# Structural enzymology of protein C

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2024 · $529,018

## Abstract

Abstract
The proposed research project continues and expands our investigation of the interaction of thrombin with the
anticoagulant protein C responsible for a key feedback regulation of the coagulation response. The project
addresses unresolved issues in the field using an innovative structural approach and plans to fill existing gaps
in basic knowledge about protein C as a substrate of the thrombin-thrombomodulin complex and activated
protein C as an enzyme that inactivates factor Va. Unraveling the architecture of multidomain factors involved in
blood coagulation, complement and fibrinolysis remains a challenging task because of the difficulty of obtaining
high resolution structures. This limitation is even more acute when considering complexes involving these factors
and their macromolecular substrates or activators. Our approach addresses this challenge directly with cryo-EM,
the new gold standard for the structural investigation of biological macromolecules. Building on our recent
success in solving the structures of human coagulation factors V and Va, the proposed research project plans
to revolutionize the structural enzymology of protein C in a way that is relevant to other multidomain proteins and
their complexes in the blood coagulation cascade. Toward this end, we have obtained preliminary cryo-EM
structures of protein C free and bound to the thrombin-thrombomodulin complex. Once fully refined as planned
under aim 1, these unprecedented structures will unravel the mechanism of protein C activation and test the
hypothesis that thrombomodulin promotes the interaction of thrombin with protein C by offering a scaffold that
changes their conformation and alleviates electrostatic clash. In addition, we have obtained a preliminary cryo-
EM structure of activated protein C free and prepared stable particles of activated protein C bound to factor Va
and protein S for cryo-EM data acquisition. Progress from these studies will elucidate how the structure of
activated protein C compares to that of its zymogen form and will further refine the mechanism of protein C
activation. Furthermore, direct information on the epitopes of recognition of factor Va will enable a structure-
based engineering of variants of activated protein C with altered specificity for potential therapeutic applications.

## Key facts

- **NIH application ID:** 10849652
- **Project number:** 5R01HL139554-07
- **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:** $529,018
- **Award type:** 5
- **Project period:** 2018-06-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10849652

## Citation

> US National Institutes of Health, RePORTER application 10849652, Structural enzymology of protein C (5R01HL139554-07). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10849652. Licensed CC0.

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