# Molecular mechanism of protein C activation

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2020 · $378,750

## Abstract

Abstract
The proposed research project focuses on the thrombomodulin-dependent activation of protein C by thrombin
as a key regulatory feedback loop of the coagulation response. Our interest in this reaction stems from its
physiological relevance, the lack of a molecular understanding of its mechanism and the translational
opportunities that might ensue from advances in basic knowledge. Investigation of protein C is motivated by
our recent success in the crystallization of prothrombin and characterization of its structure in solution, as well
as by new reagents developed in the lab, i.e., a derivative of protein C devoid of the auxiliary Gla and EGF
domains (miniPC) expressed in E. coli for isotope labeling and a fusion protein (FP) of thrombin with the
EGF456 domains of thrombomodulin that recapitulates the structural and functional properties of the thrombin-
thrombomodulin complex. Our guiding hypothesis is that thrombomodulin (the cofactor) functions by optimizing
the environment of the catalytic Ser of thrombin (the enzyme) and by exposing the Arg residue at the site of
activation of protein C (the substrate). Studies under specific aim 1 will pursue X-ray, single molecule Förster
resonance energy transfer and small angle X-ray spectroscopy of protein C free and bound to FP. Additional
details on the conformation of protein C and of its activation domain in solution will be obtained by NMR
measurements of miniPC. Success of these studies will provide unprecedented and much needed structural
information on protein C and will significantly expand our understanding of the role of conformational plasticity
in the mechanism of zymogen activation in this and other members of the trypsin family. Structural studies will
be complemented by mutagenesis studies under specific aim 2. The effect of thrombomodulin on the catalytic
Ser of thrombin will be investigated either directly through Thr, Cys and Tyr substitutions, or indirectly by
removal of potential steric hindrance in the active site region. The effect of thrombomodulin on the site of
cleavage of the activation domain of protein C will be investigated with substitutions that disengage the side
chain of R169 from neighbor interactions through perturbation of backbone and side chains. Success of our
studies will advance our basic knowledge on a key regulatory reaction of the coagulation cascade and will offer
a relevant template for the analysis of other cofactor-assisted interactions in the blood coagulation,
complement and fibrinolytic cascades.

## Key facts

- **NIH application ID:** 9918442
- **Project number:** 5R01HL139554-03
- **Recipient organization:** SAINT LOUIS UNIVERSITY
- **Principal Investigator:** Enrico Di Cera
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $378,750
- **Award type:** 5
- **Project period:** 2018-06-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9918442

## Citation

> US National Institutes of Health, RePORTER application 9918442, Molecular mechanism of protein C activation (5R01HL139554-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9918442. Licensed CC0.

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