The overall objective of our research is to understand the structure- function relationships of proteins that are involved in blood coagulation and anticoagulation. We are focusing herein on the structural biochemistry of human protein C (PC) and activated protein C (APC), an anticoagulant zymogen and enzyme, respectively. Our overall hypothesis is that by understanding the structure-function relationships of individual regions of these proteins we can employ this knowledge to effectively up- regulate or downregulate certain functions, and to incorporate features of unrelated proteins. Proteins of the class of PC and APC contain areas classified as domains, which include the gamma-carboxyglutamic acid (Gla) domain (GD), a helical stretch (HS), two consecutive epidermal growth factor-like (EGF) domains, and a serine protease (PD) homology domain. It is hypothesized that these regions incorporate different functions of these proteins. We propose to rigorously define the roles of these individual domains and to assess whether they can function independently.
Five specific aims are proposed: (l) to synthesize polypeptides containing amino acid sequences present in the [GD]-[HS], [HS]-[EGF1] and [PD] domains of PC and APC and to utilize these polypeptides to define details of their Ca2+-related properties; (2) to express cDNAs containing domain combinations of regions of PC and APC, particularly the [GD]-]HS]-[EGF1], [GD]-[HS]-[EGF1]-[EGF2], [HS]-[EGF1]- [EGF2]-[PD], [EGF2]-[PD] and [PD] domains, and to determine whether these constructs possess or inhibit functional Ca2+-dependent properties of the intact proteins; (3) to evaluate the importance of specific amino acid residues in the Ca2+-dependent structural and functional properties of r- PC and r-APC by site-directed mutagenesis strategies; (4) to construct and express r-chimeric PC, thrombomodulin (TM), fvII, and fIX mutants in which [GD] (where relevant) and/or [EGF]-like domains have been exchanged between these proteins, and to determine whether the structural and functional properties of both the chimeric protein and the newly incorporated domain have been gained or lost; and (5) to perform charge- to-alanine replacements of residues within [EGF1], [EGF2], and [PD] of PC in order to reveal locations within these modules that are involved in activation of PC by thrombin and thrombin/TM, and the loci of APC that are responsible for inactivation of fVa and fVIII. The significance of this work lies in development of a rigorous basic understanding of the functions of domains of proteins and, as a result of this knowledge, to potentially incorporate desired features of one protein into another in a rational and predictable manner. This will allow strategic molecular drug design to occur which could result in therapeutic benefits in patients with coagulation abnormalities.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL019982-22
Application #
2668632
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1976-12-01
Project End
2000-02-29
Budget Start
1998-03-01
Budget End
1999-02-28
Support Year
22
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Cheriyan, John; Balsara, Rashna D; Hansen, Kasper B et al. (2016) Pharmacology of triheteromeric N-Methyl-D-Aspartate Receptors. Neurosci Lett 617:240-6
Balsara, Rashna; Dang, Alexander; Donahue, Deborah L et al. (2015) Conantokin-G attenuates detrimental effects of NMDAR hyperactivity in an ischemic rat model of stroke. PLoS One 10:e0122840
Cheriyan, John; Mezes, Christina; Zhou, Ning et al. (2015) Heteromerization of ligand binding domains of N-methyl-D-aspartate receptor requires both coagonists, L-glutamate and glycine. Biochemistry 54:787-94
Kunda, Shailaja; Yuan, Yue; Balsara, Rashna D et al. (2015) Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors. J Biol Chem 290:18156-72
Balsara, Rashna D; Ferreira, Ashley N; Donahue, Deborah L et al. (2014) Probing NMDA receptor GluN2A and GluN2B subunit expression and distribution in cortical neurons. Neuropharmacology 79:542-9
Huang, Luoxiu; Balsara, Rashna D; Castellino, Francis J (2014) Synthetic conantokin peptides potently inhibit N-methyl-D-aspartate receptor-mediated currents of retinal ganglion cells. J Neurosci Res 92:1767-74
Balsara, Rashna D; Chapman, Sarah E; Sander, Ian M et al. (2014) Non-invasive imaging and analysis of cerebral ischemia in living rats using positron emission tomography with 18F-FDG. J Vis Exp :
Ploplis, Victoria A; Donahue, Deborah L; Sandoval-Cooper, Mayra J et al. (2014) Systemic platelet dysfunction is the result of local dysregulated coagulation and platelet activation in the brain in a rat model of isolated traumatic brain injury. J Neurotrauma 31:1672-5
Castellino, Francis J; Chapman, Michael P; Donahue, Deborah L et al. (2014) Traumatic brain injury causes platelet adenosine diphosphate and arachidonic acid receptor inhibition independent of hemorrhagic shock in humans and rats. J Trauma Acute Care Surg 76:1169-76
Donahue, Deborah L; Beck, Julia; Fritz, Braxton et al. (2014) Early platelet dysfunction in a rodent model of blunt traumatic brain injury reflects the acute traumatic coagulopathy found in humans. J Neurotrauma 31:404-10

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