Structure and function of factor XI

Abstract

Factor XI (FXI) is the zymogen of an enzyme (FXIa) that contributes to hemostasis by activating factor IX. Although bleeding associated with FXI deficiency is relatively mild, there has been resurgence of interest in FXI because of studies indicating it makes contributions to thrombosis and other processes associated with dysregulated coagulation. FXI is an unusual dimeric protease, with structural features that distinguish it from vitamin K-dependent coagulation proteases. The recent availability of crystal structures for zymogen FXI and the FXIa catalytic domain have enhanced our understanding of structure-function relationships for this molecule. FXI contains 4 "apple domains" that form a disk structure with extensive interfaces at the base of the catalytic domain. The characterization of the apple disk structure, and its relationship to the catalytic domain, have provided new insight into the mechanism of FXI activation, the interaction of FXIa with the substrate factor IX, and the binding of FXI to platelets. Analyses of missense mutations associated with FXI deficiency have provided additional clues to localization of ligand-binding sites on the protein surface. Together, these data will facilitate efforts to understand the physiology and pathology of this unusual protease, and development of therapeutics to treat thrombotic disorders.

Figure 1

Plasma coagulation protease reactions. Scheme for the cascade/waterfall model of thrombin generation triggered by activation of FXII. Conversion of FXI to FXIa is shown in red. A mechanism for initiation of coagulation by the FVIIa/TF complex through FX activation is also shown. Roman numerals indicate unactivated coagulation factors, and activated factors are indicated by “a.” Nonenzymatic cofactors are indicated by numerals in black ovals. The dotted line designated “1” indicates feedback activation of FXI by thrombin, whereas the dotted line indicated by “2” represents activation of FIX by FVIIa/TF.

Figure 2

Human FXI apple domains. Topology diagrams based on the FXI zymogen structure. For all diagrams, the first, second, third, and fourth apple domains (A1, A2, A3, and A4) are shown in cyan, light blue, orange, and yellow, respectively. (A) The apple domain consists of a 7-stranded β-sheet (blue) with a central α-helix positioned on top (red). Disulfide bonds are shown in orange. (B) Topology diagram showing the disk formed by the 4 apple domains, with the catalytic domain removed. Sites of residues implicated in ligand binding are red for thrombin, green for high-molecular-weight kininogen (HK), black for GPIb, blue for heparin, and orange for FIX. (C) Apple domain disk showing the positions of mutations identified in FXI-deficient patients that involve buried residues (green) or surface exposed residues (red). Mutations Gly155Glu, Arg184Gly, and Ser248Asn (boxed) are examples of rare CRM⁺ mutations. (D) Two A4 domains forming the FXI dimer interface. The Cys321 interchain disulfide bond is shown at the top in orange. Hydrophobic residues Leu284, Ile290, and Tyr329 are shown in black, and a salt bridge is formed between Lys331 (blue) and Glu287 (red).

Figure 3

The structure of zymogen FXI. Topology diagram of dimeric FXI viewed from 2 perspectives rotated 90 degrees. The catalytic domain is in white. Sites of residues implicated in ligand binding are red for thrombin (T), green for HK, black for GPIb, blue for heparin sites (H1 and H2), and orange for FIX. Positions for the activation loop (AL) cleavage site (Arg360-Ile370) residue Ile370 and active site (AS) residues Ser557, Asp462, and His413 are shown in purple.

Figure 4

FXI-binding site for HK. (A) The relative positions of Gly104 and Gly155 in the A2 domain are shown. Gly155 is involved in a tight β-turn forming a hydrogen bond with Thr152. The compact hydrogen bond network extends through Thr152 and Thr158 to form contacts to the main chain through residues Lys103 and Ile105. Changes at Gly104 or Gly155 will probably disrupt this network. (B) Charged surface (blue represents positive; red, negative) representation of the underside of the FXI apple domain disk, showing positions of Gly104 and Gly155. These residues are required for proper formation of a charged channel that is a probable binding site for HK. The dotted line represents the predicted binding site of HK that terminates in a pocket at the base of the A2 domain.

Figure 5

FXI activation. Each FXI subunit is activated by cleavage between Arg369 and Ile370. FXI (0-hour time point) migrates slightly faster than FXIa (top band, 6 hours) on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activation of FXI (schematic at left) by α-thrombin (shown) or FXIIa proceeds through an intermediate with 1 cleaved subunit (center), designated 1/2-FXIa. Subsequent conversion of 1/2-FXIa to fully activated FXIa (right) appears to be a slower process. In the diagrams, the A1, A2, A3, and A4 domains are shown in light blue, cyan, orange, and yellow, respectively, and the catalytic domains (CAT) are in white. Circles represent unactivated catalytic domains; three-fourths circles, activated catalytic domains. In these diagrams, the catalytic domain moves relative to the apple domain disk after cleavage of Arg369-Ile370, exposing a surface on A3 thought to contain a FIX-binding site.

Figure 6

The FXIa protease domain. (A) Topologic diagram showing the superposition of FXI (white) and FXIa (red) protease domain crystal structures. Conformational changes include a 12 Å shift in the position of Ile370 into the protease core after cleavage of the Arg369-Ile370 bond, and an unraveling of an α-helix containing Arg489 to fill a pocket left empty by removal of the A3 domain and Arg184. The Arg side chain from the FXIa ligand in panel B is shown in blue. (B) Structure of FXIa in complex with PN2-KPI domain inhibitor (blue). (C) The side chain of Arg184 is buried in the FXI zymogen through contacts with the protease domain. This residue is thought to act as a switch, which is released on zymogen activation to engage substrate FIX. (D) Stick diagram showing the side chain of Arg184 that forms 3 noncovalent interactions with the side chains of Ser268 (gray) from the A3 domain, and Asp488 (green) and Asn566 (white) from the protease domain in the FXI zymogen structure.

Figure 7

FIX activation. (A) FIX contains (from the N-terminus) a Gla domain (green), 2 epidermal growth factor (EGF) domains (red), an activation peptide (AP; gray bar), and a catalytic (CAT) domain (white). FIX is converted to FIXaβ by cleavage after Arg145 and Arg180, releasing the AP. FVIIa/TF initially cleaves FIX after Arg145 to form the intermediate FIXα, with subsequent cleavage after Arg180 to form FIXaβ (bold arrows). White three-fourths circle represents the active catalytic domain of FIXaβ. During this reaction, FIXα accumulates before formation of FIXaβ. FXIa also cleaves FIX initially after Arg145; however, no FIXα accumulates. Initial cleavage of zymogen FIX after Arg180 to generate the intermediate FIXaα appears to be a minor reaction (thin arrows). (B-C) Reducing Western blots of FIX (100nM) activated by 1nM FVIIa/TF (B) or FXIa (C) over 30 minutes (time indicated between panels). Markers at the right indicate migration of standards for zymogen FIX (FIX), the large fragment of FIXα (FIXα), the heavy chain (HC) of FIXaβ, and the light chain (LC) of FIXα or FIXaβ. No FIXα accumulates during activation by FXIa.

Figure 8

Platelet-binding site on FXI. The locations of 2 residues (Ser248 and Arg250 in black), that probably form a GPIb platelet-binding site on the FXI A3 domain (orange), are shown relative to residues that form the heparin-binding site (Ly252, Lys253, and Lys255 in blue). Ser248 forms hydrogen bonds with Asp194 and Thr249, which are probably disrupted in the hereditary FXI mutation Ser248Gln. The adjacent A2 domain is shown in light blue. The position of an N-linked glycan moiety attached to residue Asn108 of the A2 domain is shown in yellow and red.