In models of vascular injury, the engagement of blood platelets by substrate-immobilized von Willebrand Factor (vWF) under fluid flow leads to platelet activation and subsequent thrombus formation. Experiments carried out in suspension assays that apply fluid forces on platelets and blood proteins also demonstrate that mechanical forces cause changes in platelet membrane phospholipid distribution, and they augment shear-induced platelet activation and aggregation. In our recent studies on platelet activation, we proposed a two-step mechanism of cell activation to explain these observations based on the relative roles of platelet receptor Gplb, plasma vWF and fluid forces. As opposed to traditional scaling arguments, we developed rigorous computational methods to estimate the magnitude and nature of fluid forces applied on cells and molecules under these conditions. Further, we observed that vWF undergoes self-association or aggregation when mixed under defined conditions. This suggests that protein conformation may change under fluid shear flow and this may have functional consequences. Based on these observations, the specific goals of this project are: 1) To determine the physiological fluid shear conditions under which vWF unimers may self-associate. For this aspect, light scattering, chromatography, western blot analysis and surface plasmon resonance are employed to detect vWF self-association, and to determine the kinetics/affinity of this process. Studies of shear- induced platelet activation are also conducted to establish a mechanistic link between vWF self- association and platelet activation. 2) To demonstrate that the size of the vWF molecule and length of platelet Gplb receptor are critical parameters regulating platelet activation rates under fluid shear. In order to do this, we create microspheres and nanoparticles of varying sizes bearing immobilized antibodies and recombinant forms of vWF at varying densities. The ability of these particles to bind and activate cells is quantified. 3) To characterize conformational changes in vWF under physiological fluid shear conditions. Here, the solution structure of vWF and protein conformational changes under fluid shear are measured using light, neutron and X-ray scattering spectroscopy. New mathematical theories are developed to quantitatively guide the interpretation of the above experiments. Successful completion of this work will establish that fluid shear may regulate bio-molecule structure and function. Results linking self-association and platelet activation, in the long run, may also prompt in vivo examination of this phenomenon and stimulate new drug development against self-association. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL077258-02
Application #
7228989
Study Section
Bioengineering, Technology and Surgical Sciences Study Section (BTSS)
Program Officer
Sarkar, Rita
Project Start
2006-05-01
Project End
2010-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
2
Fiscal Year
2007
Total Cost
$370,285
Indirect Cost
Name
State University of New York at Buffalo
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Zhang, C; Kelkar, A; Nasirikenari, M et al. (2018) The physical spacing between the von Willebrand factor D'D3 and A1 domains regulates platelet adhesion in vitro and in vivo. J Thromb Haemost 16:571-582
Gogia, Shobhit; Kelkar, Anju; Zhang, Changjie et al. (2017) Role of calcium in regulating the intra- and extracellular cleavage of von Willebrand factor by the protease ADAMTS13. Blood Adv 1:2063-2074
Zhang, Changjie; Neelamegham, Sriram (2017) Application of microfluidic devices in studies of thrombosis and hemostasis. Platelets 28:434-440
Liu, Gang; Cheng, Kai; Lo, Chi Y et al. (2017) A Comprehensive, Open-source Platform for Mass Spectrometry-based Glycoproteomics Data Analysis. Mol Cell Proteomics 16:2032-2047
Nascimbene, Angelo; Neelamegham, Sriram; Frazier, O H et al. (2016) Acquired von Willebrand syndrome associated with left ventricular assist device. Blood 127:3133-41
Hubbard, A R; Heath, A B; Kremer Hovinga, J A et al. (2015) Establishment of the WHO 1st International Standard ADAMTS13, plasma (12/252): communication from the SSC of the ISTH. J Thromb Haemost 13:1151-3
Gogia, Shobhit; Lo, Chi Y; Neelamegham, Sriram (2015) Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13. PLoS One 10:e0126556
Gogia, Shobhit; Neelamegham, Sriram (2015) Role of fluid shear stress in regulating VWF structure, function and related blood disorders. Biorheology 52:319-35
Shao, Shuai; Geng, Jumin; Ah Yi, Hyun et al. (2015) Functionalization of cobalt porphyrin-phospholipid bilayers with his-tagged ligands and antigens. Nat Chem 7:438-46
Madabhushi, Sri R; Zhang, Changjie; Kelkar, Anju et al. (2014) Platelet GpIba binding to von Willebrand Factor under fluid shear:contributions of the D?D3-domain, A1-domain flanking peptide and O-linked glycans. J Am Heart Assoc 3:e001420

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