# Structural enzymology of factor V activation

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2024 · $537,851

## Abstract

Abstract
Work under previous support has broadened our understanding of the factors that influence the catalytic activity
of thrombin and set the stage for a structure-based characterization of how this enzyme interacts with
physiological substrates. Unraveling the architecture of factors involved in blood coagulation remains a
challenging task because of the difficulty of obtaining high resolution structures for proteins containing multiple
domains. We have recently shown how to address this challenge by using cryo-EM, the new gold standard for
the structural investigation of biological macromolecules. The proposed research project is a segue to our
pioneering cryo-EM structural work on human coagulation factors V and Va free and bound to factor Xa in the
prothrombinase complex and the structure of this complex bound to prothrombin. Specifically, we plan to solve
the cryo-EM structures of factor V in complex with thrombin (aim 1) and its active precursor meizothrombin (aim
2) with the goal of revealing the molecular basis of a key step of the initiation phase of the coagulation response
that leads to assembly of the prothrombinase complex. The proposal is supported by exciting preliminary cryo-
EM maps currently refined at 6.6 Å resolution for the thrombin-factor V complex (aim 1) and 3.8 Å resolution for
the meizothrombin-fV complex (aim 2). Once fully refined, these structures will reveal the distinct modes of
binding of thrombin and meizothrombin at preferred sites of activation and the full architecture of meizothrombin
for the first time. Underlying epitopes will be validated independently by mutagenesis of specific residues of
thrombin, meizothrombin and factor V. In addition, the structures will test the hypothesis that binding of thrombin
and meizothrombin to factor V rigidifies the disordered B domain and visualizes the structural determinants that
keep factor V in its inactive state. Success of the proposed studies will significantly advance our basic knowledge
on factor V and its activation in ways that are directly relevant to other multidomain proteins in the blood
coagulation cascade and that may benefit the development of new therapeutic strategies.

## Key facts

- **NIH application ID:** 10835071
- **Project number:** 5R01HL147821-06
- **Recipient organization:** SAINT LOUIS UNIVERSITY
- **Principal Investigator:** Enrico Di Cera
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $537,851
- **Award type:** 5
- **Project period:** 2019-06-01 → 2027-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10835071

## Citation

> US National Institutes of Health, RePORTER application 10835071, Structural enzymology of factor V activation (5R01HL147821-06). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10835071. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*