Project 1: Integrins are a family of ubiquitous transmembrane heterodimers that mediate fundamental processes requiring cell-matrix and cell-cell interactions and reside on cell surfaces in an equilibrium between resting inactive molecules and active ligand-binding molecules. Like other proteins, integrins are """"""""soft"""""""" amino acid polymers that continuously sample ensembles of conformational states. Conversion from their resting to active states occurs when allosteric modulators such the divalent cation Mn[2+] or the cytoskeletal protein talin shift the distribution of conformations from one pre-existing population to another. The resting integrin conformation is enforced by non-convalent interactions involving their membraneproximal cytoplasmic, transmembrane, and juxtamembrane extracellular domains;these interactions are disrupted when an integrin shifts to its active conformation. This project is focused on the platelet integrin allbB3 , a receptor for macromolecular ligands such as fibrinogen and von Willebr and factor (VWF) following platelet stimulation. Binding of these ligands to allbp3 is responsible for platelet aggregation and is a critical step in the formation of hemostatic platelet plugs and pathologic arterial thrombi. The overall goals of the project are gain a thermodynamic understanding of allbp3 regulation and to use this understanding to develop novel allosteric modulators to attenuate allbp3 function.
In Aim 1, we will generate a thermodynamic model for allbp3 activation, testing the hypothesis that topographically-distinct interactions between allb and B3 differentially regulate allbp3 activation. The relative contribution of the membrane-proximal cytoplasmic, transmembrane, and juxtamembrane extracellular domains of allb or B3 to maintaining allbB3 in its resting conformation will be quantitated using a novel AraC-based bacterial transcriptional reporter system, as well as optical tweezers-based force spectroscopy. The data will be used to derive a thermodynamic model of allbB3 activation. Chemical libraries and molecular databases will then be screened for potential allbB3 inhibitors that bind to the relevant extracellular hot spot regions of either allb or B3.
In Aim 2, we will use soluble anti-transmembrane domain peptides to modify the activation state of individual allbB3 molecules in situ. By destabilizing the allbB3 heterodimer, we found that an anti-allb transmembrane domain peptide causes ligand binding-independent transactivation of B3-bound c-Src, implying that allbB3 activation alone is sufficient to cause rapid allbB3 oligomerization. Conversely, by stabilizing the allbB3 heterodimer, transmembrane-domain targeted peptides would act as novel allbB3 antagonists. Thus, we propose using computational methods to design peptides that stabilize the allbB3 TM domain heterodimer, preventing allbB3 activation and serving as lead compounds for the development of novel antithrombotic agents.

Public Health Relevance

Fibrinogen and von Willebr and factor binding to the active form of the integrin allbB3 is responsible for the platelet stickiness that quenches bleeding after trauma and causes the thrombi that complicate atherosclerosis. However, current intravenous allbB3 inhibitors have limited clinical applicability and oral inhibitors were associated with excess mortality. The goals of this project are to determine how platelets regulate allbB3 function and to use this information to design novel, effective, and safer allbB3 inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
2P01HL040387-26A1
Application #
8694330
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2014-05-16
Budget End
2015-04-30
Support Year
26
Fiscal Year
2014
Total Cost
$777,944
Indirect Cost
$218,295
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Greineder, Colin F; Johnston, Ian H; Villa, Carlos H et al. (2017) ICAM-1-targeted thrombomodulin mitigates tissue factor-driven inflammatory thrombosis in a human endothelialized microfluidic model. Blood Adv 1:1452-1465
Tomaiuolo, Maurizio; Brass, Lawrence F; Stalker, Timothy J (2017) Regulation of Platelet Activation and Coagulation and Its Role in Vascular Injury and Arterial Thrombosis. Interv Cardiol Clin 6:1-12
Zhao, Liang; Thorsheim, Chelsea L; Suzuki, Aae et al. (2017) Phosphatidylinositol transfer protein-? in platelets is inconsequential for thrombosis yet is utilized for tumor metastasis. Nat Commun 8:1216
Shen, Jian; Sampietro, Sara; Wu, Jie et al. (2017) Coordination of platelet agonist signaling during the hemostatic response in vivo. Blood Adv 1:2767-2775
Kononova, Olga; Litvinov, Rustem I; Blokhin, Dmitry S et al. (2017) Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin ?IIb?3. Biochemistry 56:1932-1942
Höök, Peter; Litvinov, Rustem I; Kim, Oleg V et al. (2017) Strong Binding of Platelet Integrin ?IIb?3 to Fibrin Clots: Potential Target to Destabilize Thrombi. Sci Rep 7:13001
Welsh, J D; Poventud-Fuentes, I; Sampietro, S et al. (2017) Hierarchical organization of the hemostatic response to penetrating injuries in the mouse macrovasculature. J Thromb Haemost 15:526-537
Cuker, Adam; Husseinzadeh, Holleh; Lebedeva, Tatiana et al. (2016) Rapid Evaluation of Platelet Function With T2 Magnetic Resonance. Am J Clin Pathol 146:681-693
Bennett, Joel S (2016) Shedding New Light on the Platelet Storage Lesion. Arterioscler Thromb Vasc Biol 36:1715-6
Zhu, S; Welsh, J D; Brass, L F et al. (2016) Platelet-targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow. J Thromb Haemost 14:1070-81

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