This project involves basic and clinical research on regulation of blood coagulation and on venous thromboembolic disease (VTE). The three Specific Aims involve biochemical, biophysical and clinical studies of endogenous plasma lipids and lipid-binding proteins that are newly discovered modulators of coagulation pathways and thrombin generation.
Aim 1 is focused on minor abundance, plasma anticoagulant sphingolipids while Aim 2 is focused on minor abundance, plasma procoagulant lipids.
Aim 3 is centered on studies of the procoagulant activities of cholesteryl ester transfer protein (CETP).
For aims 1 and 2, we hypothesize that thrombin generation can be directly influenced by minor abundance single chain plasma lipids carried by albumin, so-called "soluble" lipids. The anticoagulant soluble lipids include palmitoyl carnitine and related long chain acyl carnitines and lyso-sulfatide. Based on Surface Plasmon Resonance (SPR) binding studies and on prothrombinase assays, we hypothesize that the anticoagulant soluble lipids bind and directly inhibit factor Xa. Preliminary data show that certain anticoagulant plasma lipids bind and inhibit Gla-domainless-factor Xa, showing for the first time that plasma lipids can bind to a clotting factor outside its Gla domain, thereby altering factor Xa activity. This mechanism for the potential effects of anticoagulant plasma lipids on coagulation provides a novel paradigm for the effects of plasma lipids on coagulation.
For aim 2, we hypothesize that thrombin generation can be stimulated by lyso-phosphatidic acid (lyso-PA) and by oxidized fatty acids. Moreover, based on preliminary data, we hypothesize that the procoagulant action of lyso-PA is greatly augmented by LDL, Lp(a) and apolipoprotein (a). We discovered that factor Xa has a strong affinity for apo(a) which may contribute to the procoagulant mechanism(s). We hypothesize that specific amino acid sequences in fXa interact with specific domains and residues of apo(a) to potentiate the procoagulant activity of lyso-PA. Recombinant factor X/IX chimeras will be used to determine apo(a) binding domain on fXa while a panel of apo(a) deletion mutants will be use to identify the essential domain(s) for fXa binding and lyso-PA procoagulant effects.
For aim 3, we hypothesize that cholesteryl ester transfer protein (CETP) can influence thrombin generation by novel direct interactions with clotting factors. SPR preliminary data show that CETP binds to factor Xa. Biochemical studies using recombinant wild type and mutant CETP molecules will be used to show that CETP binds directly to factors Xa and alters its activity. We will characterize the ability for CETP to transfer lipids from lipid-rich donor particles to factor Xa, thereby altering the functional properties of factor Xa. Based on the hypothesis that imbalances in plasma procoagulant or anticoagulant minor abundance lipids, CETP activity, or elevated serum amyloid A (SAA) are linked to VTE risk, we will use existing VTE patient registries and determine if these plasma analytes are biomarkers for VTE. This project will reveal new insights into the pathophysiology of thrombosis and may improve diagnosis and treatment of thrombosis.

Public Health Relevance

Excessive, uncontrolled blood clotting causes thrombosis where blood clots occlude arteries or a vein, resulting in life-threatening heart attacks, ischemic stokes, or venous blood clots in the legs or lungs. Thrombosis is linked to an imbalance of anticoagulant and procoagulant mechanisms. The proposed studies of fat molecules and fat associated-protein in the blood that control blood clotting and thrombosis will provide new basic and clinical knowledge that will hopefully improve diagnosis and treatment of thrombosis.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01HL021544-38
Application #
8720042
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
Project End
Budget Start
Budget End
Support Year
38
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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