The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function

Abstract

In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fold increase in the rate of thrombin generation compared with factor Xa alone. Structurally, little is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a crystal structure of protein C inactivated factor Va (A1.A3-C1-C2) that depicts a previously uncharacterized domain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copper-binding site have both been identified. Surprisingly, neither shows a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffold for the future generation of models of coagulation cofactors.

Fig. 1.

The structure of bovine factor Va_i.(A) Schematic drawing of the structure of bovine factor Va. The extent and names of the five domains, metal-binding sites, and phosphorylation sites are indicated. Dashed lines and outlined fonts depict the A2 domain that is removed in factor Va_i.(B) Ribbon diagram of bovine factor Va_i indicating the positions of the carbohydrates (orange) and the metals (Ca²⁺, gray; Cu²⁺, pink). A van der Waals surface representation is shown in the background. Domains are color coded throughout all figures as follows: A1, red; A3, blue; C1, green; and C2, yellow. All structural figures were prepared by using pymol (46).

Fig. 2.

Comparison of homologous cofactor models. Domain orientation of the model of factor Va (PDB ID code 1FV4) (A) (45), the cryoEM structure of factor VIIIa (B) (47), and the crystal structure of bovine Va_i (C). The sizes and orientation of the ovals were scaled to match the cryoEM C2 domain.

Fig. 3.

Stereo images of the metal-binding sites in factor Va_i. (A) The copper-binding site in the A3 domain (blue) with anomalous density for the copper is shown at 3 σ. The trigonal planar coordination geometry is shown with dashed lines. Nearby residues from the A1 domain (backbone shaded red) are shown, and the distance to the closest residue is shown in red. (B) The octahedral coordination geometry (dashed lines) of the calcium-binding site in the A1 domain (red).

Fig. 4.

Potential C domain membrane interactions. (A) The membrane-binding spikes of the C1 (Left) and C2 (Right) domains. The domains are displayed in similar orientations with respect to the overall β-barrel fold. Residues potentially involved in membrane binding are shown. (B) Packing interactions of the tryptophans from spike C2-1 (2050 and 2051) with a hydrophobic pocket in the A3 domain (white, hydrophobic; blue, polar) from a neighboring molecule.

Fig. 5.

Model of factor Va. Overlaid structure of ceruloplasmin (PDB 1KCW, yellow) on the bovine Va_i structure (black). For clarity, the ceruloplasmin A domain representing the A2 domain is depicted in red as a surface representation. Measurements do not include extended loops. (Right) Image rotated 90° about a vertical axis.