was supported by the British Heart Foundation (PG/2001005).

Contribution: S.R.F. performed the research and analyzed data; N.A.B. supervised the research and wrote the manuscript; and N.J.M. supervised the research, analyzed data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Dr Nicola J. Mutch, School of Medicine & Dentistry, Institute of Medical Sciences, Foresterhill, University of Aberdeen, Aberdeen, AB25 2ZD, UK; e-mail: n.j.mutch@abdn.ac.uk.