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

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.

Double Heterozygous Mutations (Cys247Tyr and 252delAsn) Cause Factor XII Deficiency in a Chinese Family

“…15 There were other three mutations reported in this region: Gly259Glu, 15 Arg267Gly, 15 and Trp268Arg. 16 In vitro expression study of Gly259Glu and Arg267Gly indicated that an extensive intracellular degradation of these two mutations occurred in the pre-Golgi compartment, resulting in insufficient secretion. 15 Since Cys247 and Asn252 were adjacent to Gly259/Arg267, we speculated that the reduction mechanism of FXII:C and FXII:Ag caused by Cys247Tyr and 252delAsn was the same as that of Gly259Glu/Arg267Gly.…”

Factor XII’s autoactivation and cell biology interdigitate in disease states

“…The nexus of FXII as a both a serine protease zymogen and a mediator of cell biology related to inflammation and leukocyte biology comes together in several novel recent reports from the Maas laboratory in Utrecht on a familial hereditary autoinflammatory disease. [38][39][40] These reports describe a four-generation family with an autosomal dominant cold-induced urticaria associated with chills, headache, and malaise. 38 On whole genome sequencing a novel p.W268R (T859A) mutation was found in the kringle region (exon 9) of FXII.…”

“…Hofman et al show that in an experimental cell system in HEK293 cells, FXII W268R is activated intracellularly and is then secreted into medium. 39 Presumably the "secreted" W268R FXIIa activates PK allowing for PKa-C1INH complexes, W268R FXIIa-C1INH complexes, cHK, and BK liberation. It is not entirely clear how the intracellular FXII W268R becomes activated, but it may proceed by autoactivation.…”

“…The investigators made great efforts to demonstrate an intracellular protease, but the studies were negative. 39 The W268R mutation in FXII itself (a) uncovers a cryptic plasminogen binding site possibly upstream to the mutation and (b) exposes the active site of FXII for easier access for activation (Figure 2). 39 In support of that assessment, Clark et al from the same laboratory show that a FXII mutant of fibronectin type II (FXII Δ1-71) is more susceptible to autoactivation and cleavage by PKa than wild type FXII.…”

“…39 The W268R mutation in FXII itself (a) uncovers a cryptic plasminogen binding site possibly upstream to the mutation and (b) exposes the active site of FXII for easier access for activation (Figure 2). 39 In support of that assessment, Clark et al from the same laboratory show that a FXII mutant of fibronectin type II (FXII Δ1-71) is more susceptible to autoactivation and cleavage by PKa than wild type FXII. 40 These data indicate that the fibronectin type II domain shields the activation loop of FXII (compare Figures 1 and 2).…”