by Alisa Wolberg (University of North Carolina at Chapel Hill, USA)
Venous thrombosis/thromboembolism is a major public health crisis.
Among cardiovascular cases of death, venous thrombosis/thromboembolism (VTE) is the third most common after coronary heart disease and ischaemic stroke. Estimates suggest VTE is responsible for more than 500,000 deaths in both the European Union and United States every year.1
Venous obstruction causes venous valve damage and valvular incompetence, incomplete recanalization impedes venous outflow, and thrombi may dislodge and travel to the lungs, resulting in pulmonary embolism. Even with treatment, 30-50% of VTE patients develop recurrent VTE or suffer debilitating long-term morbidity including chronic pain, edema, and intractable venous leg ulcers (so-called “post-thrombotic syndrome”) or chronic pulmonary hypertension.2-4
VTE is triggered by intravascular activation of coagulation resulting in thrombin-mediated intraluminal fibrin deposition (reviewed in 4
). Red blood cells (RBCs) become incorporated into the fibrin network, and undergo consolidation as the thrombus contracts. This process culminates in the production of a fibrin- and RBC-rich thrombus characteristic of venous thrombi.
Current methods of anticoagulation to reduce VTE target plasma procoagulant activity via broad (e.g., vitamin K antagonists that reduce the circulating levels of γ-carboxylated clotting factors) or specific (e.g., new/direct oral anticoagulants that target factor Xa or thrombin) approaches. However, treatment with vitamin K antagonists requires frequent monitoring, and both traditional and newer anticoagulants carry a significant risk of catastrophic bleeding.4
Efforts to understand the pathophysiology of VTE and develop new approaches to safely reduce VTE are highly clinically significant.Factor XIII(a) crosslinking of fibrin α-chains mediates RBC retention in venous thrombi, and consequently, thrombus size.
Factor XIII (FXIII) is a protransglutaminase that, once activated to FXIIIa, crosslinks fibrin and other proteins to the clot. We recently investigated the contribution of FXIII to clot formation and found that FXIIIa activity is a major determinant of both clot RBC content and clot size.5,6
Briefly, we observed that in the absence of plasma FXIII, RBCs are extruded from contracting human whole blood clots, resulting in the production of small clots.5
These observations were consistent with the “excessive red cell fallout” reported many years ago in a study of a family with congenital FXIII deficiency7
, and suggested a previously-unrecognized role for FXIII(a) in mediating clot composition.
Since whole blood clots from mice with full or even partial (heterozygous) deficiency of the FXIII catalytic A subunit show similarly increased extrusion of RBCs during clot contraction5, these mice provided an experimental model to investigate the contribution of FXIII to VTE in vivo. Following inferior vena cava ligation, thrombi from FXIII-deficient mice had lower RBC content and were smaller than thrombi from WT mice5
, indicating that FXIII contributes to venous thrombus formation in vivo. Interestingly, mice carrying alanine mutations in fibrinogen residues γ390-396 (Fibγ390-396A
exhibit decreased binding of FXIII to fibrinogen, delayed FXIII activation, and delayed fibrin crosslinking, and phenocopied FXIII-deficient mice, producing smaller venous thrombi with reduced RBC content.5
These data demonstrate the importance of FXIII localization on fibrinogen for normal FXIII activation and fibrin crosslinking, and indicate that disrupting FXIII-fibrinogen interactions reduces RBC retention in clots and clot size.5
More recently, we found that FXIIIa does not directly crosslink RBCs to the clot, but rather, promotes RBC retention via its effects on the fibrin network. During coagulation, FXIIIa crosslinks residues in the fibrin(ogen) γ- and α-chains, increasing clot biochemical and biophysical stability. Using recombinant fibrinogen variants that lack γ- or α-chain crosslinking residues and FXIIIa inhibitor concentrations that preferentially block formation of α-chain-rich high-molecular weight crosslinked species but not γ-γ dimers, we showed that the effect of FXIIIa on RBC retention is mediated by its ability to crosslink fibrin α-chains.6
Together, these findings reveal a previously undescribed role for FXIII-fibrinogen interactions in VTE, and suggest the FXIII(a)-fibrinogen axis is a novel therapeutic target for reducing VTE.Implications.
These findings raise intriguing questions about the possibility of targeting FXIII(a) to reduce VTE. Notably, FXIII has a wide normal range in humans, and neither FXIII-heterozygous mice (50% FXIII) nor Fibγ390-396A
mice show signs of excessive bleeding5,8,9
, suggesting there may be a therapeutic window in which inhibiting FXIII does not significantly increase bleeding risk. In vitro experiments show that treating normal human blood with a transglutaminase active site inhibitor dose-dependently increases RBC extrusion from contracting clots and reduces clot size5
, providing proof-of-concept for this approach. However, it remains unclear whether FXIIIa inhibition is a viable approach for anticoagulation in vivo. At present, these studies are hindered, in part, by the lack of specific FXIIIa inhibitors with appropriate pharmacokinetic and pharmacodynamic characteristics for in vivo experiments. Efforts to generate these molecules are underway.
These observations also have important implications for understanding fibrin function during coagulation. These findings appear to unite observations on the (patho)physiologic effects of FXIIIa during thrombosis with decades of elegant studies elucidating the remarkable biophysical characteristics of fibrin fibers. Fibrin has impressive extensibility akin to that seen in spider silk, with the ability of fibers to be stretched over 2.5 times and recover elastically.10
Notably, these characteristics are strongly influenced by fibrin α-chain crosslinking, which increases fibrin fiber elasticity and stiffness.11
Thus, fibrin networks may require stiff fibers to retain RBCs within the contracting thrombus. It is therefore interesting to consider that in addition to maintaining clot integrity during blood flow and wound healing, crosslinked fibrin is also critical for determining thrombus composition (RBC content). The potential impact of thrombus RBC content on thrombus resolution remains to be determined.
Further studies of mechanisms mediating both individual fibrin fiber and whole clot integrity are likely to reveal new information on the contribution of fibrin crosslinking to hemostasis and thrombosis, and the feasibility of modulating this activity to decrease VTE.
- Thrombosis: a major contributor to the global disease burden. J Thromb Haemost. 2014;12:1580-1590.
- Heit JA. Venous thromboembolism epidemiology: implications for prevention and management. Semin Thromb Hemost. 2002;28 Suppl 2:3-13.
- Ashrani AA, Heit JA. Incidence and cost burden of post-thrombotic syndrome. J Thromb Thrombolysis. 2009;28:465-476.
- Wolberg AS, Rosendaal FR, Weitz JI, Jaffer IH, Agnelli G, Baglin T, Mackman N. Venous thrombosis. Nat Rev Dis Pri. 2015;1:http://www.nature.com/articles/nrdp20156.
- Aleman MM, Byrnes JR, Wang J-G, Tran R, Lam WA, Di Paola J, Mackman N, Degen JL, Flick MJ, Wolberg AS. Factor XIII activity mediates red blood cell retention in venous thrombi. J Clin Invest. 2014;124:3590-3600.
- Byrnes JR, Duval C, Wang Y, Hansen CE, Ahn B, Mooberry MJ, Clark MA, Johnsen JM, Lord ST, Lam WA, Meijers JC, Ni H, Ariens RA, Wolberg AS. Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin alpha-chain crosslinking. Blood. 2015;126:1940-1948.
- Hanna M. Congenital deficiency of factor 13: report of a family from Newfoundland with associated mild deficiency of factor XII. Pediatrics. 1970;46:611-619.
- Flick MJ, Du X, Witte DP, Jirouskova M, Soloviev DA, Busuttil SJ, Plow EF, Degen JL. Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-1 is critical for host inflammatory response in vivo. J Clin Invest. 2004;113:1596-1606.
- Lauer P, Metzner HJ, Zettlmeissl G, Li M, Smith AG, Lathe R, Dickneite G. Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit. Thromb Haemost. 2002;88:967-974.
- Liu W, Jawerth LM, Sparks EA, Falvo MR, Hantgan RR, Superfine R, Lord ST, Guthold M. Fibrin fibers have extraordinary extensibillity and elasticity. Science. 2006;313:634.
- Helms CC, Ariëns RA, Uitte de Willige S, Standeven KF, Guthold M. alpha-alpha cross-links increase fibrin fiber elasticity and stiffness. Biophys J. 2012;102:168-175.
Prof. Alisa Wolberg is an Associate Professor in the Department of Pathology and Laboratory Medicine at the University of North Carolina at Chapel Hill. Prof. Wolberg’s research interests focus on (patho)physiologic mechanisms that contribute to hemostasis and thrombosis, with a particular focus on mechanisms that mediate fibrin formation, structure, and stability.