Recent advances in factor XII structure and function

Abstract

Purpose of review: Factor XII (FXII), the precursor of the protease FXIIa, contributes to pathologic processes including angioedema and thrombosis. Here, we review recent work on structure-function relationships for FXII based on studies using recombinant FXII variants.

Recent findings: FXII is a homolog of pro-hepatocyte growth factor activator (Pro-HGFA). We prepared FXII in which domains are replaced by corresponding parts of Pro-HGA, and tested them in FXII activation and activity assays. In solution, FXII and prekallikrein undergo reciprocal activation to FXIIa and kallikrein. The rate of this process is restricted by the FXII fibronectin type-2 and kringle domains. Pro-HGA replacements for these domains accelerate FXII and prekallikrein activation. When FXII and prekallikrein bind to negatively charged surfaces, reciprocal activation is enhanced. The FXII EGF1 domain is required for surface binding.

Summary: We propose a model in which FXII is normally maintained in a closed conformation resistant to activation by intramolecular interactions involving the fibronectin type-2 and kringle domains. These interactions are disrupted when FXII binds to a surface through EGF1, enhancing FXII activation and prekallikrein activation by FXIIa. These observations have important implications for understanding the contributions of FXII to disease, and for developing therapies to treat thrombo-inflammatory disorders.

Figure 1.. The Kallikrein-Kinin System and Contact Activation.

Shown are western blots of time courses for reactions containing FXII (200 nM), PK (300 nM) and/or FXI (30 nM) in 20mM HEPES, pH7.4, 100mM NaCl, 0.1% PEG-8000, 10 μM ZnCl₂ incubated at 37 °C. At the indicated times, samples were removed into reducing SDS-sample buffer. Samples were size fractionated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose and developed with goat polyclonal antibodies against human FXII, PK or FXI. (A) Reciprocal activation of FXII and PK in the absence of a surface. (B) Reciprocal activation of FXII and PK in the presence of 100 μg/ml leukocyte DNA. (C) Autoactivation of FXII in the absence (left panel) or presence (right panel) of 70 μM polyphosphate (60–100 unit chain length). (D) Reciprocal activation of FXII and FXI in the absence (no surface) or presence of 70 μM polyphosphate (+ polyphosphate). For all panels, positions of markers for FXII, PK and FXI, and the heavy chains (HC) and light chains (LC) of FXIIa, PKa, and FXIa are shown on the right. Blots in panel A and B are from reference , and panel C is from reference . Images in Panel D have not been previously published.

Figure 2.. Factor XII Structure.

(A) Amino acid sequence and disulfide bonds for human plasma FXII. The heavy chain contains a fibronectin type 2 (purple), epidermal growth factor 1 (blue), fibronectin type 1 (orange), epidermal growth factor 2 (green), and kringle (brown) domain, and a proline-rich regions (magenta). The catalytic triad His393, Asp442 Ser544 in the protease domain (light chain) are indicated in red. Positions of O- and N-linked glycosylation sites are indicated by blue and orange diamonds. Positions of disease-associated mutations are shown in green circles. Image adapted from reference . (B) Schematic diagrams of human FXII (white), Pro-HGFA (gray), and their activated forms FXIIa and HGFA. The FXII fibronectin type 2 (FN2), epidermal growth factor 1 (EGF1), fibronectin type 1 (FN1), epidermal growth factor 2 (EGF2), and kringle (KNG) domains, the proline-rich region (PRR), and the protease domain are indicated. Pro-HGFA is organized similarly except that it does not have a PRR, and the corresponding sequence is not assigned a name. Positions of FXII and Pro-HGFA active site serine residues (Ser544 and Ser563, respectively) are indicated by black bars, and sites for proteolytic activation (after Arg353 and Arg372, respectively) are indicated by black arrows. Image adapted from reference .

Figure 3.. Activity in Factor XII Zymogen and ΔFXII.

(A & B) FXII activity. (A) FXII and FXII-T (200 nM) were incubated in the absence of an activator (control, left column), with 70 μM Poly-P (center), or with 50 nM kallikrein without a surface (right). (B) FXII, FXII-T or FXII with the active site serine changed to alanine (FXII-S554A), 200 nM, and PK (200 nM) were incubated at 37°C. For panels A and B, at indicated time points, samples were removed into reducing sample buffer. Samples were size fractionated by SDS-PAGE, followed by western blot using a polyclonal anti-human FXII or anti-PK IgG. Positions of standards for FXII (XII) and PK and the heavy chain (HC) and light chain (LC) of FXIIa and PKa are indicated at the right of each image. Data is from reference . (C & D) ΔFXII activation and activity. (C) FXII (○), the full-length FXII precursor used to generate ΔFXII (◻), or ΔFXII (△), 200 nM, were incubated at 37°C with PKa (10 nM). FXIIa activity was measured by chromogenic substrate assay. (D) PK (200 nM) was mixed with 12.5 nM FXII (▽) or FXII-Lys309 (▼). The FXII preparations were preincubated with thrombin, which removes the heavy chain from FXII-Lys309 but not from FXII, prior to addition to PK. PKa generation was determined with a chromogenic substrate assay. Data for panels C and D are from reference . (E&F) Effects of ΔFXII on high molecular weight kininogen (HK). (E) Western blots of human FXII-deficient plasma supplemented with FXII or ΔFXII (400 nM). At indicated times, samples were removed into non-reducing sample buffer. Western blots were probed with goat anti-human HK IgG (HK). Positions of standards for HK and cleaved HK (HKa) are shown on the right. (F) Bradykinin generation in normal plasma after addition of ΔFXII (160 nM; black), ΔFXII and an inhibitor of kallikrein (KV999272 10 mM; gray), or vehicle (white). Bradykinin was measured by ELISA (Enzo Life Sciences). Data for panels E and F are from reference .

Figure 4.. Factor XII/Pro-HGFA Chimeras.

(A) Schematic diagrams for FXII, Pro-HGFA and FXII with domains replaced with corresponding domains from Pro-HGFA. Domain abbreviations are explained in the legend for figure 2. (B&C) Reciprocal activation of FXII and PK. (B) PK (60 nM) was mixed with 12.5 nM FXII, HGFAHC/FXIILC, or ΔFXII (left panel) or FXII-Lys253 (right panel), and 200 mM chromogenic substrate S-2302 at 37°C. Changes in OD of 405 nm were continuously monitored on a spectrophotometer. (C) Reactions set up as in panel B, except that FXII/Pro-HGFA chimeras were used in place of FXII. Data are from reference .

Figure 5.. Surface-Dependent FXII activation.

(A) FXII (100 nM) incubated with vehicle (black), 6–10 kDa dextran sulfate (2.5 ug/ml, blue) or 500 kDa dextran sulfate (0.5 ug/ml, red). At indicated times samples were removed and FXIIa activity was detected with a chromogenic substrate. (B) FXII (100 nM) were incubated with varying concentrations of 6–10 kDa dextran sulfate (blue) or 500 kDa dextran sulfate (red). After 30 minutes, FXIIa activity was detected with a chromogenic substrate. (C) FXII autoactivation. FXII or FXII-Pro-HGFA chimeras (figure 4), 200 nM, were incubated with chromogenic substrate S-2302 (200 mM) and polyphosphate (70 μM). Changes in optical density at 405 nm were monitored on a spectrophotometer. Comparable reactions without polyphosphate are indicated by dashed lines. (D) FXII, Pro-HGFA or FXII-Pro-HGFA chimeras (20 μg), were applied to a 1 ml heparin-Sepharose column in buffer containing 100 mM NaCl at pH 7.4. Elution was with a linear NaCl gradient (150–1000 mM). Shown are NaCl concentrations required to elute protein from the column. (E) FXII autoactivation, as in panel C, except that polyphosphate was replaced with 130 nM polyethyleneimine. (F) Model for FXII surface-dependent activation. The image on the left proposes a model for FXII in a closed conformation with the KNG domain binding to lysine/arginine residues elsewhere in the heavy chain through an Asp-X-Asp/Glu lysine-binding motif (represented by the white triangle). In the diagram, the interaction is with residues in the FN2 domain; however, the binding site(s) could reside elsewhere in the molecule. When FXII binds to a negatively charged surface, the internal binding interactions involving KNG are disrupted, exposing the activation cleavage site (indicated by scissors). Surface binding in this model is through the EGF1 domain, although different domains may contribute, depending on the surface. Data in panels, C-F are from reference .