Abstract
Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215-217 in the activation domain and the other pathway terminating at the N terminus insertion site. To stabilize segment 215-217, we replaced the flexible 170 loop of FVIIa with the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VYT). The FVIIa-VYT variant exhibited 60-fold higher amidolytic activity than FVIIa, and displayed similar FX activation and antithrombin inhibition kinetics to the FVIIa.sTF complex. The sTF-independent activity of FVIIa-VYT was partly mediated by an increase in the N terminus insertion and, as shown by X-ray crystallography, partly by Tyr-172 inserting into a cavity in the activation domain stabilizing the S1 substrate-binding pocket. The combination with L163V likely drove additional changes in a delicate hydrogen-bonding network that further stabilized S1-S3 sites. In summary, we report the first FVIIa variant that is catalytically independent of sTF and provide evidence supporting the existence of two TF-mediated allosteric activation pathways.
Highlights
Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation
Because FVIIa variants incorporating the 170 loop from trypsin have previously shown compromised soluble ectodomain of TF (sTF) affinity, we evaluated sTF binding to all FVIIa variants using both a functional activity-based assay and surface plasmon resonance (SPR) steady-state kinetics to ensure saturation with sTF in relevant experiments (Fig. 2A)
Values obtained for FVIIa-WT and previously reported variants were in agreement with published results [21, 28, 33]
Summary
Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. The grafted loop provided significant stabilization of the activation domain, mainly by insertion of residue Tyr-172 into a cavity of the FVIIa protease domain promoting pathway I (Fig. 1, A–C) This orientation and interaction are highly similar to that observed in constitutively active trypsin, and we hypothesized that this variant could serve as a starting point for generating a fully matured FVIIa variant independent of TF-induced allosteric enhancement. We combined this variant with previously described point mutations shown to increase activation domain stability and N terminus insertion (pathway II) (Fig. 1D). Based on a combined structural, functional, and biophysical evaluation, we show that a FVIIa variant with the 170 loop from trypsin and the L163V point mutation has obtained full catalytic activity and is functionally independent of sTF in several aspects
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