# Structure and dynamics of prothrombin

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2022 · $378,750

## Abstract

Abstract
The proposed research project continues and expands a line of investigation on the structure and function of
thrombin and its zymogen precursors that has been continuously funded by the NIH since 1994. We now propose
to expand our investigation of the molecular architecture of prothrombin with 19F NMR and cryo-EM, two powerful
techniques never before used in the study of this important clotting factor. Our goal is to characterize the
structural plasticity of prothrombin free and bound to its physiological activator prothrombinase and to
significantly advance our molecular understanding of how this prototypic, multi-domain clotting factor functions
as a substrate in the most important reaction of the blood coagulation cascade. Studies under specific aim 1 will
characterize the conformational landscape of prothrombin and its derivatives prethrombin-2 and thrombin using
19F NMR. We hypothesize that the recently discovered open-closed equilibrium of prothrombin is controlled by
the conformational properties of two critical residues of the protease domain, i.e., W547 in the active site and
W468 in the flexible autolysis loop, and their cross-talk with the auxiliary Gla domain, kringles and sites of
activation at R271 and R320. To test this hypothesis, we will carry out pioneering 19F NMR measurements
targeting the nine Trp residues of the protease domain of prethrombin-2 and thrombin and will then extend this
approach to full length prothrombin, free and bound to prothrombinase, by labeling the additional five Trp
residues of the Gla domain and kringles. Site-specific labeling of W547, W468, R271 and R320 for 19F NMR
measurements of environment and dynamics will be obtained by replacement with Cys and conjugation with
BFTA. These measurements will report changes that accompany activation (prethrombin-2 vs thrombin), binding
of prothrombinase (prothrombin free vs bound) and role of auxiliary domains (prothrombin vs prethrombin-2).
Studies under specific aim 2 will solve the structures of prothrombin free and bound to prothrombinase by cryo-
EM. We will test a mechanism of activation where prothrombin exists predominantly in the closed form when
free and retains this conformation when bound to prothrombinase to promote cleavage at R320 and initiate the
meizothrombin pathway. Meizothrombin then switches to the open form and promotes cleavage at R271 leading
to the mature enzyme thrombin. We have developed reagents in quantities and purity to test this hypothesis by
direct visualization with cryo-EM and obtained preliminary cryo-EM structures of prothrombin, cofactor Va and
the prothrombin-factor Xai complex. Stable particles of the prothrombin-prothrombinase complex have been
imaged by EM and are currently in the queue for cryo-EM data collection. Results from our proposed research
project will significantly advance our basic knowledge of a key reaction of the coagulation cascade and will impact
the study of other trypsin-like zymogens with modu...

## Key facts

- **NIH application ID:** 10428591
- **Project number:** 5R01HL049413-26
- **Recipient organization:** SAINT LOUIS UNIVERSITY
- **Principal Investigator:** Enrico Di Cera
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $378,750
- **Award type:** 5
- **Project period:** 1994-12-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10428591

## Citation

> US National Institutes of Health, RePORTER application 10428591, Structure and dynamics of prothrombin (5R01HL049413-26). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10428591. Licensed CC0.

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