Coagulation stops the bleeding at sites of vessel wall injuries, but excess coagulation is equally dangerous as it can lead to thrombosis. Blood vessel blockages can occur in the arterial and venous circulation and cause myocardial infarction, ischemic stroke, and pulmonary embolism, which collectively are the leading causes of disability and death in the industrialized world. In all of the anticoagulants widely used for the acute and prophylactic treatment of thrombosis, bleeding is a common side effect, and the initiation of any therapy to treat thrombotic disorders must always weigh these risks and benefits, considering the severity of the potential side effects. Thus, there is a growing need for the development of effective anticoagulants that ideally would not impair hemostatic ability.
A promising novel perspective for developing safe anticoagulants with reduced or no bleeding risks is the inhibition of coagulation factors XII (FXII) and XI (FXI), both proteases of the intrinsic coagulation pathway. FXII is the initiating protease of the procoagulant and proinflammatory contact system, and it drives both the intrinsic pathway of coagulation and the kallikrein-kinin system. Mice lacking FXII display reduced thrombosis in mouse models of injury-induced arterial and venous thrombosis and are protected from pathological thrombosis in cerebral ischemia. At the same time, humans naturally lacking FXII and FXII-knockout mice have a normal hemostatic capacity and do not bleed abnormally, which supports the idea that drugs targeting the protease could potentially display antithrombotic effects without significantly compromising hemostasis. Concordantly, the reduction of FXII expression by antisense oligonucleotides suppresses thrombosis in arterial and venous thrombosis mouse models and catheter thrombosis in rabbits. The inhibition of FXII by protein-based inhibitors, such as antibodies or insect- and plant-derived proteins, also reduces thrombosis in mouse, rat, rabbit, and primate models of induced arterial or venous thrombosis and shows potential avenues for therapeutic anticoagulation.
FXII-driven blood coagulation is a major challenge in cardiopulmonary bypass (CPB) surgeries in which a heart-lung machine temporarily supports the circulation. Contact between FXII and artificial surfaces, such as the oxygenator membrane or tubing, induces a conformational change that leads to the proteolytic activation of the FXII zymogen that turns on the contact system. Activation of the procoagulant intrinsic coagulation pathway and the proinflammatory kallikrein-kinin system leads to blood clotting and inflammation. Contact activation also poses a problem in extracorporeal membrane oxygenation (ECMO), a medical procedure in which an artificial lung system is used for longer periods of time as a life support for patients with severe cardiac and/or pulmonary failure. The standard strategy for suppressing contact system activation in extracorporeal circuits relies on high doses of heparin, which also inhibit proteases of the extrinsic and common coagulation pathway and thus bear an inherent risk of bleeding. Several strategies for suppressing contact activation were established in experimental models, but heparin remains the anticoagulation strategy of choice. In a recent study, Renné and co-workers showed that a human FXIIa-inhibiting antibody, 3F7 (IC 50 = 13 nM), prevents clotting and thrombosis in a cardiopulmonary bypass system in rabbits without increasing therapy-associated bleeding, indicating the usefulness of targeting FXII.
FXII is implicated in several other medical conditions, including hereditary angioedema (HAE), reperfusion injury, Alzheimer’s disease, and multiple sclerosis, meaning that potential FXIIa inhibitors could be used in a variety of treatments not limited to thrombosis prevention. In Type III HAE, for example, a mutation in FXII facilitates its activation, leading to excessive bradykinin release that causes edema. An improved variant of the above-mentioned FXIIa inhibitory antibody 3F7 has entered a phase I clinical evaluation for the treatment of HAE.
While high-affinity protein-based FXIIa inhibitors were successfully generated over the last years, the development of synthetic, small-molecule inhibitors has been more challenging. Small molecules have a number of strengths that make them attractive for drug development, including a uniform composition, efficient tissue penetration, high stability, low immunoreactivity, and ease of production by chemical synthesis. The best small molecule FXIIa inhibitors reported to date are the coumarin derivative 44 (IC 50 = 4.4 μM) and H-D-Pro-Phe-Arg-chloromethylketone (PCK; IC 50 = 0.18 μM). While they have proven to be useful as research compounds in FXIIa inhibition studies, their covalent inhibition mechanism and their moderate potency and selectivity limit their drug development potential.
We recently identified high-affinity FXIIa inhibitors based on cyclic peptides using phage display, including the bicyclic peptide FXII618 (K i = 8.1 nM). In previous work, the substitution of individual amino acids in FXII618 to unnatural ones further improved the affinity, though due to their limited binding to animal FXIIa homologs and low proteolytic stability in plasma, these particular inhibitors could not be evaluated in vivo. For this reason, we describe herein the further improvement of the inhibitor to achieve sub-nanomolar potency towards human, mouse, and rabbit FXIIa, and we enhanced the stability in plasma to several days (half-life in human plasma ex vivo). These properties allowed for the pre-clinical evaluation of the inhibitor in two animal models. This work shows
