The Fibronectin Type II Domain of Factor XII Ensures Zymogen Quiescence.

Factor XII (FXII) zymogen activation requires cleavage after arginine 353 located in the activation loop. This cleavage can be executed by activated FXII (autoactivation), plasma kallikrein (PKa), or plasmin. Previous studies proposed that the activation loop of FXII is shielded to regulate FXII activation and subsequent contact activation. In this study, we aimed to elucidate this mechanism by expressing and characterizing seven consecutive N-terminally truncated FXII variants as well as full-length wild-type (WT) FXII. As soon as the fibronectin type II domain is lacking (FXII Δ1-71), FXII cleavage products appear on Western blot. These fragments display spontaneous amidolytic activity, indicating that FXII without the fibronectin type II domain is susceptible to autoactivation. Additionally, truncated FXII Δ1-71 is more easily activated by PKa or plasmin than full-length WT FXII. To exclude a contribution of autoactivation, we expressed active-site incapacitated FXII truncation variants (S544A). FXII S544A Δ1-71 is highly susceptible to cleavage by PKa, indicating exposure of the activation loop. In surface binding experiments, we found that the fibronectin type II domain is non-essential for binding to kaolin or polyphosphate, whereas the following epidermal growth factor-like domain is indispensable. Binding of full-length FXII S544A to kaolin or polyphosphate increases its susceptibility to cleavage by PKa. Moreover, the activation of full-length WT FXII by PKa increases approximately threefold in the presence of kaolin. Deletion of the fibronectin type II domain eliminates this effect. Combined, these findings suggest that the fibronectin type II domain shields the activation loop of FXII, ensuring zymogen quiescence.

Cold-induced urticarial autoinflammatory syndrome related to factor XII activation.

Hereditary autoinflammatory diseases are caused by gene mutations of the innate immune pathway, e.g. nucleotide receptor protein 3 (NLRP3). Here, we report a four-generation family with cold-induced urticarial rash, arthralgia, chills, headache and malaise associated with an autosomal-dominant inheritance. Genetic studies identify a substitution mutation in gene F12 (T859A, resulting in p.W268R) which encodes coagulation factor XII (FXII). Functional analysis reveals enhanced autocatalytic cleavage of the mutated protein and spontaneous FXII activation in patient plasma and in supernatant of transfected HEK293 cells expressing recombinant W268R-mutated proteins. Furthermore, we observe reduced plasma prekallikrein, cleaved high molecular weight kininogen and elevated plasma bradykinin. Neutrophils are identified as a local source of FXII. Interleukin-1ß (IL-1ß) is upregulated in lesional skin and mononuclear donor cells exposed to recombinant mutant proteins. Treatment with icatibant (bradykinin-B2-antagonist) or anakinra (interleukin-1-antagonist) reduces disease activity in patients. In conclusion, our findings provide a link between contact system activation and cytokine-mediated inflammation.

Evidence for bradykinin release in chronic spontaneous urticaria.

BACKGROUND: Chronic spontaneous urticaria (CSU) is characterized by recurrent itchy weals and/or angioedema and is believed to be driven by mast cell activation. It was shown that excessive mast cell activation during anaphylaxis initiates contact activation, resulting in bradykinin release. Evidence for bradykinin release was never demonstrated in CSU. OBJECTIVE: To study biomarkers of bradykinin release in CSU. METHODS: Plasma samples of CSU patients were collected during routine visits at the outpatient clinic. Cleaved high molecular weight kininogen (cHK) was used as a biomarker for bradykinin release. cHK, factor XIIa-C1-inhibitor (FXIIa-C1-INH), kallikrein-C1-INH, plasmin-antiplasmin (PAP) complexes and soluble urokinase-type plasminogen activator receptor (suPAR) levels were determined by ELISA. Clinical data and data on tryptase levels were collected from medical records. cHK levels were compared to previously determined levels in hereditary angioedema (HAE). RESULTS: One hundred seventeen samples from 88 CSU patients and 28 samples from healthy controls were analysed. Median cHK level in CSU was 9.1% (range: 1.4%-21.5%), significantly increased compared to healthy controls (median 6.0% range: 0%-19.9%; P = .0005) and comparable to HAE (n = 46, median 10.3%, range 0%-44.3%, P > .9999). cHK levels normalized in patients during disease remission (median 6.5% range 1.5%-20.8%) but were not dependent on the presence of angioedema, acute angioedema attacks or response to antihistamines. Surprisingly, cHK levels were inversely correlated to serum tryptase (r = -0.65 P = .0137). C1-INH complexes and suPAR levels were not elevated in patients compared to healthy controls. PAP-complex levels in patients were elevated compared to healthy controls but there was no correlation between PAP-complex and cHK levels. CONCLUSIONS: cHK levels are elevated in symptomatic CSU patients compared to healthy controls, indicating increased bradykinin production. Increased cHK levels are not limited to patients with angioedema. CLINICAL RELEVANCE: If elevated bradykinin generation has clinical implications in the pathology of CSU is open to debate.

A mutation in the kringle domain of human factor XII that causes autoinflammation, disturbs zymogen quiescence, and accelerates activation.

Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin. Pathological mutations in the F12 gene, which encodes FXII, provoke acute tissue swelling in hereditary angioedema (HAE). Interestingly, a recently identified F12 mutation, causing a W268R substitution, is not associated with HAE. Instead, FXII-W268R carriers experience cold-inducible urticarial rash, arthralgia, fever, and fatigue. Here, we aimed to investigate the molecular characteristics of the FXII-W268R variant. We expressed wild type FXII (FXII-WT), FXII-W268R, and FXII-T309R (which causes HAE), as well as other FXII variants in HEK293 freestyle cells. Using chromogenic substrate assays, immunoblotting, and ELISA, we analyzed expression media, cell lysates, and purified proteins for FXII activation. Recombinant FXII-W268R forms increased amounts of intracellular cleavage products that are also present in expression medium and display enzymatic activity. The active site-incapacitated variant FXII-W268R/S544A reveals that intracellular fragmentation is largely dependent on autoactivation. Purified FXII-W268R is highly sensitive to activation by plasma kallikrein and plasmin, compared with FXII-WT or FXII-T309R. Furthermore, binding studies indicated that the FXII-W268R variant leads to the exposure of a plasminogen-binding site that is cryptic in FXII-WT. In plasma, recombinant FXII-W268R spontaneously triggers high-molecular-weight kininogen cleavage. Our findings suggest that the W268R substitution influences FXII protein conformation and exposure of the activation loop, which is concealed in FXII-WT. This results in intracellular autoactivation and constitutive low-grade secretion of activated FXII. These findings help to explain the chronically increased contact activation in carriers of the FXII-W268R variant.

High occurrence of antihistamine resistance in patients with recurrent idiopathic angioedema.

Antihistamines are the most prescribed therapy in recurrent idiopathic angioedema, yet little is known about their efficacy. Herein, we report on clinical improvement with antihistamine therapy in 120 patients evaluating angioedema attack frequency. A high incidence (36%) of antihistamine refractory cases was observed. Forty percent of patients on antihistamine prophylaxis suffered from 1 or more angioedema attacks per month. Our findings stress the need for additional treatment options for recurrent idiopathic angioedema.

Angioedema attacks in patients with hereditary angioedema: Local manifestations of a systemic activation process.

Hereditary angioedema (HAE) caused by a deficiency of functional C1-inhibitor (C1INH) becomes clinically manifest as attacks of angioedema. C1INH is the main inhibitor of the contact system. Poor control of a local activation process of this system at the site of the attack is believed to lead to the formation of bradykinin (BK), which increases local vasopermeability and mediates angioedema on interaction with BK receptor 2 on the endothelium. However, several observations in patients with HAE are difficult to explain from a pathogenic model claiming a local activation process at the site of the angioedema attack. Therefore we postulate an alternative model for angioedema attacks in patients with HAE, which assumes a systemic, fluid-phase activation of the contact system to generate BK and its breakdown products. Interaction of these peptides with endothelial receptors that are locally expressed in the affected tissues rather than with receptors constitutively expressed by the endothelium throughout the whole body explains that such a systemic activation process results in local manifestations of an attack. In particular, BK receptor 1, which is induced on the endothelium by inflammatory stimuli, such as kinins and cytokines, meets the specifications of the involved receptor. The pathogenic model discussed here also provides an explanation for why angioedema can occur at multiple sites during an attack and why HAE attacks respond well to modest increases of circulating C1INH activity levels because inhibition of fluid-phase Factor XIIa and kallikrein requires lower C1INH levels than inhibition of activator-bound factors.

Hereditary Angioedema Attacks: Local Swelling at Multiple Sites.

Hereditary angioedema (HAE) patients experience recurrent local swelling in various parts of the body including painful swelling of the intestine and life-threatening laryngeal oedema. Most HAE literature is about attacks located in one anatomical site, though it is mentioned that HAE attacks may also involve multiple anatomical sites simultaneously. A detailed description of such multi-location attacks is currently lacking. This study investigated the occurrence, severity and clinical course of HAE attacks with multiple anatomical locations. HAE patients included in a clinical database of recombinant human C1-inhibitor (rhC1INH) studies were evaluated. Visual analog scale scores filled out by the patients for various symptoms at various locations and investigator symptoms scores during the attack were analysed. Data of 219 eligible attacks in 119 patients was analysed. Thirty-three patients (28%) had symptoms at multiple locations in anatomically unrelated regions at the same time during their first attack. Up to five simultaneously affected locations were reported. The observation that severe HAE attacks often affect multiple sites in the body suggests that HAE symptoms result from a systemic rather than from a local process as is currently believed.

Grayanotoxin poisoning: 'mad honey disease' and beyond.

Many plants of the Ericaceae family, Rhododendron, Pieris, Agarista and Kalmia, contain diterpene grayanotoxins. Consumption of grayanotoxin containing leaves, flowers or secondary products as honey may result in intoxication specifically characterized by dizziness, hypotension and atrial-ventricular block. Symptoms are caused by an inability to inactivate neural sodium ion channels resulting in continuous increased vagal tone. Grayanotoxin containing products are currently sold online, which may pose an increasing risk. In humans, intoxication is rarely lethal, in contrast to cattle and pet poisoning cases. Scientific evidence for the medicinal properties of grayanotoxin containing preparations, such as honey or herbal preparation in use in folk medicine, is scarce, and such use may even be harmful.