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

Award Number: R01HL158007
ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 04/01/2021
PERIOD OF PERFORMANCE END DATE: 03/31/2025

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Mechanisms controlling the efficiency of hemostatic vitamin K-dependent protein activation - 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 of these aims will reveal the mechanisms of efficient VKD protein carboxylation, and provide important new information for warfarin therapy.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $538,321 )
2024	2024	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE OF CASE WESTERN RESERVE UNIVERSITY	9500 EUCLID AVE.	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	001	4	7/5/2024	NON-COMPETING CONTINUATION	$43,943
2024	2024	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE OF CASE WESTERN RESERVE UNIVERSITY	9500 EUCLID AVE.	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	000	4	3/20/2024	NON-COMPETING CONTINUATION	$494,378
														Subtotal = $538,321

Issue Date FY: 2023 ( Subtotal = $549,308 )
2023	2023	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE OF CASE WESTERN RESERVE UNIVERSITY	9500 EUCLID AVE.	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	000	3	3/30/2023	NON-COMPETING CONTINUATION	$549,308
														Subtotal = $549,308

Issue Date FY: 2022 ( Subtotal = $564,308 )
2022	2022	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE/CWRU	9500 EUCLID AVE	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	000	2	3/18/2022	NON-COMPETING CONTINUATION	$507,876
2022	2022	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE/CWRU	9500 EUCLID AVE	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	001	2	6/8/2022	NON-COMPETING CONTINUATION	$56,432
														Subtotal = $564,308

Issue Date FY: 2021 ( Subtotal = $549,308 )
2021	2021	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE/CWRU	9500 EUCLID AVE	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	001	1	6/27/2021	NEW	$54,930
2021	2021	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE/CWRU	9500 EUCLID AVE	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	000	1	3/22/2021	NEW	$494,378
														Subtotal = $549,308

Grand Total All Awards = $2,201,245

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

Award Number: R01HL158007

ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 04/01/2021

PERIOD OF PERFORMANCE END DATE: 03/31/2025

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