Fig 2: FXII is expressed by skin and blood neutrophils in FACAS.a Representative image from serial sections of the lesional skin of FACAS patient ca. 1 h after cold exposure. FXII immunoreactivity is restricted to neutrophils at perivascular sites and within blood vessels (white arrows), n = 2 technical replicates. b Exemplary images from cytospins isolated from the peripheral blood of FACAS patients and healthy control subjects. Only single FACAS neutrophils (0.72%), but not healthy control neutrophils (0%), present with double-positive cytoplasmatic staining for FXII and MPO. Original magnification ×400, n = 3 technical replicates. c Peripheral blood mononuclear cells were isolated from healthy control subjects by density-gradient centrifugation and stimulated with increasing doses of bradykinin. IL-8 secretion was assessed by ELISA. Pooled data of n = 3 different donors, n = 2 technical replicates. The graph shows individual data points and the mean ± s.e.m. Statistical significance is indicated by one-way ANOVA with Sidak’s multiple comparisons. Source data are provided as a source data file.

Fig 3: Phenotype and genotype in patients with FXII-associated cold autoinflammatory syndrome (FACAS).a Urticarial rash of the face of a 9-year-old girl and the leg of her 35-year-old mother. b Skin histopathology reveals dermal edema (H.E. yellow arrow) and perivascular infiltrates composed of macrophages (CD163) and few intravascular neutrophils (myeloperoxidase [MPO], yellow arrow). Original magnification ×400 (n = 2 technical replicates). c Family pedigree over four generations identifying affected (shaded black) and unaffected (unshaded) individuals. Exome sequenced individuals are underlined. Where conducted, the results of Sanger sequencing of p.W286R are shown to top left of each individual whereby “+” denotes heterozygote for variant and wild-type, and “-” denotes healthy reference. d Representative sequencing chromatogram of the heterozygous genotype in an affected individual (top) and chromatogram of healthy individual from the same region (bottom).

Fig 4: FXII W268R induces and increases IL-1ß secretion and expression.a–c Peripheral blood mononuclear cells were isolated from healthy control subjects by density-gradient centrifugation. Interleukin-1ß (IL-1ß) secretion requires a two-step process: (i) transcriptional upregulation of pro-IL-1ß via toll-like receptors and (ii) conversion of pro-IL-1ß into its active form by inflammasome activation. Cells were pre-stimulated with toll-like receptor agonist lipopolysaccharide (LPS), and nucleotide binding like receptor protein 3 (NLRP3) inflammasome activation was induced by the danger molecule ATP (LPS + ATP = blue symbols). Red symbols denote LPS-stimulated cells only, and green symbols represent ATP-activated cells without priming by LPS. Experiments were performed in the presence of FXII-depleted plasma (control) and addition of recombinant FXII wild-type (rFXII wt) and W268R (rFXII W268R) protein (a) or rFXII W268R-S544A (b). Experiments in (c) were conducted with purified FXII, FXIIa, and different doses of the small peptide-based inhibitor PPACK. Black arrows indicate priming effect by the mutant rFXII W268R (a) and the active site incapacitated rFXII W268R-S544A (b), which was absent in rFXII wt and control. Also, the presence of FXIIa (with or without PPACK) did not induce changes in IL-1ß release (c). IL-1ß secretion was assessed by ELISA. Pooled data of n = 3 different (a) or n = 2 (b, c) experiments. Bar graphs show individual data points and the mean ± s.e.m. Statistical significance in is indicated by one-way ANOVA (a, c) with Tukey’s multiple comparisons test for ATP between the groups control vs. rFXII W268R and rFXII wt vs. rFXII W268R (a) or unpaired Student’s t test (b). d, e Representative image from serial sections of the lesional skin from FACAS patient following cold exposure exhibits IL-1ß immunoreactivity. IL-1ß expression is pronounced around blood vessels (yellow arrows) and co-localizes with neutrophils (d) and macrophages (e) (white arrows). Original magnification ×400, n = 2 technical replicates. Source data are provided as a source data file.

Fig 5: FXII W268R results in fragmentation and spontaneous activation of FXII.a FXII fragmentation in FACAS plasma samples and FXII W268R mutant proteins. Citrate plasma of affected (#1, #2, #5) and non-affected (#3, #4, #6) family members, recombinant wild-type and W268R FXII expressed in HEK293 cells and plasma from an FXII-HAE patient was separated by SDS-PAGE (reducing blot) and immunoblotted for FXII. Red arrows at 50 kDa indicate heavy chain of cleaved FXII in the plasma of affected patients and recombinant W268R FXII (rFXII W268R), which is barely visible in non-affected and hereditary angioedema (HAE) III plasma or wild-type FXII (rFXII wt). Recombinant W268R and wild-type FXII were expressed in HEK293 cells. Black arrows point to rFXII W268R fragments observed by mass spectrometry in c, n = 5 technical replicates. b Immunoblot (reduced conditions) of recombinant mutant proteins (in the presence or absence of surface activator kaolin) and controls (rFXII wt and purified aFXIIa). In addition to 50 kDa (heavy chain, HC) and 25 kDa (light chain, LC) bands, a further band appears at 60 kDa (II) that is absent in rFXII wt and aFXIIa. Black arrows point to rFXII W268R fragments observed by mass spectrometry in c, n = 3 technical replicates. c Schematic presentation of FXII W268R procession. Red arrows point to mutation at position W268R. Proteolytic sites are indicated by blue arrows. Cleavage after position 353 results in FXIIa W268R HC (50 kDa) and FXIIa W268R LC (25 kDa) fragments. Additional cleavage results in a truncated FXII W268R fragment (60 kDa) cleaved at position 447. d FXIIa activity was measured by a chromogenic assay demonstrating considerable spontaneous FXIIa activity in plasma of affected individuals (n = 3), but less in healthy donors (n = 3 healthy family members). e Spontaneous FXIIa activity was also observed in culture supernatants from HEK293 cells expressing W268R, but not wild-type FXII. Graphs show individual data points and the mean ± s.e.m. Statistical significance is indicated by unpaired Student’s t test with Welch’s correction (d, e). Source data are provided as a source data file.