This project studies mechanotransduction of platelets in health and diseases. Novel single-cell and single-molecule biophysical analyses will be employed to elucidate the mechanisms of mechanoreception by platelet glycoprotein (GP) Ib-IX-V complex. GPIb? mediates platelet rolling by binding to von Willebrand factor (VWF). Our preliminary studies showed that mechanical force exerted on GPIb?-VWF bonds can induce conformational changes in two regions of the GPIb? molecule. Importantly, these conformational change events were followed by induction of intraplatelet calcium fluxes. The duration, the magnitude, and the frequency of force were found to be important parameters in triggering platelet Ca2+ signaling. Furthermore, we identified an intermediate affinity state of integrin ?IIb?3 as induced by GPIb?-VWF interaction under force. We hypothesize that GPIb? functions as a mechanoreceptor such that force regulates its ligand binding and signaling processes. Dysregulation of GPIb? mechanoreception results in aberrant adhesion and signaling, leading to diseases in hemostasis and thrombosis. Previous methods of studying platelet signaling are of low spatial, temporal, and force resolutions, with insufficient capabilites for direct characterization of single- molecule dynamics, and unable to synchronize the adhesion and signaling observations at the single-platelet level. Our newly developed fluorescence biomembrane force probe technology overcomes these limitations. We will measure the kinetics of GPIb? interacting with VWF and simultaneously monitor signaling events including intracellular Ca2+ and cGMP levels as well as integrin activation.
Three specific aims are proposed to test our hypothesis: 1) signal initiation by platelet mechanoreceptor GPIb?; 2) interplay between force-induced Ca2+ and cGMP signaling, and 3) dysregulated platelet mechanotransduction in human diseases. The proposed studies are innovative and unprecedented in several ways: in the ability to quantify signaling in single platelets in real-time concurrently with analysis of binding kinetics, and in defining the relationships among force attributes, bond characteristics, and signaling outcomes at the single-platelet and single-molecule levels. Importantly, we will elucidate the underlying mechanisms of the biophysical observation using mouse models targeting key molecular players of signaling pathways and relate dysregulation of mechanoreception by GPIb? with human diseases, including the bleeding disorder von Willebrand diseases (VWD) and thrombotic disorder thrombotic thrombocytopenic purpura (TTP). These studies will elucidate the force-regulated mechanreception of an important receptor on the platelet surface and thus provide key insights into vascular physiology and pathology, which will serve as a foundation for designing new therapeutic approaches to inhibiting pathological platelet function during thrombosis and/or intervention to VWD and TTP.

Public Health Relevance

This project analyzes the mechanoreception of a major platelet surface receptor from both healthy donors and patients with von Willebrand diseases and with thrombotic thrombocytopenic purpura. This molecule is crucial in the initiation of the adhesion and signaling cascade in the hemostatic and thrombotic processes; thus the proposed work helps provide insights to these bleeding and thrombotic disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL132019-03
Application #
9462219
Study Section
Intercellular Interactions Study Section (ICI)
Program Officer
Sarkar, Rita
Project Start
2016-04-01
Project End
2020-03-31
Budget Start
2018-04-01
Budget End
2019-03-31
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Georgia Institute of Technology
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
097394084
City
Atlanta
State
GA
Country
United States
Zip Code
30318
Chen, Yunfeng; Ruggeri, Zaverio M; Du, Xiaoping (2018) 14-3-3 proteins in platelet biology and glycoprotein Ib-IX signaling. Blood 131:2436-2448
Ju, Lining; McFadyen, James D; Al-Daher, Saheb et al. (2018) Compression force sensing regulates integrin ?IIb?3 adhesive function on diabetic platelets. Nat Commun 9:1087
Xu, Xiaohong Ruby; Wang, Yiming; Adili, Reheman et al. (2018) Apolipoprotein A-IV binds ?IIb?3 integrin and inhibits thrombosis. Nat Commun 9:3608
Zhou, F; Chen, Y; Felner, E I et al. (2018) Microfluidic auto-alignment of protein patterns for dissecting multi-receptor crosstalk in platelets. Lab Chip 18:2966-2974
Pang, Aiming; Cui, Yujie; Chen, Yunfeng et al. (2018) Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation. Blood 132:533-543
Zhu, Guangheng; Zhang, Qing; Reddy, Emily C et al. (2017) The integrin PSI domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy. Blood 129:1840-1854
Ju, Lining; Zhu, Cheng (2017) Benchmarks of Biomembrane Force Probe Spring Constant Models. Biophys J 113:2842-2845
Ju, Lining; Chen, Yunfeng; Li, Kaitao et al. (2017) Dual Biomembrane Force Probe enables single-cell mechanical analysis of signal crosstalk between multiple molecular species. Sci Rep 7:14185
Mehta-D'souza, Padmaja; Klopocki, Arkadiusz G; Oganesyan, Vaheh et al. (2017) Glycan Bound to the Selectin Low Affinity State Engages Glu-88 to Stabilize the High Affinity State under Force. J Biol Chem 292:2510-2518
Chen, Yunfeng; Ju, Lining; Rushdi, Muaz et al. (2017) Receptor-mediated cell mechanosensing. Mol Biol Cell 28:3134-3155

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