Restoring the procofactor state of factor Va-like variants by complementation with B-domain peptides

Abstract

Coagulation factor V (FV) circulates as an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domain. Conserved basic and acidic sequences within the B-domain appear to play an important role in keeping FV as an inactive procofactor. Here, we utilized recombinant B-domain fragments to elucidate the mechanism of this FV autoinhibition. We show that a fragment encoding the basic region (BR) of the B-domain binds with high affinity to cofactor-like FV(a) variants that harbor an intact acidic region. Furthermore, the BR inhibits procoagulant function of the variants, thereby restoring the procofactor state. The BR competes with FXa for binding to FV(a), and limited proteolysis of the B-domain, specifically at Arg(1545), ablates BR binding to promote high affinity association between FVa and FXa. These results provide new insight into the mechanism by which the B-domain stabilizes FV as an inactive procofactor and reveal how limited proteolysis of FV progressively destabilizes key regulatory regions of the B-domain to produce an active form of the molecule.

Keywords: Autoinhibition; Coagulation Factors; Enzyme Mechanisms; Factor V; Factor Va; Hemostasis; Protein Engineering; Prothrombinase; Recombinant Protein Expression.

FIGURE 1.

Design and expression of FV B-domain fragments. A, structural organization of FV. The BR (Lys⁹⁶³–Lys¹⁰⁰⁸) is indicated in blue, and the AR (Thr¹⁴⁹²–Asn¹⁵³⁸) is indicated in red. Amino acid sequences of the BR and AR used to design B-domain fragments are shown below. B, SDS-PAGE analysis of purified B-domain fragments. Purified proteins (2 μg/lane) were resolved on 4–12% gels under reducing conditions and stained with Coomassie Brilliant Blue. First lane, the BR peptide; second lane, the acetylated BR peptide (Ac-BR); third lane, the BR+AR peptide. The apparent molecular weights of the protein standards are indicated.

FIGURE 2.

Inhibition of FV variants by B-domain fragments. A, structures of FV, FV-810 and FVa, indicating the presence of the BR (blue) and AR (red). B, the BR peptide (0–8 μm) was titrated into reaction mixtures containing 1.4 μm prothrombin, 3 μm DAPA, 50 μm PCPS, and 0.1 nm rFVa (○) or FV-810 (●) in assay buffer at 25 °C. Reactions were initiated with 2 nm FXa, and prothrombin activation was measured as described under “Experimental Procedures.” C and D, specific clotting activity was measured in FV-deficient plasma supplemented with 0.25 nm FV-810 (C) or rFVa (D) and the indicated peptides at 5 μm. Ac-BR, the acetylated BR peptide.

FIGURE 3.

Direct binding of the BR peptide to FV-810. FV-810 was titrated into reaction mixtures containing 20 nm (●) or 40 nm (○) OG488-BR peptide and 50 μm PCPS in assay buffer at 25 °C. Changes in fluorescence anisotropy of OG488-BR were measured as described under “Experimental Procedures.” Lines were drawn after analysis to independent, non-interacting sites with the fitted constants K_d = 2.07 ± 0.2 nm and n = 1.27 ± 0.02 mol of FV-810/mol of OG488-BR at saturation. Control experiments were performed by titrating FV-810 into buffer containing 10 mm EDTA (×) or by titrating rFVa (▴). Inset, the unlabeled BR peptide was titrated into reaction mixtures containing 30 nm OG488-BR, 20 nm FV-810, and 50 μm PCPS. The fitted constants for the unlabeled BR peptide were determined as K_d = 2.1 ± 0.2 nm and n = 1.0 ± 0.06 mol of BR/mol of FV-810 assuming the constants determined above for OG488-BR.

FIGURE 4.

Sedimentation velocity of the BR peptide. The sedimentation velocity of 5 μm QSY7-BR was measured as described under “Experimental Procedures” either alone (A) or in the presence of 7 μm FV-810 (B). The panels show 14 scans taken at 8-min intervals.

FIGURE 5.

Effect of BR peptides from several species on FV-810 activity. A, multiple sequence alignment of the BR elements from human (Homo sapiens), bovine (B. taurus), lizard (A. carolinensis), and zebrafish (D. rerio) FV proteins. Lysine and arginine residues are shaded in blue. B, purified BR fragments (2 μg each) were resolved by reducing SDS-PAGE and stained with Coomassie Brilliant Blue. C, human (●), bovine (♦), lizard (▴), and zebrafish (■) BR peptides were titrated into reactions containing 1.4 μm prothrombin, 3 μm DAPA, 50 μm PCPS, and 0.1 nm FV-810 in assay buffer at 25 °C. Prothrombin activation was measured as described in the legend to Fig. 1A. D, human (●) and bovine (♦) BR peptides were titrated into reactions containing 30 nm OG488-BR, 20 nm FV-810, and 50 μm PCPS at 25 °C. Fluorescence anisotropy was measured, and equilibrium binding constants were determined assuming a stoichiometry of 1 mol of FV-810/mol of BR peptide: the human BR, K_d = 2.2 ± 0.2 nm; and the bovine BR, K_d = 28.3 ± 0.6 nm.

FIGURE 6.

Competitive binding of the BR peptide and FXa to FV-810. A, FXa^S195A (●) or zymogen FX^S195A (▴) was titrated into reaction mixtures containing 30 nm OG488-BR, 20 nm FV-810, and 50 μm PCPS in assay buffer at 25 °C. Changes in OG488-BR anisotropy were measured, and lines were drawn as described with the fitted constants K_d = 1.8 ± 0.2 nm for FXa and n = 1.1 ± 0.07 mol of FXa/mol of FV-810 assuming the binding constants for OG488-BR determined in Fig. 2A. B, reactions containing 1.4 μm prethrombin-2, 3 μm DAPA, 50 μm PCPS, 5 nm FV-810, and 0 nm (■), 125 nm (●), 250 nm (▴), 500 nm (♦), or 1 μm (▾) BR peptide were prepared in assay buffer at 25 °C. Reactions were initiated by the addition of 1–50 nm FXa, and thrombin generation was monitored as described under “Experimental Procedures.” Experimental data were fitted to a model for tight binding with calculated values of K_d = 2.0 ± 0.2 nm for FXa and K_d = 34.2 ± 3.6 nm for the BR.

FIGURE 7.

Effect of the BR peptide on FV-810 thrombin cleavage site variants. A, schematic of the partial cleavage products generated by incubating FV-810 containing the R709Q and/or R1545Q mutation with thrombin. B, reactions containing 600 nm FV-810, FV-810^R709Q, FV-810^R1545Q, FV-810^QQ, or rFVa were incubated for 15 min at 37 °C with buffer or 10 nm thrombin and then quenched with 20 nm hirudin. Samples were resolved by 4–12% gradient SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue. C and D, specific clotting activities of FV-810 variants after incubation with buffer (C) or thrombin (D). FV-deficient plasma was supplemented with the quenched reaction products at 0.25 nm, and specific activity was measured in the absence (white bars) or presence (gray bars) of 5 μm BR peptide.

FIGURE 8.

Direct binding of OG488-BR to thrombin-cleaved FV-810 variants. FV-810 (●), FV-810^R709Q (▴), FV-810^R1545Q (♦), and FV-810^QQ (■) were pretreated with buffer (A) or thrombin (B) as described in the legend to Fig. 7. Quenched FV-810 species were titrated into reaction mixtures containing 30 nm OG488-BR and 50 μm PCPS in assay buffer, and changes in the fluorescence anisotropy signal were measured. Binding constants were calculated assuming a stoichiometry of n = 1 of mol FV-810/mol of OG488-BR: FV-810, K_d = 2.1 ± 0.3 nm; FV-810^R709Q, K_d = 7.1 ± 1.6 nm; FV-810^R1545Q, K_d = 2.0 ± 0.4 nm; and FV-810^QQ, K_d = 0.31 ± 0.29 nm. After incubation with thrombin, neither FV-810 nor FV-810^R709Q had detectable binding to OG488-BR, whereas FV-810^QQ bound with a calculated K_d of 1.3 ± 0.1 nm, and FV-810^R1545Q bound with a K_d of 30.3 ± 4.1 nm.