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

控制止血维生素 K 依赖性蛋白激活效率的机制

基本信息

批准号：
10594567
负责人：
KATHLEEN Lucile BERKNER
金额：
$ 54.93万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10594567
关键词：
Affinity Chromatography Anticoagulants Apoptosis Artificial Heart Binding Biological Biological Assay Blood Coagulation Factor Blood coagulation Bone Development CRISPR/Cas technology Calcium Binding Cell Line Cells Complex Data Defect Diet Dimerization Disease Dose Drug usage Embryonic Development Epoxy Compounds Excretory function Extrahepatic Functional disorder Growth Growth and Development function Health Heart Valves Hemorrhage Hemostatic Agents Hemostatic function Human Hydroquinones Inflammation Ingestion Mammalian Cell Mediating Metabolism Modeling Monitor Mus Mutation Oxidoreductase Persons Pharmaceutical Preparations Physiology Platelet Activation Play Production Proteins Quinone Reductases Quinones Reaction Recycling Regulation Resistance Role Signal Transduction Testing Therapeutic Tissues Ubiquitin Vitamin K Vitamin K 2 Warfarin Yeasts calcification carboxylate carboxylation cell growth dietary dimer gamma-glutamyl carboxylase in vivo mutant mutant mouse model oxidation paralogous gene protein activation protein complex protein protein interaction reduced vitamin K virtual vitamin K1 oxide

项目摘要

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.

项目总结