Factor XII Structure-Function Relationships

Abstract

Factor XII (FXII), the zymogen of the protease FXIIa, contributes to pathologic processes such as bradykinin-dependent angioedema and thrombosis through its capacity to convert the homologs prekallikrein and factor XI to the proteases plasma kallikrein and factor XIa. FXII activation and FXIIa activity are enhanced when the protein binds to a surface. Here, we review recent work on the structure and enzymology of FXII with an emphasis on how they relate to pathology. FXII is a homolog of pro-hepatocyte growth factor activator (pro-HGFA). We prepared a panel of FXII molecules in which individual domains were replaced with corresponding pro-HGFA domains and tested them in FXII activation and activity assays. When in fluid phase (not surface bound), FXII and prekallikrein undergo reciprocal activation. The FXII heavy chain restricts reciprocal activation, setting limits on the rate of this process. Pro-HGFA replacements for the FXII fibronectin type 2 or kringle domains markedly accelerate reciprocal activation, indicating disruption of the normal regulatory function of the heavy chain. Surface binding also enhances FXII activation and activity. This effect is lost if the FXII first epidermal growth factor (EGF1) domain is replaced with pro-HGFA EGF1. These results suggest that FXII circulates in blood in a "closed" form that is resistant to activation. Intramolecular interactions involving the fibronectin type 2 and kringle domains maintain the closed form. FXII binding to a surface through the EGF1 domain disrupts these interactions, resulting in an open conformation that facilitates FXII activation. These observations have implications for understanding FXII contributions to diseases such as hereditary angioedema and surface-triggered thrombosis, and for developing treatments for thrombo-inflammatory disorders.

Fig. 1

The kallikrein-kinin system and contact activation. (A) Reciprocal activation of factor XII (FXII) and prekallikrein (PK) in the absence of a surface. (B) Contact activation on a negatively charged surface (gray cloud) includes autoactivation of FXII, subsequent reciprocal activation of FXII and PK, and FXIIa activation of factor XI (FXI). High-molecular-weight kininogen (HK) facilitates PK and FXI binding to the surface. (C) Schematic diagram of HK showing the domain (D1–D6) structure and the position of bradykinin (BK) within domain 4. PKa cleaves HK at two locations to release BK.

Fig. 2

Factor XII. Shown are the amino acid sequence and domain structure of human plasma FXII. The FXII heavy chain contains fibronectin type 2, epidermal growth factor 1, fibronectin type 1, epidermal growth factor 2 and kringle domains, and a proline-rich region. The protease domain is a trypsin-like catalytic unit. Disulfide bonds between cysteine residues are shown in yellow circles. The catalytic triad (His393, Asp442, and Ser544) are indicated by red circles. Cleavage sites for PKa (Arg334, Arg343, Arg353) are indicated by dark blue circles. Residues in green are sites of mutations discussed in the text. Plasmin cleavage sites (Arg291, Lys326, Arg334) are indicated in light blue. Anion-binding residues (Lys73, Lys74, Lys76, and Lys81) are indicated by purple circles.

Fig. 3

Factor XII and pro-hepatocyte growth factor activator structures. (A) Schematic diagrams of FXII (white), pro-HGFA (gray), and their activated and truncated forms. 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. AlphaFold predictions for (B) human FXII and (C) human pro-HGFA. The fibronectin type 2 (FN2, purple), epidermal growth factor 1 (EGF1, brown), fibronectin type 1 (FN1, magenta), epidermal growth factor 2 (EGF2, gray), kringle (KNG, green), and protease (PD, yellow) domains are shown. The proline-rich region of FXII and corresponding area for pro-HGFA have been removed because AlphaFold did not assign a structure to them.

Fig. 4

Proline-rich region variability. FXII PRR sequences between cysteine residues 276 and 340 (human sequence) are shown from representative organisms for different classes of vertebrates. The corresponding regions from human, mouse, and lung fish pro-HGFA are also shown. Proline residues are in red.

Fig. 5

Properties of factor XII-T and factor XII truncated forms. (A) Reciprocal FXII-PK activation. PK (60 nM) was mixed with 12.5 nM FXII or ΔFXII-310, and 200 uM chromogenic substrate S-2302 at 37 °C. Changes in optical density (OD) at 405 nm were continuously monitored. (B) PK activation by FXII and FXII-T. FXII or FXII-T (200 nM) and PK (200 nM) were incubated at 37 °C. At indicated times, samples were evaluated by Western blot using anti-FXII or anti-PK polyclonal IgGs. Positions of standards for FXII (XII) and PK and the heavy chain (HC) and light chain (LC) of FXIIa and PKa are indicated on the right. (C) FXII activation. FXII (○), the full-length FXII precursor used to generate ΔFXII-Lys³⁰⁹ (□), and ΔFXII-310 (4), 200 nM each, were incubated at 37 °C with PKa (10 nM). FXIIa activity was measured by chromogenic assay. (D) FXII-induced HK cleavage. Western blots of human FXII-deficient plasma supplemented with FXII or ΔFXII-310 (400 nM). At indicated times, samples were removed into a nonreducing sample buffer. Western blots were probed with anti-HK IgG. Positions of standards for HK and cleaved HK (HKa) are shown on the right. (E) Bradykinin generation in normal plasma after addition of 160 nM ΔFXII-310 (●), ΔFXII-310 and kallikrein inhibitor (KV999272 10 nM; ●), or vehicle (○). Bradykinin was measured by ELISA. (F) FXII cleavage by plasmin. FXII was incubated with plasmin and analyzed by Western blot using a polyclonal anti-FXII IgG under nonreducing conditions. Positions of three truncated forms are indicated by arrows. (G) Nonreducing Coomassie Blue-stained SDS-PAGE of purified recombinant wild-type FXII and truncated forms. (H) PK (60 nM) was mixed with 12.5 nM FXII (○), Δ292 (□), Δ327 (◊), or Δ335 (Δ). PKa generation was determined with a chromogenic assay. (I) HK cleavage in human FXII-deficient plasma after supplementing with FXII, ΔFXII-292, ΔFXII-327, and ΔFXII-335. The procedure is the same as in panel D. Panels A, C, D, and E are from Shamanaev et al. Panel B is from Ivanov et al.

Fig. 6

Pro-HGFA activation by thrombin. (A) Schematic diagrams of pro-HGFA, Δ-pro-HGFA, and FXII-HC/HGFA-LC. Abbreviations for domains are the same as in ►Fig. 3. (B) Activation of pro-HGFA. Pro-HGFA (260 nM) was incubated with thrombin (26 nM) in the absence (●) or presence (■) of dextran sulfate (10 μg/mL) at 37 °C. (C) Pro-HGFA (●), Δpro-HGFA (▲), or FXII-HC/HGFA-LC (♦), 260 nM each, was incubated with thrombin (26 nM) in the absence of a surface at 37 °C. For panels B and C, at indicated times, samples of reaction were stopped and HGFA generation was determined by chromogenic assay. (D) Nonreducing Coomassie Blue-stained SDS-PAGE of recombinant wild-type pro-HGFA, pro-HGFA-Lys309, pro-HGFA-Ala563, and pro-HGFA-Lys309, Ala563. (E) Pro-HGFA-Ala⁵⁶³ and pro-HGFA-Lys309, Ala563, each 260 nM, were incubated with 26 nM thrombin in the absence of a surface. At indicated times, samples were removed, and size fractionated by reducing SDS-PAGE. Western blots were probed with anti-hemagglutinin IgG which recognizes a tag on the C-terminus of the HGFA light chain. Panels are from Shamanaev et al.

Fig. 7

Importance of the factor XII FN2 and KNG domains. (A) Schematic diagrams of FXII, Δ-FXII, and HGFA-HC/FXII-LC and FXII/pro-HGFA individual domain chimeras. Abbreviations are the same as in the legend for ►Fig. 3. (B–D) FXII-PK reciprocal activation. PK (60 nM) was mixed with (B) 12.5 nM FXII, HGFA-HC/FXII-LC, ΔFXII-310 or control; (C) 12.5 nM FXII, FXII-pro-HGFA domain chimeras, or control; or (D) 6 nM FXII, HGFA-HC/FXII-LC, FXII-Lys253 or control. For B–D, 200 uM S-2302 was added and changes in OD of 405 nm were continuously monitored on a spectrophotometer. The color coding in panel C matches that in panel A. (E) HK cleavage after supplementing FXII-deficient human plasma with 160 nM FXII or FXII-Lys253. The procedure is the same as in ►Fig. 5D. Panels B–E are from Shamanaev et al.

Fig. 8

Factor XII activation and activity. (A, B) FXII activation on polyphosphate. (A) FXII (200 nM) was incubated without (left) or with (right) 70 μM polyphosphate at 37 °C. (B) FXII (100 nM) was mixed with 12.5 nM PKa without (left) or with (right) 70 μM polyphosphate at 37 °C. For panels A and B, samples were removed at indicated time points into reducing sample buffer, and evaluated by Western blot using a polyclonal anti-FXII IgG. Positions of standards for FXII (XII), and the heavy chain (HC) and light chain (LC) of FXIIa are indicated at the right. (C, D) FXII activation with dextran sulfate. (C) FXII (100 nM) and PKa (12.5 nM) were incubated with short-chain dextran sulfate (6–10 kDa, blue), long-chain dextran sulfate (500 kDa, red), or vehicle (black). At indicated times, FXIIa activity was determined with a chromogenic substrate. (D) FXII (100 nM) was incubated with varying concentrations of short-chain (6–10 kDa, blue) or long-chain (500 kDa, red) dextran sulfate. After 30 minutes, FXIIa activity was measured with a chromogenic substrate. (E) FXII, pro-HGFA, HGFA-HC/FXII-LC, FXII-HC/HGFA-LC, or FXII/pro-HGFA chimeras, each 200 nM, were incubated with 70 μM polyphosphate at 37 °C and chromogenic substrate S-2302 (200 uM). Changes in OD at 405 nm were monitored on a spectrophotometer. Comparable reactions without polyphosphate are indicated by dashed lines. The color code is the same as in ►Fig. 7A. (F) FXII, pro-HGFA, or FXII-pro-HGFA chimeras (20 μg) were applied to a heparin-Sepharose column and eluted with a linear NaCl gradient (50–1,000 mmol/L). Shown are NaCl concentrations required to elute protein from the column. (G, H) PK (60 nM) was incubated with 60 pM (G) FXIIa, βFXIIa, HGFA-HC/FXIIa-LC, or (H) activated forms of FXII/pro-HGFA domain chimeras in the absence (G) or presence (G) of 70 μM polyphosphate. At indicated times, aliquots were removed and PKa measured by chromogenic assay. Panels A, C, and D are from Shamanaev et al (with permission) and panels E–H are from Shamanaev et al.

Fig. 9

A model for factor XII activation. Left. The schematic diagram representing FXII in its closed form in solution, with access to the activation cleavage site at Arg353 (black circle) restricted by components of the heavy chain. Right. When FXII binds to a negatively charged surface, the internal binding interactions involving FN2 and KNG (and perhaps other domains) are disrupted, and the activation cleavage site (black circle) becomes more accessible to cleavage by a protease such as PKa or FXIIa (indicated by scissors). Surface binding in this model is through the EGF1 domain, although other domains may also contribute, and surface-binding domains may vary depending on the surface.