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

Zonne L.M Hofman,Chantal C Clark,Wariya Sanrattana,Aziz Nosairi,Naomi M.J Parr,Minka Živkovic,Karoline Krause,Niklas A Mahnke,Jörg Scheffel,C Erik Hack,Marcus Maurer,Steven De Maat,Coen Maas

doi:10.1074/jbc.ra119.009788

Abstract

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.

Highlights

Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin
Our findings suggest that the W268R substitution influences FXII protein conformation and exposure of the activation loop, which is concealed in FXII-WT
Western blotting analyses of expression medium containing FXII-W268R showed that it is partly fragmented (Fig. 1A). This fragmentation is predominantly seen under reducing conditions and is much less pronounced during expression of FXII-WT or FXII-T309R

Summary

Introduction

Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin. Factor XII (FXII) is a 78-kDa liver-expressed serine protease zymogen that circulates in blood plasma (30 ␮g/ml; 375 nM). It consists of five N-terminal domains that are connected to the C-terminal protease domain by a proline-rich region. INH), the main plasma inhibitor of FXIIa and PKa. In addition, bradykinin-mediated HAE can be caused by mutations in the F12 gene (encodes FXII). All of these are located in the proline-rich region, a 53-amino acid sequence that is unique to FXII Three of these mutations alter the enzymatic processing of FXII during activation through introduction of cleavage sites that are sensitive to plasmin [10] and thrombin [11]. The resulting truncated FXII products display an enhanced sensitivity for activation (10 –12)

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