# Mechanisms controlling the efficiency of hemostatic vitamin K-dependent protein activation

> **NIH NIH R01** · CLEVELAND CLINIC LERNER COM-CWRU · 2022 · $564,308

## Abstract

Project Summary
Dietary vitamin K is used in virtually all tissues to convert clusters of Glus to gamma-carboxylated Glus (Glas)
in vitamin K-dependent (VKD) proteins. Carboxylation activates VKD proteins by generating a calcium-binding
module required for their function. The first VKD proteins identified were coagulation factors, which also have
signaling roles that impact other physiologies (e.g. inflammation). Additional extrahepatic VKD proteins also
regulate calcification, growth control, apoptosis and signal transduction. Defining Gla formation is therefore
essential for understanding the impact of VKD proteins on human health and disease. A single gamma-
glutamyl carboxylase generates Gla by oxygenating vitamin K hydroquinone (KH2) to an epoxide (KO). KO is
then recycled by the vitamin K oxidoreductase (VKORC1) in two steps: from epoxide to vitamin K quinone, and
then quinone to hydroquinone. We showed that VKORC1 forms a dimer that is important in accomplishing
these two reactions. VKORC1 is the target of warfarin, a drug used by millions of people worldwide to control
blood clotting, for example with mechanical heart valves. We made the surprising discovery that warfarin
uncouples normal KO reduction, necessitating a second reductase during therapy to generate KH2 for VKD
protein carboxylation. The results are highly significant because extrahepatic VKD proteins may be poorly
carboxylated and dysfunctional if the second reductase is not ubiquitously expressed like VKORC1.
 We showed that a VKORC1 dimer is important to KO recycling to KH2, and our recent preliminary data
suggest that VKORC1 and the carboxylase form a complex. We hypothesize that vitamin K sequestration by
these protein-protein interactions promotes efficient vitamin K recycling. Some VKORC1 mutations cause
warfarin resistance, i.e. the requirement for higher warfarin doses to manage hemostasis, and we hypothesize
that these mutations disrupt dimer integrity. Naturally occurring carboxylase mutations cause severe bleeding,
and some mutants appear to be defective in VKORC1-carboxylase interaction. The aims in this application will
define the protein-protein interactions that make VKD protein carboxylation so efficient and what role they play
in warfarin inhibition. Aim 1 will test whether vitamin K sequestration mediates VKORC1 reduction by
identifying VKORC1 dimerization domains and testing their function in CRISPR/Cas9 edited cell lines deleted
for endogenous VKORC1. Aim 2 will test the importance of VKORC1-carboxylase association in vitamin K
recycling by determining whether human carboxylase mutations that cause severe bleeding disrupt normal
vitamin K recycling, and by studying the efficiency of vitamin K recycling in a carboxylase mutant mouse model.
Aim 3 will test the hypothesis that a quinone reductase distinct from VKORC1 supports VKD carboxylation
during warfarin therapy by testing candidate reductases we have identified in cell line models. Successful
completion...

## Key facts

- **NIH application ID:** 10376350
- **Project number:** 5R01HL158007-02
- **Recipient organization:** CLEVELAND CLINIC LERNER COM-CWRU
- **Principal Investigator:** KATHLEEN Lucile BERKNER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $564,308
- **Award type:** 5
- **Project period:** 2021-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10376350

## Citation

> US National Institutes of Health, RePORTER application 10376350, Mechanisms controlling the efficiency of hemostatic vitamin K-dependent protein activation (5R01HL158007-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10376350. Licensed CC0.

---

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