Protein C, a naturally occurring plasma protein, is a serine protease precursor that can be converted to the active enzyme, activated protein C (APC). Current knowledge supports the broad concept that the protein C system provides tissue homeostasis when many types of cells and organs are subjected to various injuries. APC exerts two major, distinct activities: (1) anticoagulant activity and (2) cytoprotective direct effects on cells. APC's anticoagulant activity targets factors Va and VIIIa in reactions requiring the cofactor, plasma protein S. Based on APC-initiated cell signaling, the cytoprotective activities of APC potentially comprise anti-apoptotic and anti-inflammatory activities, alterations of gene expression, and stabilization of endothelial and epithelial barriers. For cell signaling initiation activation of protease activated receptor1 (PAR1) by APC bound to another receptor (endothelial protein C receptor) is a key paradigm. Other receptors play key roles, and there is a major gap in basic knowledge about APC receptors and mechanisms that directly bind APC. This project is focused on providing key basic knowledge about APC's interactions with three of its key receptors, PAR1, Mac-1 (?M?2, CD11b/CD18, C3 receptor), and apoER2 (LRP8) and with its anticoagulant cofactor, protein S. The project is based on several hypotheses, each of which is strongly supported by preliminary data. We hypothesize that PAR1 cleavage at Arg46 mediates APC-initiated biased, cytoprotective signaling. Using the latest methods for obtaining GPCR x-ray structures, we expect to obtain 3-dimensional x-ray crystallographic structures of at least three different conformations of PAR1 to define the basis for biased signaling. The N- terminal tail of PAR1 provides tethered ligands are agonists for biased signaling. We will use synthetic peptides combined with studies of PAR1 mutants to decipher the tale of the various tails of PAR1 that can cause intra- molecular agonism. We expect to obtain insights into where and how the novel PAR1 Asn47-tail, compared to the Ser-42 tail, binds to PAR1. We hypothesize that the alpha-M I-domain of Mac-1 and multiple domains of apoER2 can bind APC on partially overlapping, extended surfaces of APC. Strong preliminary data delineated the areas on APC that merit interrogation. Using mutagenesis and appropriate binding protocols, we will identify the surfaces on APC that bind each receptor. Using mutagenesis of receptors, we will identify residues and areas on each receptor that bind APC. Based on preliminary studies of novel APC mutants, we hypothesize that protein S binds to an extended APC surface involving multiple residues that span an area from the top of the Gla domain over the EGF1 domain and onto the top of the EGF2 domain. We expect to map completely the protein S binding surface on APC. New knowledge about APC-receptor interactions could give rise to novel biologics involving 2nd or 3rd generation APC mutants with beneficially modified specificities.

Public Health Relevance

This project is focused on obtaining basic knowledge about protein-protein interactions that are responsible for the beneficial, protective activities of the enzyme, activated protein C, which can provide multiple activities that reduce damage to cells and tissue. Based on preclinical studies in animals, activated protein C shows beneficial, often mortality reducing effects in the settings of various pathological conditions including ischemia/reperfusion injury of the heart, kidney and lungs, ischemic stroke, kidney transplant, inflammatory lung injury, gastrointestinal inflammatory diseases, pseudomonas lung infection, acute neurotrauma, pancreatic islet transplantation, diabetes, and severe whole body radiation. Thus, new knowledge about activated protein C variants may lead to improved 2nd or 3rd generation biologics for man.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL052246-20
Application #
8639235
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
1994-12-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
20
Fiscal Year
2014
Total Cost
$579,398
Indirect Cost
$273,647
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
von Drygalski, Annette; Bhat, Vikas; Gale, Andrew J et al. (2014) An engineered factor Va prevents bleeding induced by anticoagulant wt activated protein C. PLoS One 9:e104304
Winkler, Ethan A; Sengillo, Jesse D; Sagare, Abhay P et al. (2014) Blood-spinal cord barrier disruption contributes to early motor-neuron degeneration in ALS-model mice. Proc Natl Acad Sci U S A 111:E1035-42
Deguchi, H; Elias, D J; Griffin, J H (2014) Gain in translation: heme oxygenase-1 induced by activated protein C promotes thrombus resolution. J Thromb Haemost 12:90-2
von Drygalski, A; Cramer, T J; Bhat, V et al. (2014) Improved hemostasis in hemophilia mice by means of an engineered factor Va mutant. J Thromb Haemost 12:363-72
von Drygalski, Annette; Furlan-Freguia, Christian; Ruf, Wolfram et al. (2013) Organ-specific protection against lipopolysaccharide-induced vascular leak is dependent on the endothelial protein C receptor. Arterioscler Thromb Vasc Biol 33:769-76
Wang, Yaoming; Zhao, Zhen; Chow, Nienwen et al. (2013) Activated protein C analog promotes neurogenesis and improves neurological outcome after focal ischemic stroke in mice via protease activated receptor 1. Brain Res 1507:97-104
Wang, Yaoming; Zhao, Zhen; Chow, Nienwen et al. (2013) Activated protein C analog protects from ischemic stroke and extends the therapeutic window of tissue-type plasminogen activator in aged female mice and hypertensive rats. Stroke 44:3529-36
Wang, Yaoming; Sinha, Ranjeet Kumar; Mosnier, Laurent O et al. (2013) Neurotoxicity of the anticoagulant-selective E149A-activated protein C variant after focal ischemic stroke in mice. Blood Cells Mol Dis 51:104-8
Burnier, Laurent; Fernandez, Jose A; Griffin, John H (2013) Antibody SPC-54 provides acute in vivo blockage of the murine protein C system. Blood Cells Mol Dis 50:252-8
Hansen, John-Bjarne; Fernandez, Jose A; Borch, Knut H et al. (2012) Activated protein C plasma levels in the fasting and postprandial states among patients with previous unprovoked venous thromboembolism. Thromb Res 129:502-7

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