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Development of vascular injury models to measure the interactions between platelets, endothelial cells and nitric oxide under physiological flow conditions
Sylman, Joanna
Sylman, Joanna
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2015
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2016-09-24
Abstract
The formation of a stable clot is a balance between pro- and antithrombotic biochemical mechanisms coupled to biophysical mechanisms mediated by local hemodynamics. A disruption in this balance leads to excessive clotting, or thrombosis, which is the leading cause of death in the United States. Blood clots form at the site of a vascular injury by platelet adhesion, activation, and aggregation coupled to a network of reactions called coagulation. Endothelial cells (EC) that line blood vessels also help regulate clot formation by secretion of platelet inhibitors such as nitric oxide (NO), supporting the activated protein C anticoagulation pathway, and expression of adhesive ligands that promote blood cell adhesion. The relative roles of these EC-mediated pro- and antithrombotic pathways for different types of injuries and in different vascular beds are largely unknown, despite detailed knowledge of the biochemistry and molecular biology of these pathways. Specifically, there is little known about the distribution of EC-secreted platelet inhibitor at the site of a focal injury, and how the transport of those inhibitors is influenced by blood flow. In this thesis, microfluidic vascular injury models and the finite element method were used to measure and model how the spatial and temporal distribution of NO and EC activation affect platelet function. NO is a free radical synthesized and released by the endothelium in a shear stress dependent manner that modulates platelet function. The flux of NO at the site of an injury is unknown. In my first study, synthetic NO-releasing polymers were used to mimic endothelial function, in which the NO flux was decoupled from the shear stress. I studied collagen-mediated platelet adhesion and aggregation over a range of physiological shear rates and NO fluxes. NO was found to induce measureable inhibition of platelet aggregation at fluxes of 0.33× 10-10 mol cm-2 min-1 to 2.5× 10-10 mol cm-2 min-1 at shear rates of 200-500 s-1. A computational model of NO transport was developed to determine the mass transfer limitations of NO in mediating platelet inhibition. The NO distribution was found to be reaction limited in the platelet rich layer, formed when RBC develop a core and there is margination of platelets to the vessel wall, and transport limited in the RBC core. The outcome of this study was the first report to isolate the shear rate dependent effect of NO on platelet aggregation. It is generally accepted that NO inhibition of platelets occurs primarily through soluble guanylyl cyclase (sGC)-dependent pathways. But, recent studies suggested that sGC-independent mechanisms might also mediate inhibition at millimolar NO donor concentration. In this study, I used the NO-releasing polymer system described above to determine the relative role of sGC dependent and independent signaling. Platelets treated with a small molecule inhibitor of sGC showed the existence of an independent pathway at an NO flux of 6.8×10-10 mol cm-2 min-1 at 200-500 s-1 which corresponded to an NO concentration of 65 – 240 nM. This outcome of this study is the first report of a sGC independent pathway in flowing whole blood. Endothelial cells (EC) inhibit and promote platelet activation in a shear stress dependent manner. Previous studies of EC-platelet interactions use chemical activators to indiscriminately activate entire monolayers of EC. In this study, I focally activated EC with heat using surface microelectrodes to create a well-defined injury zone of activated EC supporting platelet adhesion surrounded by quiescent EC inhibiting platelet activation. A computational model of the heat transfer was built to determine the in situ temperature increases directly under the cells at each voltage, which dictated whether the EC remained quiescent, became activated, or were killed. Platelet adhesion was supported between activated EC by EC-derived von Willebrand factor and laminin. We expect this model will be useful in ongoing studies of platelet-EC interactions. EC-derived NO contributes to platelet inhibition but it is unknown under which conditions it is the most important. Using the previously established EC-based focal injury model, the platelet aggregate formation was measured in the presence and absence of an endothelial nitric oxide synthase inhibitor. It was hypothesized that NO inhibits the cross-talk between upstream and downstream injuries by inhibiting platelet activation in a shear stress dependent manner. NO was found to have a pronounced effect in a dual injury configuration and when EC were conditioned by shear stress for days prior to the injury. Overall, the relationship between clot size and EC function under physiological flow conditions was determined by creation of a series of microfluidic focal vascular injury models which allowed for spatial and temporal control of NO release and EC activation.
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