Platelet Biomechanics and the Composition of Structural Elements Are Influenced by Mechanical Stimulation and Affect Mechanoresponse
AdvisorSlepian, Marvin J.
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PublisherThe University of Arizona.
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AbstractThrombosis is a significant cause of morbidity and mortality in patients with ventricular assist devices. The activation of platelets by physical forces in these devices is understood to be a major driver of thrombosis in this patient population, and is an event which is not blocked by current anti-platelet or anticoagulant pharmaceuticals. Such cellular reactivity to physical forces requires some form of sensation mechanism on the exterior of the cell and the transmittance of the physical signal into a chemical signal on the interior of the cell. The membrane and the cytoskeleton are interconnected elements which are known to allow other cell types to interact with their mechanical environment. These two elements maintain the “architectural” aspects of structure and shape as well as playing roles in cellular function and signal transduction. As structural molecules, these components are capable of re-distributing forces applied to the cell. Both are furthermore known to undergo dramatic rearrangement on platelet activation. Therefore, we hypothesize that the constitutive composition and arrangement of the membrane and cytoskeleton play an important role in platelet biomechanics such that these properties are altered by shear stress and that changing these properties will result in an altered mechanotransductive response.To this end, we examined the effect of shear on platelet lipid composition and mobility, evaluated the effect of membrane organization on platelet response to shear, quantified the effect of mechanical stimulation on platelet mechanical properties and cytoskeletal arrangement, and considered the potential of membrane or cytoskeletal re-organization as a “mechanoceutical” strategy for the inhibition of platelet shear response. In order to examine the effect of shear on lipid composition and mobility, we used mass spectrometry to perform a lipidomic analysis of lipids associated with and outside the platelet, and fluorescence anisotropy to determine lipid mobility within non-activated and activated platelets. We identified a unique lipid profile in shear-activated platelets indicative of non-specific loss of pro-coagulant lipid species. To investigate the influence of membrane organization on shear response, platelets exposed to the membrane modulator dimethylsulfoxide (DMSO) were examined by descriptive and functional metrics and for its ability to inhibit platelet response to shear. DMSO was determined to successfully inhibit shear-mediated activation at low doses by non-specifically decreasing membrane organization and inhibiting signal transduction. The effect of force on mechanical properties and cytoskeletal arrangement was determined using atomic force microscopy as both an imaging method and a mechanical stimuli. Platelets were found to rearrange their cytoskeleton upon the application of physical force, however this capability broke down at forces either high in magnitude or frequency. Finally, we examined potential molecules for further “mechanoceutical” applications. Although cytoskeletal modification could be successful in preventing responsiveness to shear, membrane modulation, particularly using cholesterol-like molecules, was determined to be the most promising strategy for inhibiting response to physical forces while maintaining a biochemical response.
Degree ProgramGraduate College