Contact system sends defensins to the rescue

In this issue of Blood, Abu-Fanne et al identify a link between activation of the contact system of coagulation involving factor XIIa (FXIIa) and kallikrein, promoting neutrophils to release the antimicrobial peptide α-defensin-1, which enhances fibrin polymerization kinetics, alters fibrin morphology, and inhibits fibrinolysis. These reactions may serve as an extension or consequence of innate immunity and are shown to affect in vivo murine thrombosis.¹ 

The contact factors, including FXI, FXII, high-molecular-weight kininogen, and plasma prekallikrein, serve as a nidus connecting the activation of coagulation to inflammation and have long been suspected of playing a role in the pathologic host response seen in sepsis. As contact activation contributes to a number of proinflammatory and procoagulant effects, and its absence does not significantly compromise hemostasis, inhibiting activation of the contact system may be a safe and efficacious method of attenuating the systemic inflammatory response.²  To date, much of the focus has centered on understanding the triggers of contact activation, including bacterial components such as long-chain polyphosphates and neutrophil extracellular traps, and the consequence of contact activation on thrombin generation, fibrin formation, and thrombosis.^3-6  However, it was unknown whether a mechanism exists by which contact activation directly stimulates the procoagulant activity of neutrophils, and whether subsequent neutrophil activity impeded clot resolution.

Abu-Fanne et al now elegantly demonstrate that stimulation of neutrophils with the contact activation members kallikrein and FXIIa promotes the release of the antimicrobial cationic peptide α-defensin-1 from neutrophil azurophilic granules. The secretion of α-defensin-1 was found to promote the kinetics and stability of fibrin formation, increase fibrin mass, and by incorporating into nascent fibrin clots, impede fibrinolysis. Open questions remain as to the region of fibrin(ogen) that mediates α-defensin-1 binding, as well as whether α-defensin-1 differentially regulates fibrin formation under arterial vs venous flow conditions. Regardless, this finding extends the known functions for α-defensin-1 beyond its roles as a potent microbicidal peptide and modulator of macrophage-driven inflammation, acting as a “molecular brake” for messenger RNA translation and promotion of resolution.^7,8 

As mouse neutrophils do not express α-defensins, the authors make use of transgenic mice expressing human α-defensin-1 (Def⁺⁺), the primary defensin in neutrophils.⁹  In a model of thrombus formation induced by partial ligation of the inferior vena cava, Def⁺⁺ mice were shown to have larger, neutrophil-rich occlusive clots that were resistant to heparin treatment and fibrinolysis as compared with wild-type mice. Neutrophil depletion and prolonged treatment with colchicine, an inhibitor of microtubule polymerization used in autoinflammatory diseases, inhibited release of α-defensin-1 as well as reduced thrombus formation. This suggests that inhibiting neutrophil activation and presumably α-defensin-1 release may represent an effective antithrombotic strategy to prevent immunothrombosis without compromising hemostasis. Traditional anticoagulants or newer direct oral anticoagulants might not suffice in the absence of adjuvant therapy. Use of the activated FXII inhibitor corn trypsin inhibitor was likewise antithrombotic in this model, consistent with a role for contact activation in thrombosis; yet this result does not explicitly identify the relative contribution of neutrophil α-defensin-1 release in contact activation-mediated thrombosis. It remains to be clarified to what extent α-defensin-1 plays a role in other neutrophil processes and the relative amount of α-defensin-1 released by neutrophils during thrombus formation in humans.

The large, dense fibrin clots generated in part by α-defensin-1 raise basic questions whether this process is integral to (patho)physiologic host defense mechanisms. As fibrin can act as a protective film to prevent microbes from entering and proliferating in a clot,¹⁰  α-defensin-1 enhancement of the dense fibrin network suggests that α-defensin-1 may act as an antimicrobial agent through direct and indirect physiological mechanisms. Alternatively, microbes like Staphylococcus aureus secrete coagulases, encapsulating the bacteria in fibrin to evade the immune response. Could further generation of fibrin by α-defensin-1 pathologically contribute to immune evasion or alternatively incite disseminated intravascular coagulopathy, a thrombohemorrhagic state seen in overt sepsis? The case may be that although too much is pathologic, a little is better than none at all (ie, the French red wine paradox).

In conclusion, Abu-Fanne et al present a novel mechanistic finding that contact activation stimulates neutrophil release of α-defensin-1, which exerts direct effects on fibrin polymerization and thrombus formation (see figure). It is important to note that although present in the paneth cells of the small intestine in mice, α-defensins are absent from mouse neutrophils. In contrast, α-defensins constitute 50% of the total content of azurophil granules in human neutrophils and are even still expressed in neutrophils of patients with immune-impaired diseases, including serine protease-deficient neutrophils in Papillon-Lefévre syndrome patients. Although in mice the α-defensin-1 pathway may be dispensable for the physiological function of neutrophils, it may be required for physiology in humans as an extension of innate immunity. Thus, future studies will need to mechanistically resolve whether the direct inhibition of α-defensin-1 before or after its release might be therapeutic. This exciting study provides rationale to target and inhibit contact activation, limiting immunothrombosis without compromising hemostasis and potentially preserving other immune host defense mechanisms.