This revised renewal two year ARRA-funded project extends the PI's basic and clinical research on regulation of blood coagulation and on translational clinical research of venous thrombosis. Thrombosis is strongly linked to an imbalance of anticoagulant and procoagulant mechanisms. Three of four specific aims involve basic studies of novel plasma molecules that regulate clotting while the last aim involves translational research to identify novel biomarkers for thrombosis.
For aims 1 and 2, we hypothesize that thrombin generation can be influenced by minor abundance single chain plasma lipids, so-called """"""""soluble"""""""" lipids. Based on Surface Plasmon Resonance (SPR) binding studies and on clotting assays, we hypothesize that anticoagulant acyl carnitines, including palmitoyl carnitine, directly inhibit coagulation factor Xa by binding to Xa. In preliminary studies, palmitoyl carnitine binds to Gla-domainless-factor Xa and inhibits its activity. These preliminary data and the proposed experimentation thus relate to a novel paradigm for the effects of plasma lipids on coagulation pathways. Recombinant factor X/IX chimeras and variant factor X molecules from various species will be used to identify putative lipid binding domains on factor Xa.
For aim 2, we hypothesize that plasma phospholipid transfer protein (PLTP) can directly influence plasma coagulability and thrombin generation by novel direct interactions with clotting factors. SPR preliminary data show that PLTP binds to specific clotting factors.
For aim 3, we will determine some of the three dimensional structural properties of the prothrombinase complex comprising factors Xa, Va and prothrombin on phospholipid membranes. First, we will introduce Cys mutations and prepare fluorescently labeled factor Va. Then we will use Forster Resonance Energy Transfer (FRET) to generate a set of multiple point- to-point and point-to-plane distances that can be used to generate and then interpret FRET-derived distances for the prothrombinase complex.
For aim 4, based on the hypothesis that imbalances of plasma anticoagulant minor abundance lipids are linked to thrombosis risk, we will use already available frozen plasma samples from thrombosis patients and matched controls to determine if certain targeted plasma lipids or two lipid binding plasma proteins (PLTP or serum amyloid A) are biomarkers for thrombosis. This project will increase insights into the pathophysiology of thrombosis and may improve diagnosis and treatment of thrombosis.
Excessive, uncontrolled blood clotting causes thrombosis where blood clots occlude arteries or veins, 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 protein and fat molecules 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.
|Deguchi, Hiroshi; Elias, Darlene J; Griffin, John H (2017) Minor Plasma Lipids Modulate Clotting Factor Activities and May Affect Thrombosis Risk. Res Pract Thromb Haemost 1:93-102|
|Deguchi, Hiroshi; Navarro, Silvia; Payne, Amanda B et al. (2017) Low level of the plasma sphingolipid, glucosylceramide, is associated with thrombotic diseases. Res Pract Thromb Haemost 1:33-40|
|Deguchi, Hiroshi; Banerjee, Yajnavalka; Elias, Darlene J et al. (2016) Elevated CETP Lipid Transfer Activity is Associated with the Risk of Venous Thromboembolism. J Atheroscler Thromb 23:1159-1167|
|Deguchi, Hiroshi; Sinha, Ranjeet K; Marchese, Patrizia et al. (2016) Prothrombotic skeletal muscle myosin directly enhances prothrombin activation by binding factors Xa and Va. Blood 128:1870-1878|
|Gale, Andrew J; Bhat, Vikas; Pellequer, Jean-Luc et al. (2016) Safety, Stability and Pharmacokinetic Properties of (super)Factor Va, a Novel Engineered Coagulation Factor V for Treatment of Severe Bleeding. Pharm Res 33:1517-26|
|Sinha, Ranjeet K; Yang, Xia V; Fernández, José A et al. (2016) Apolipoprotein E Receptor 2 Mediates Activated Protein C-Induced Endothelial Akt Activation and Endothelial Barrier Stabilization. Arterioscler Thromb Vasc Biol 36:518-24|
|Wang, Yaoming; Zhao, Zhen; Rege, Sanket V et al. (2016) 3K3A-activated protein C stimulates postischemic neuronal repair by human neural stem cells in mice. Nat Med 22:1050-5|
|Alsultan, Abdulrahman; Gale, Andrew J; Kurban, Kadijah et al. (2016) Activation-resistant homozygous protein C R229W mutation causing familial perinatal intracranial hemorrhage and delayed onset of thrombosis. Thromb Res 143:17-21|
|Griffin, John H; Fernández, José A; Lyden, Patrick D et al. (2016) Activated protein C promotes neuroprotection: mechanisms and translation to the clinic. Thromb Res 141 Suppl 2:S62-4|
|Banno, Fumiaki; Kita, Toshiyuki; Fernández, José A et al. (2015) Exacerbated venous thromboembolism in mice carrying a protein S K196E mutation. Blood 126:2247-53|
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