Better with poorly performing fibrin(ogen)

In this issue of Blood, Hur et al¹ demonstrate that nonpolymerizable fibrinogen, Fga^EK, can preserve hemostatic function without promoting occlusive thrombosis. These findings shed new light on the role of fibrin and fibrin polymerization in thrombosis and hemostasis, with potential implications for developing safer therapeutic agents for thrombotic diseases.

Fibrin clots form through thrombin-mediated conversion of fibrinogen into fibrin (see figure).² In this process, thrombin enzymatically cleaves FpA and FpB from fibrinogen, exposing “A” knobs (GPR; high affinity) and “B” knobs (GHR; low affinity) at the N-termini of the α and β chains located within the central E region. These “A” and “B” knobs subsequently interact with holes “a” and “b” in the γ- and β-nodules, respectively, resulting in the polymerization of monomeric fibrin into an extensive fiber network. Activated coagulation factor XIII (FXIII) crosslinks the nascent fibrin fiber, bolstering its stability and resistance to fibrinolysis. This dense fibrin clot seals the damaged tissue and halts blood loss. Once bleeding is under control, fibrin recruits inflammatory cells, initiates the fibrinolytic pathway, and contributes to the wound-healing process.² In both human and mice, complete lack of fibrinogen leads to severe bleeding, lack of platelet aggregation, absence of FXIII activation, delayed wound healing, and an elevated risk of perinatal death, as reported in Fga^−/− mice.³ 

Given the vital role of fibrin polymerization in hemostasis and wound healing, the findings by Hur et al revealing that Fga^EK mice, which are incapable of generating polymerized fibrin, display normal hemostasis, platelet aggregation, and wound-healing responses are surprising. Several factors may contribute to the ability of Fga^EK to preserve hemostasis and wound healing. Although Fga^EK cannot support fibrin polymerization, it contains a functional FpB that can be cleaved by thrombin. Once the "B" knob is exposed, Fga^EK can form multimeric complexes through the “B”:“b” knob-to-hole interaction and the intermolecular αC-domain:αC-domain interaction as well as support platelet aggregation through γ404-411 and FXIII activation through the αC-domain.² Hence, the results from this report support the notion that fibrin, rather than fibrin polymerization per se, is necessary for maintaining hemostatic and wound-healing responses, as long as the "B" knob remains functional.

Fibrinogen is believed to exist in an inert state with low reactivity in light of its high circulating concentration (2-4 mg/mL). Thrombin-mediated removal of FpA and FpB transforms fibrinogen into a reactive molecule capable of interacting with various protein and nonprotein ligands, including the αC-domain of another fibrin(ogen), tPA, plasminogen (Plg), FXIII, Mac-1, α_Vβ₃, and more (see figure). For instance, tPA's catalytic efficiency for plasminogen is significantly enhanced 100- to 1000-fold in the presence of fibrin,⁴ and patients with congenital afibrinogenemia exhibit impaired FXIII activation.⁵ In view of the pivotal roles of fibrin in these processes, it is intriguing that Fga^EK mice exhibit normal FXIII activation, fibrinolysis, and wound-healing responses.

One plausible explanation is the functional FpB present in nonpolymerizable Fga^EK, which can facilitate a conformational switch when cleaved by thrombin. FpB is known to interact with the αC-domain, potentially masking binding sites within the αC-domains and the βN-domain⁶ (see figure). Removing FpB leads to the dissociation of the intramolecular αC-domain:αC-domain complex, thereby exposing cryptic sites within these domains for tPA, Plg, FXIII, α_Vβ₃, and others. Once released from its intramolecular interaction, the αC-domains are available to form intermolecular interactions between different fibrinogen molecules.⁷ Additionally, the unmasked cryptic binding sites within the fibrin βN-domain can facilitate fibrin binding to the endothelium via VE-cadherin and VLDLR.⁶ One potential exception to this model is the cryptic binding site for Mac-1 located within the γ-module. Mac-1 is known to preferentially recognize fibrin, and its interaction with fibrin plays a crucial role in the pathogenesis of several cardiovascular and neurological diseases.⁸ Disrupting Mac-1 binding to fibrin, by mutating the Mac-1-recognition motif γ377-395 within the γ-module (the Fibγ^390-396A mice), mitigates most fibrin-associated pathology.⁸ Curiously, Fga^EK mice exhibit compromised antimicrobial host defense, which mirrors the defective host defense phenotype observed in Fibγ^390-396A mice.⁹ The similarity in their antimicrobial responses between the Fga^EK and Fibγ^390-396A mice suggests that the Mac-1 binding site within Fga^EK fibrin likely remains cryptic, a notion that warrants further investigation.

In summary, this report highlights the potential of targeting fibrin polymerization, rather than the blood coagulation cascade or platelet function, as a means to strike a delicate balance between thrombosis prevention and hemostasis maintenance. It also showcases the feasibility of harnessing the unique properties of nonpolymerizable Fga^EK as a promising antithrombotic agent. These include its poor capability to form a dense fibrin network yet its retention of other functionalities to support platelet aggregation, endothelial cell binding,^2,6 interactions with various protein and nonprotein ligands, and facilitation of fibrinolysis. Fortuitously, the Mac-1 binding site within this nonpolymerizable fibrinogen appears to remain concealed even after thrombin-mediated activation, potentially reducing its pathological activity in various diseases.

Previous research has explored strategies targeting fibrin polymerization, such as the use of knob-mimic peptides (GPRP and GHRP) to suppress fibrin polymerization. However, their low affinity and short circulating half-life, combined with high concentrations of fibrinogen in circulation, renders them unsuitable as antithrombotic drugs. Compared with these small peptides, Fga^EK could be more efficient at suppressing fibrin polymerization, as it can cap the growing end of fibrin polymer, thereby terminating the polymerization process. Indeed, Fga^WT/EK mice, which carry both normal (WT) and nonpolymerizable (Fga^EK) fibrinogen, experience a significant reduction in thrombosis incidence and thrombus size while retaining normal hemostasis. Furthermore, human patients with dysfibrinogenemia (fibrinogen Detroit, Metz, Frankfurt XIII, etc), all of which harbor a mutation at the thrombin cleavage site Aα(16)Arg, are either asymptomatic or only suffer mild bleeding.¹⁰ These clinical insights suggest that nonpolymerizable fibrinogen can be used safely in human patients to mitigate thrombotic tendencies.

It will undoubtedly be a long road to translate nonpolymerizable fibrinogen into a safe and efficient antithrombotic agent. Nonetheless, this work offers a glimmer of hope that the elusive balance between preventing thrombosis and maintaining hemostasis may be attainable in the future.

Conflict-of-interest disclosure: The author declares no competing financial interests.