Interdomain engineered disulfide bond permitting elucidation of mechanisms of inactivation of coagulation factor Va by activated protein C

Abstract

Procoagulant factor Va (FVa) is inactivated via limited proteolysis at three Arg residues in the A2 domain by the anticoagulant serine protease, activated protein C (APC). Cleavage by APC at Arg306 in FVa causes dissociation of the A2 domain from the heterotrimeric A1:A2:A3 structure and complete loss of procoagulant activity. To help distinguish inactivation mechanisms involving A2 domain dissociation from inactivation mechanisms involving unfavorable changes in factor Xa (FXa) affinity, we used our FVa homology model to engineer recombinant FVa mutants containing an interdomain disulfide bond (Cys609-Cys1691) between the A2 and A3 domains (A2-SS-A3 mutants) in addition to cleavage site mutations, Arg506Gln and Arg679Gln. SDS-PAGE analysis showed that the disulfide bond in A2-SS-A3 mutants prevented dissociation of the A2 domain. In the absence of A2 domain dissociation from the A1:A2:A3 trimer, APC cleavage at Arg306 alone caused a sevenfold decrease in affinity for FXa, whereas APC cleavages at Arg306, Arg506, and Arg679 caused a 70-fold decrease in affinity for FXa and a 10-fold decrease in the k(cat) of the prothrombinase complex for prothrombin without any effect on the apparent K(m) for prothrombin. Therefore, for FVa inactivation by APC, dissociation of the A2 domain may provide only a modest final step, whereas the critical events are the cleavages at Arg506 and Arg306, which effectively inactivate FVa before A2 dissociation can take place. Nonetheless, for FVa Leiden (Gln506-FVa) inactivation by APC, A2 domain dissociation may become mechanistically important, depending on the ambient FXa concentration.

Fig. 1.

Structural model of the A-domains trimer of FVa. Panels A and B show CPK space-filling models shown in two orientations with B rotated approximately 90 degrees relative to A. Panels C and D show ribbon schematic models in the same orientations as A and B, respectively. The A2 domain is highlighted in white. The A1 and A3 domains are gray; Cys609 and Cys1691 are black; Arg306 is white. The surface loop from residue 301 to residue 315, which contains Arg306, is in black. Panels A and B were created with WebLab Viewer Pro and panels C and D were made with Molscript (Kraulis 1991) and rendered with Raster3D (Merritt and Bacon 1997).

Fig. 2.

Schematic of recombinant B domain-deleted FV molecules and SDS-PAGE analysis of recombinant proteins. (A) The top schematic is of the primary sequence of FVΔB (see Materials and Methods) with the locations of the different domains indicated. The second schematic shows activated FVΔB (FVa), a heterodimer of the N-terminal heavy chain and the C-terminal light chain associated in the presence of Ca²⁺ ions. Arrows indicate sites of cleavage in FVa by APC. The third schematic shows the fragments produced following cleavage of FVa (FVai) by APC and also shows the sites of the Cys mutations that did or did not result in disulfide bond formation. (B) Silver-stained gel of purified recombinant B-domain-deleted FV and plasma-derived FV. Lane 1, plasma-derived FV; lane 2, 2183A-FV; lane 3, A2-SS-A3-FV; lane 4, Q506-A2-SS-A3-FV. (C) Immunoblot analysis of thrombin-activated FVa with an anti-FV light chain monoclonal antibody. Lane 1, plasma-derived FV; lane 2, thrombin-activated plasma-derived FVa; lane 3, 2183A-FV; lane 4, thrombin-activated 2183A-FVa.

Fig. 3.

Immunoblots of various FVa and FVai mutants. (A) Immunoblot developed with an anti-FV light chain monoclonal antibody. Samples in lanes 1–6 were not reduced, and those in lanes 7–12 were reduced. Lanes 1 and 7, 2183A-FVa; lanes 2 and 8, 2183A-FVai; lanes 3 and 9, A2-SS-A3-FVa; lanes 4 and 10, A2-SS-A3-FVai; lanes 5 and 11, Q506-A2-SS-A3-FVa,; lanes 6 and 12, Q506-A2-SS-A3-FVai. (B) Immunoblots developed with anti-FV heavy chain polyclonal antibodies. Lane 1, nonreduced A2-SS-A3-FVa; lane 2, nonreduced A2-SS-A3-FVai; lane 3, reduced A2-SS-A3-FVa; lane 4, reduced A2-SS-A3-FVai; lane 5, nonreduced Q506-A2-SS-A3-FVa; lane 6, nonreduced Q506-A2-SS-A3-FVai; lane 7, reduced Q506-A2-SS-A3-FVa; lane 8, reduced Q506-A2-SS-A3-FVai. Band positions for crosslinked and noncrosslinked fragments are indicated at the right side of each blot. LC, light chain; HC, heavy chain; A1, A1 domain; A2, A2 domain; A2c, C-terminal fragment of the A2 domain (residues 507–679). Molecular weight marker positions (kD, Novex SeeBlue standards) are indicated on the left side.

Fig. 4.

Time course of FVa mutant inactivation by APC. Various recombinant FVa mutants were incubated with APC, and aliquots taken at the indicated timepoints were assayed for remaining FVa activity and also saved for SDS-PAGE analysis. Inactivation reactions were performed with 2.5 nM APC and 4 nM FVa. FVa was assayed in the prothrombinase assay at 20 pM concentration with Xa at 1 nM. (A) 2183A-FVa (▵), A2-SS-A3-FVa (), Q506-FVa (○), Q506-A2-SS-A3-FVa (▪), Q506/Q679-A2-SS-A3-FVa (⋄), and control Q506-A2-SS-A3-FVa without APC (□). (B) Data from A expanded to allow comparison of APC-treated Q506-A2-SS-A3-FVa (▪) and Q506/Q679-A2-SS-A3-FVa (⋄) with control Q506-A2-SS-A3-FVa without APC (□). (C) Immunoblot of aliquots from the timecourse of APC inactivation of Q506-A2-SS-A3-FVa. Lanes are labeled with the time (min) of incubation with APC. Crosslinked and noncrosslinked fragments are indicated on the right side of the blot. LC, light chain; HC, heavy chain; A2, A2 domain. Molecular weight markers are indicated on the left (kD).

Fig. 5.

Effects of FXa concentration on cofactor activity of intact FVa and APC-cleaved FVai mutants. 2183A-FVa (▵), A2-SS-A3-FVai (•), Q506-A2-SS-A3-FVa (□) and -FVai (▪) and Q506/Q679-A2-SS-A3-FVa (⋄) and -FVai (♦) were assayed for cofactor activity in a prothrombinase assay with variations in FXa. FVa or FVai was at 2 pM except for A2-SS-A3-FVai, which was at 100 pM. FXa was varied from 5 to 600 pM, except with A2-SS-A3-FVai, when it was varied from 300 to 4000 pM. Data are the average of two or more experiments, and the K_1/2Xa was derived by best fit to a hyperbolic equation.

Fig. 6.

Determination of kinetic constants of the prothrombinase complex with FVa variants. Prothrombin was titrated into the prothrombinase complex, and K_m and k_cat were derived by best fit to a hyperbolic equation. Symbols are the same as in Fig. 4 ▶. FXa was at 20 pM, and FVa or FVai was 1 nM except for A2-SS-A3-FVai, which was 5 nM. Prothrombin was varied from 25 to 1500 nM. Data are the average of two or more experiments.

Fig. 7.

Model of the mechanism of inactivation of FVa by APC. The schematic representation of the windmill-like structure of factor Va is based on a three-dimensional molecular model of factor Va (Pellequer et al. 2000). APC cleaves FVa at three Arg residues in the heavy chain: Arg306, Arg506, and Arg679. In the model depicted, cleavages at Arg506 and Arg306 are random but cleavage at Arg506 is faster (indicated by the heavier arrow), and therefore cleavage at Arg506 predominantly takes place first. K_d reflects affinity of phospholipid-bound FVa for FXa, and k_cat is the value for the prothrombinase complex (FXa:FVa:phospholipid). Cleavage at Arg506 results in a 40-fold loss of affinity for FXa. Cleavage at Arg306 also results in a loss of affinity for FXa. When both cleavages have taken place, the affinity for FXa is reduced by about 100-fold and the k_cat for prothrombin is reduced by about 10-fold. Cleavage of Arg679 is likely not significant because it occurs much more slowly than the other cleavages, and it is thus not presented in this scheme. This model is compiled from published work (Kalafatis et al. 1994a; Nicolaes et al. 1995; Mann et al. 1997; Hockin et al. 1999) and the data presented in this paper.