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.
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.
|Fong, Karen P; Zhu, Hua; Span, Lisa M et al. (2016) Directly Activating the Integrin Î±IIbÎ²3 Initiates Outside-In Signaling by Causing Î±IIbÎ²3 Clustering. J Biol Chem 291:11706-16|
|Litvinov, Rustem I; Farrell, David H; Weisel, John W et al. (2016) The Platelet Integrin Î±IIbÎ²3 Differentially Interacts with Fibrin Versus Fibrinogen. J Biol Chem 291:7858-67|
|Bennett, Joel S (2016) Shedding New Light on the Platelet Storage Lesion. Arterioscler Thromb Vasc Biol 36:1715-6|
|Fuentes, Rudy E; Zaitsev, Sergei; Ahn, Hyun Sook et al. (2016) A chimeric platelet-targeted urokinase prodrug selectively blocks new thrombus formation. J Clin Invest 126:483-94|
|Armstead, William M; Riley, John; Yarovoi, Serge et al. (2016) Tissue-Type Plasminogen Activator-A296-299 Prevents Impairment of Cerebral Autoregulation After Stroke Through Lipoprotein-Related Receptor-Dependent Increase in cAMP and p38. Stroke 47:2096-102|
|Welsh, John D; Muthard, Ryan W; Stalker, Timothy J et al. (2016) A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature. Blood 127:1598-605|
|Sayani, Farzana A; Abrams, Charles S (2015) How I treat refractory thrombotic thrombocytopenic purpura. Blood 125:3860-7|
|Meng, Ronghua; Wu, Jie; Harper, Dawn C et al. (2015) Defective release of Î± granule and lysosome contents from platelets in mouse Hermansky-Pudlak syndrome models. Blood 125:1623-32|
|Ma, Peisong; Ou, Kristy; Sinnamon, Andrew J et al. (2015) Modulating platelet reactivity through control of RGS18 availability. Blood 126:2611-20|
|Muthard, Ryan W; Welsh, John D; Brass, Lawrence F et al. (2015) Fibrin, Î³'-fibrinogen, and transclot pressure gradient control hemostatic clot growth during human blood flow over a collagen/tissue factor wound. Arterioscler Thromb Vasc Biol 35:645-54|
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