Bradykinin: Inflammatory Product of the Coagulation System

The available genetic evidence of HAE-related mutations clearly points towards a central role of the plasma contact system in this disease. Most HAE patients have SERPING1 gene mutations (encoding for C1-inhibitor (C1-INH) production) [14, 15] while a small minority have mutations in the F12 gene, with normal C1-INH activity [16‐20].

Hereditary angioedema is hallmarked by recurrent attacks of angioedema. Attacks can be life-threatening when swelling compromises the airways, and extremely painful when located in the intestine [21, 22]. Therapy targeting the contact system has been successful in HAE, strongly supporting the concept that angioedema is mediated via bradykinin production [23‐25]. Evidence for bradykinin involvement in angioedema is not limited to HAE. First, a comparable phenotype can be observed in patients that have acquired C1-INH deficiency due to underlying auto-immune or lymphoproliferative disease [26, 27]. Second, anti-hypertensive drugs that inhibit bradykinin breakdown, such as angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPPIV) or neprilysin (NEP), can induce angioedema. During clinical trials of NEP inhibitors [28], up to 2.17 % of patients and 0.2–0.65 % of patients prescribed ACE inhibitors developed angioedema [29, 30].

Evidently, contact activation is closely linked to the coagulation system [31‐33]. Activation of coagulation and fibrinolysis during HAE attacks has been repeatedly reported [34‐47]. Yet, HAE patients present with swellings but not with thrombotic tendency [37]. Combined genetic and clinical findings suggest that a subset of coagulation factors are actively involved in angioedema attacks.

Coagulation factors are readily available throughout the blood circulation to initiate fibrin formation and reinforce platelet plugs at sites of injury. These interactions are essential to ensure a properly functioning hemostatic system (Fig. 1). This system consists of a set of precursor proteins (zymogens) that circulate in the blood and has to be activated to become biologically active. The key initiator of the coagulation system, tissue factor (TF), is normally not present in the circulation. Cells that surround the vessel wall express TF so that only when the endothelial layer is compromised will locally active coagulation take place [48, 49]. After binding to TF, activated factor VII (FVII) of the extrinsic pathway activates factor X (FX), and to a lesser extent factor IX (FIX) [50]. Activated FX next triggers the formation of a small amount of active factor II (thrombin). Thrombin accelerates coagulation via positive feedback mechanisms. Factor VIII (FVIII), activated by thrombin, forms a complex together with FIX that strongly increases additional FXa and thrombin generation. Thrombin activates factor V ( FV) that, in a similar manner, contributes to thrombin formation in complex with FXa. Furthermore, FXI of the intrinsic pathway is also activated by thrombin [51], and additional FIX is being amplified by thrombin activation. Ultimately, thrombin converts fibrinogen into fibrin. Fibrin strands are reinforced through cross-linking by activated factor XIII (FXIII) (also activated by thrombin). The fibrin lattice, together with platelet aggregates and trapped red blood cells, forms a thrombus to seal the damaged area and prevent further haemorrhage. The initiator of this cascade of events, TF, is of vital importance since TF deficiency is lethal and incompatible with normal embryonic development or may cause early death in the perinatal period [52].

As formerly discussed, the most important function of FXII appears to be activation of PPK. Besides that, FXII is also capable of activation of plasminogen [33, 64, 65]. Compared to tPA and uPA, FXIIa is a relatively weak plasminogen activator. However, population studies have proposed that FXIIa may protect against cardiovascular disease via plasminogen activation [66, 67]. This may be attributable to additional interactions between the contact system and the fibrinolytic system: FXIIa can enzymatically inactivate PAI-1 [68], whereas PK stimulates uPA activation on the endothelium [65].

Efforts to identify the initial spark of contact activation focused on the presence of in vivo negatively charged compounds (i.e. glycosaminoglycans, misfolded protein aggregates, etc.). The discovery that plasmin can enzymatically activate FXII, in the absence of a surface, provides an alternative mechanism for this activation process. The importance is underscored by observations in HAE and patients receiving r-tPA therapy. Under physiological circumstances, this mechanism may take place at sites of vascular obstruction, or slow perfusion, where the tissues are endangered by hypoxia. Alternatively, minor tissue trauma or infection may provide signals to the vascular endothelium, resulting in local sequestration and activation of the contact system. We postulate that FXII functions as a ‘sensor molecule’ that interacts with the environment to detect conditions where increased vasopermeability is required. Excitingly, it is possible that a variety of enzymes other than plasmin can fulfil a similar role and contribute to FXII activation.

Recent work shows that HAE attacks are associated with increasing neutrophil counts and neutrophil elastase levels [96]. Neutrophils are important cells for the innate immunity. They migrate out of the bloodstream within hours after a pathogen is detected. Migration of neutrophils to sites of inflammation is orchestrated by several mechanisms, such as chemotaxis by interleukins and complement factors and interaction of neutrophils with endothelial cell receptors [97]. It can be envisioned that neutrophils interact with the contact system to boost neutrophil extravasation by bradykinin-mediated vasodilatation. First of all, neutrophil elastase, released by active neutrophils, inactivates C1-INH thereby allowing contact activation to take place [98]. Second, in vitro studies show that the bradykinin B1 receptor (BKB1R) regulates neutrophil trafficking [99, 100]. Third, both kallikrein and FXIIa can induce neutrophil degranulation [101]. Finally, neutrophil extracellular traps (NETs) have been shown to activate FXII. NETs exist of DNA, and the negative charge of DNA is believed to induce auto-activation of FXII [102]. At the very least, NETs can sequester FXII and present it for activating cleavage. NETs are a binding site for antimicrobial proteins such as histones, neutrophil elastase and cathepsins [99]. To what extent the proteins loaded on NETs contribute to neutrophil-induced FXII activation is not yet elucidated. The negative charge of NETs, in combination with potential FXII-activating enzymes, would make neutrophils a plausible platform for contact activation. Further research to neutrophil and FXII activation may help understand its possible relevance for HAE patients.

Angioedema as a symptom of anaphylaxis and chronic spontaneous urticaria is likely to be mediated by mast cell/basophil activation. Histamine is the central chemical mediator released by mast cells, inducing hyperpermeability and vasodilatation, and is therefore held responsible for the angioedema seen in allergic responses. Indeed, anti-histamine therapy reduces the occurrence of angioedema in urticaria patients [2]. It should be noted that plasma bradykinin levels were not found elevated in four patients with an acute attack of anti-histamine-sensitive angioedema [103]. Yet, evidence accumulates that angioedema in allergic reactions is also accompanied by bradykinin release. FXIIa-C1-INH complexes and kallikrein-C1-INH complexes were increased up to tenfold, within minutes after experimental insect stings in six allergic patients who developed shock or angioedema [1]. In contrast, patients who only developed urticarial rash in reaction to the venom showed a non-significant increase in C1-INH complexes. In line with these results, it was shown that in the patients with angioedema or shock, a third up to half of the total pool of plasma HK was cleaved within minutes [1]. These results were confirmed in another study where HK was analysed in patients with an anaphylactic reaction (mainly induced by food allergens) [104]. In this study, even patients with a mild reaction (i.e. gastro-intestinal complaints) showed 60 % cleavage of their HK pool. Evidence of the clinical relevance of bradykinin during anaphylaxis with hypotension was further demonstrated in animal models. Hypotension after IgE-mediated, antigen-induced anaphylaxis was reduced in FXII-deficient mice compared to wild-type mice [104]. The same protection toward IgE-mediated hypotension was reported in BKB2R knockout mice and HK- and PPK-deficient mice [105]. Although direct release of bradykinin from HK by tryptase could also contribute to these findings, it would be reasonable to propose that bradykinin production during anaphylaxis is for a major part FXII-driven.

Moreover, mast cell degranulation may trigger FXII activation via the release of heparin, which activates FXII in vitro and induces FXII-dependent hypotension in mice models [10]. Heparin-induced vascular leakage in mice was diminished by BKB2R antagonist (icatibant) and exaggerated in C1-INH-deficient mice [10]. Heparin is a negatively charged compound, capable of inducing FXII auto-activation. However, like the NETs of neutrophils, heparin binds a large variety of proteins [106]. Contrary to this, a study of 14 HAE patients showed that tryptase levels do not increase during HAE attacks [107]. It might be helpful to keep in mind that in HAE patients that also suffer from allergies, attacks may be aggravated by an allergic trigger. What do these studies mean for HAE? Angioedema attacks in HAE are currently not believed to be mast cell-driven (“allergic”), and anti-histamine therapy has no effect on symptoms. So far, there is no solid evidence for an association between allergic responses and angioedema attacks. Additionally, heparin-protein interactions are usually studied in an isolated in vitro manner. However, the possibility of protein-protein interactions on heparin extracellular matrix may be of importance. In the case of FXII, both the net charge of heparin and nearby proteins might synergistically activate FXII. Further research into mast cell and FXII interactions may help to understand the pathological mechanisms behind in vivo FXII activation and angioedema.

The contribution of bradykinin to angioedema with normal levels of C1-INH (i.e. chronic spontaneous urticaria with angioedema, IH-AAE, InH-AAE, U-HAE, etc.) is still uncertain. It might be speculated that since FXII activation can result from mast cell degranulation, bradykinin may also play a supportive role in forms of angioedema that are currently classified as ‘histaminergic’, based on the presence of wheals, pruritus or (partial) response to anti-histamine therapy. Current studies were unable so far to demonstrate bradykinin or an increased HK cleaving in such patients [103, 104, 108]. Considering the strong evidence of interaction between mast cells and bradykinin production, patients with idiopathic angioedema and recurrent swellings who fail to respond to high-dose anti-histamine treatment might benefit from therapy targeting the contact activation.