As the second most expressed membrane receptor complex on the platelet surface, the glycoprotein (GP) Ib-IX-V complex is essential to platelet physiology. It is identified primarily as the receptor for a number of hemostatically important proteins including von Willebrand factor and thrombin. It has also been implicated in the genesis and clearance of platelets. Malfunction of this multi-subunit receptor complex can lead to severe bleeding diathesis and contribute to many cardiovascular diseases. However, how this complex carries out its multi-faceted functions is not clear, primarily due to the lack of understanding of its structure and organization. The GPIb-IX-V complex comprises of 4 different type I transmembrane proteins, with the GPIb-IX complex possessing the ligand-binding activity and signaling capability. We hypothesize that inter-subunit interactions are important to the functions and regulation of the GPIb-IX complex. In this project, we aim to elucidate its organizing principle and to explore functional roles of inter-subunit interactions.
In Specific Aim 1, to characterize the postulated weak interaction between the ectodomains of GPIb1 and GPIX subunits, we will define the interfacial region and residues in both domains and determine their roles in the assembly and inside-out regulation of the receptor complex.
Specific Aim 2 is to elucidate the structural basis for the inter-ectodomain interactions. Structure of the recombinant GPIb2 ectodomain will be determined by X-ray crystallography. Engineered GPIX variants with improved stability that retain the native binding region to GPIb2 and GPIb1 will be produced for structural determination and functional analysis.
Specific Aim 3 is to elucidate the structural basis for the interaction among transmembrane helices in the GPIb-IX complex by heteronuclear triple resonance NMR spectroscopy. Overall, careful biochemical and structural dissection of individual interactions in isolated protein domains, in transfected mammalian cells and in human platelets will help to elucidate the likely structural changes in the GPIb-IX complex in response to intracellular regulatory signals and ligand binding, and to provide insights on the molecular basis for GPIb-IX-related diseases. This project will also afford us the opportunity to develop tools and reagents to specifically perturb the inter-subunit interaction of interest, which may lead to novel therapeutic strategies.

Public Health Relevance

The glycoprotein Ib-IX-V complex in the platelet helps the blood to clot properly. Malfunction of this complex leads to severe bleeding and can contribute to many cardiovascular diseases. This project seeks to determine the structure of this complex and to learn how it functions and how it is regulated.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL082808-07
Application #
8207976
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Sarkar, Rita
Project Start
2006-02-01
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
7
Fiscal Year
2012
Total Cost
$359,550
Indirect Cost
$120,980
Name
Emory University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Zhou, L; Yang, W; Li, R (2014) Analysis of inter-subunit contacts reveals the structural malleability of extracellular domains in platelet glycoprotein Ib-IX complex. J Thromb Haemost 12:82-9
Li, R; Emsley, J (2013) The organizing principle of the platelet glycoprotein Ib-IX-V complex. J Thromb Haemost 11:605-14
Liang, X; Russell, S R; Estelle, S et al. (2013) Specific inhibition of ectodomain shedding of glycoprotein Ib? by targeting its juxtamembrane shedding cleavage site. J Thromb Haemost 11:2155-62
Zhou, Zhou; Yeh, Hui-Chun; Jing, Hua et al. (2011) Cysteine residues in CUB-1 domain are critical for ADAMTS13 secretion and stability. Thromb Haemost 105:21-30
McEwan, Paul A; Yang, Wenjun; Carr, Katherine H et al. (2011) Quaternary organization of GPIb-IX complex and insights into Bernard-Soulier syndrome revealed by the structures of GPIb? and a GPIb?/GPIX chimera. Blood 118:5292-301
Yan, Rong; Mo, Xi; Paredes, Angel M et al. (2011) Reconstitution of the platelet glycoprotein Ib-IX complex in phospholipid bilayer Nanodiscs. Biochemistry 50:10598-606
Li, Renhao (2010) A hypothesis that explains the heterogeneity of drug-induced immune thrombocytopenia. Blood 115:914
Mo, Xi; Nguyen, Nam X; Mu, Fi-Tjen et al. (2010) Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha. J Biol Chem 285:32096-104
Mo, X; Nguyen, N X; McEwan, P A et al. (2009) Binding of platelet glycoprotein Ibbeta through the convex surface of leucine-rich repeats domain of glycoprotein IX. J Thromb Haemost 7:1533-40
Zhou, Zhou; Jing, Hua; Tao, Zhenyin et al. (2009) Effects of naturally occurring mutations in CUB-1 domain on synthesis, stability, and activity of ADAMTS-13. Thromb Res 124:323-7

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