# Structural basis of prothrombin activation

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2024 · $512,608

## Abstract

Abstract
Work under previous support has continued to advance basic knowledge in the field of hemostasis and
thrombosis with significant contributions to the structural understanding of thrombin dynamics and prothrombin
activation. Unraveling the architecture of coagulation factors and their complexes remains a challenging task
because of the difficulty of obtaining high resolution structures for proteins containing multiple domains. We have
addressed this challenge by being among the pioneers of using cryo-EM in the field of hemostasis and
thrombosis. We have solved the structures of factors V, Va, V short and most notably that of the prothrombin-
prothrombinase complex that has documented, for the first time, the initial step of prothrombin activation along
the meizothrombin pathway. We will build upon the progress made under previous support and address the
structural basis of prothrombin activation, that remains an important unresolved issue in the field. Using cryo-
EM, we will dissect the entire kinetic scheme that defines how prothrombinase activates prothrombin along the
meizothrombin (cleavage at R320 first) and prethrombin-2 (cleavage at R271 first) pathways. We will
characterize the interaction of prothrombinase with prothrombin and meizothrombin (aim 1), and solve the
structures of prothrombin, meizothrombin, and fXa in the free form (aim 2). These studies will unravel the nature
of conformational changes and underlying mechanism leading to assembly of the various complexes in the
kinetic scheme of activation. We will test the hypothesis that cleavage at R320 takes place with prothrombin in
the collapsed closed form whilst cleavage at R271 requires meizothrombin or prothrombin to switch to the
elongated open form. We also hypothesize that prothrombinase cleaves R320 and R271 in two different
conformations. The proposal is supported by solid preliminary cryo-EM data of the prothrombin-prothrombinase
complex on nanodiscs, the meizothrombin-prothrombinase complex, as well as of prothrombin, meizothrombin,
and fXa in the free form. Success of the project will significantly advance basic knowledge on the most important
step of the coagulation response and provide a strategy for the study of other interactions of biological relevance
to blood physiopathology.

## Key facts

- **NIH application ID:** 10874276
- **Project number:** 2R01HL049413-28
- **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:** $512,608
- **Award type:** 2
- **Project period:** 1994-12-01 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10874276

## Citation

> US National Institutes of Health, RePORTER application 10874276, Structural basis of prothrombin activation (2R01HL049413-28). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10874276. Licensed CC0.

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