Antibody-induced enhancement of factor VIIa activity through distinct allosteric pathways

Abstract

In the absence of its cofactor tissue factor (TF), coagulation factor VIIa (FVIIa) predominantly exists in a zymogen-like, catalytically incompetent state. Here we demonstrate that conformation-specific monoclonal antibodies (mAbs) can be used to characterize structural features determining the activity of FVIIa. We isolated two classes of mAbs, which both increased the catalytic efficiency of FVIIa more than 150-fold. The effects of the antibodies were retained with a FVIIa variant, which has been shown to be inert to allosteric activation by the natural activator TF, suggesting that the antibodies and TF employ distinct mechanisms of activation. The antibodies could be classified into two groups based on their patterns of affinities for different conformations of FVIIa. Whereas one class of antibodies affected both the K(m) and k(cat), the other class mainly affected the K(m). The antibody-induced activity enhancement could be traced to maturation of the S1 substrate binding pocket and the oxyanion hole, evident by an increased affinity for p-aminobenzamidine, an increased rate of antithrombin inhibition, an increased rate of incorporation of diisopropylfluorophosphate, and an enhanced fraction of molecules with a buried N terminus of the catalytic domain in the presence of antibodies. As demonstrated by site-directed mutagenesis, the two groups of antibodies appear to have overlapping, although clearly different, epitopes in the 170-loop. Our findings suggest that binding of ligands to specific residues in the 170-loop or its spatial vicinity may stabilize the S1 pocket and the oxyanion hole, and they may have general implications for the molecular understanding of FVIIa regulatory mechanisms.

FIGURE 1.

Biacore sensorgrams show the binding of different conformational states of FVIIa to F36 and F37. Representative sensorgrams for the binding of FFR-FVIIa, FVIIa, FVII, and FVIIa-sTF to F36 (top panel) or F37 (bottom panel) are depicted. F36 and F37 were captured on a CM5 chip coated with anti-mouse IgG, and 2-fold serial dilutions of ligands (3.125–200 nm) were injected. The association and dissociation phases following the injection of 200 nm ligand are shown.

FIGURE 2.

The rate of inactivation of FVIIa by carbamylation in the presence of sTF, F36, and F37. The relative activities were plotted against the incubation times in a semi-logarithmic plot. The slopes were derived from linear regression to obtain the rate constants for the decay of enzyme activity. The curves shown are representative examples of three independent experiments.

FIGURE 3.

Time-dependent [1,3-³H]DFP incorporation into FVIIa in the presence of sTF, F36, and F37. FVIIa (0.6 μm) was incubated alone or together with sTF, F36, or F37 (3 μm) in the presence of [1,3-³H]DFP (50 μm). Samples were withdrawn at different time points and fractionated by nondenaturing polyacrylamide gel electrophoresis. The content of [1,3-³H]DFP in the gel pieces containing FVIIa was determined by scintillation measurements. The maximum theoretical incorporation of [1,3-³H]DFP with the FVIIa concentration used is 9400 cpm.

FIGURE 4.

Epitope analysis using surface plasmon resonance. mAbs F36 or F37 were captured on a CM5 chip coated with anti-mouse IgG antibody and the binding, measured as maximum RUs obtained after a single injection of wild-type FVIIa and FVIIa variants (100 nm), was monitored. The data were normalized to the binding level obtained for wild-type FVIIa, which was arbitrarily set to 1. The binding of ligands to F36 is depicted by blue bars; dark blue is in the absence of FFR-CMK, and light blue is in the presence of FFR-CMK. Likewise, binding of ligands to F37 is shown by gray bars. Dark and light gray is in the absence and presence of FFR-CMK, respectively.

FIGURE 5.

Three-dimensional ribbon representation of the serine protease domain of FVIIa. The TF-binding helix (residues 165(307)-170(312)), the 170-loop (residues 170a(313)-170i(321)), and the 99-loop (residues 93(233)-102(242)) are colored red. The activation domain, i.e. the activation loop (residues 16(153)-21(158)), the autolysis loop (residues 142(285)-152(294)), the oxyanion stabilizing loop (residues 184(332)-194(343)), and the S1 entrance frame (residues 216(365)-223(372)), is colored cyan. The FFR-CMK inhibitor occupying the substrate binding pockets S1-S3 is shown in yellow colored sticks. The side chains of the N-terminal Ile-16(153) and Asp-194(343) are depicted in CPK colored sticks. Furthermore, the side chains of Met-164(306), which is a key-mediator of the TF-induced allostery, and Asp-189(338), located in the S1 pocket, are depicted in CPK colored sticks. The side chains of residues important for binding of F36 and F37 are shown in green sticks. The figure was constructed using PyMOL and the protein data base entry code 1dan. Residues highlighted in the figure have only been labeled according to chymotrypsinogen numbering.