# Molecular and Structural Recognition in Factor VIIa Induced Coagulation

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2021 · $390,000

## Abstract

Project Summary—Membrane associated or soluble tissue factor (sTF) in complex with factor (F) VIIa
activates FX to FXa and FIX to FIXa. In this process, the gamma-carboxyglutamic acid (Gla) domain and the
epidermal growth factor-like domain1 (EGF1) of FX and that of FIX play important roles in interactions with
TF/FVIIa complex. This activation process is regulated by tissue factor pathway inhibitor (TFPI), which consists
of three Kunitz domains; the first domain (K1) binds to FVIIa and the second domain (K2) binds to FXa (but not
to FIXa). Thus, formation of the quaternary TF:VIIa:Xa:TFPI inhibitory complex effectively shuts down the TF-
induced FX activation. However, uninhibited TF/FVIIa continues to activate FIX for persistent coagulation. The
molecular recognitions involved in the activation of FIX and FX by TF/FVIIa and in the formation of inhibitory
complex (TF:VIIa:Xa:TFPI) are incompletely understood. Further, large discrepancies exist between the
reported affinity values of sTF (but not for membrane TF) for zymogen FVII, FVIIa and active site inhibited-
FVIIa. Our preliminary data indicate that the sTF affinity increases ~7-fold at each step during the conversion
of zymogen FVII to FVIIa and to active site inhibited-FVIIa. Further, our NH2-Ile153-carbamylation data support
that S1-subsite occupancy and sTF binding to FVIIa protease domain are thermodynamically linked. In
Specific Aim 1, we propose to obtain crystal structures of the zymogen FVIIR152E, and two naturally occurring
activated mutants (FVIIaR304Q and FVIIaR304W) each in complex with sTF. We will further biochemically
characterize the unactivated as well as activated FVIIR304Q and FVIIR304W mutants. Such data should provide the
structure of zymogen FVIIR152E and role of Arg304 in relaying the allosteric signal to the active site of FVIIa for
enhanced activity upon TF binding. In Specific Aims 2 and 3, we propose crystallography and mutagenesis
studies to delineate molecular interactions involved in binding of Gla and EGF1 domains of FXa and FIXa to
TF/FVIIa, and in binding of FVIIa to the K1 domain and of FXa to the K2 domain of TFPI. We will use two-
domain TFPI (TFPI161) that binds to FXa and to FVIIa. Wild-type FXa after binding to TFPI via its active site
serine slowly cleaves TFPI. Thus we will use FXa in which its active site Ser195 is mutated to Ala (XaS195A).
FXaS195A binds to TFPI very tightly. To understand interactions of Gla/EGF1 domains of FIXa with FVIIa/sTF,
we will use a hybrid XaS195A in which its Gla and EGF1 domains are replaced from FIX, termed XaS195A-IXGE.
 For the proposed studies, we have expressed and purified zymogen FVIIR152E, FVIIR304Q and FVIIR304W. We
have also successfully purified the quaternary complexes of (XaS195A)-VIIa-sTF-TFPI161 and (XaS195A-IXGE)-VIIa-
sTF-TFPI161 and obtained crystals of (XaS195A)-VIIa-sTF-TFPI161 that diffract to 3.5 Å but decay after few
frames. Crystallization trials/structure determinations of quaternary complexes a...

## Key facts

- **NIH application ID:** 10147748
- **Project number:** 5R01HL141850-04
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Kanagasabai Vadivel
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $390,000
- **Award type:** 5
- **Project period:** 2018-05-18 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10147748

## Citation

> US National Institutes of Health, RePORTER application 10147748, Molecular and Structural Recognition in Factor VIIa Induced Coagulation (5R01HL141850-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10147748. Licensed CC0.

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