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.
Keywords: autoinflammatory syndrome; bradykinin; coagulation factor; enzyme mutation; factor XII; hereditary angioedema; inflammation; kringle domain; serine protease; vascular biology.
© 2020 Hofman et al.
Conflict of interest statement
The authors declare that they have no conflicts of interest with the contents of this article
Cold-induced Urticarial Autoinflammatory Syndrome Related to Factor XII ActivationJ Scheffel et al. Nat Commun 11 (1), 179. PMID 31924766.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-generat …
The Fibronectin Type II Domain of Factor XII Ensures Zymogen QuiescenceCC Clark et al. Thromb Haemost. PMID 31940673.Factor XII (FXII) zymogen activation requires cleavage after arginine 353 located in the activation loop. This cleavage can be executed by activated FXII (autoactivation) …
Plasmin Is a Natural Trigger for Bradykinin Production in Patients With Hereditary Angioedema With Factor XII MutationsS de Maat et al. J Allergy Clin Immunol 138 (5), 1414-1423.e9. PMID 27130860.Our findings indicate a new pathway for bradykinin formation in patients with HAE, in which FXII is cleaved and activated by plasmin. This should lead to the identificati …
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