Date of Award

Winter 2-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Steven A. Jones

Abstract

Platelet adhesion is regulated by both activators, such as adenosine diphosphate, and inhibitors, such as nitric oxide (NO). Both agents are released on platelet activation, so that platelets initiate both positive and negative feedback systems. In vivo platelet adhesion models generally consider the effects of platelet activators and inhibitors separately. The goal of this study was to create an environment in which interplay between positive and negative feedback can be observed together and in which the roles of endogenous and exogenous platelet activators are distinguishable. The results are expected to be applicable to the design of stents, which are susceptible to thrombus formation, and which provide multiple adjacent regions where platelet-released agents can interact with one another.

To distinguish between the role of exogenous and endogenous agents, microchannels were produced that had multiple thrombogenic (fibrinogen) regions separated by non-thrombogenic (BSA-coated) regions. This geometry reveals the effect of agents released from different thrombogenic regions on one another. Adhesion was quantified by percent platelet surface area coverage.

Surface area coverage differed between the upstream and downstream sides of the thrombogenic regions. Positive and negative feedback effects were enhanced by increased platelet production of activator and inhibitor. In contrast, when the NO donor DPTA NONOate was added, with the intent to overwhelm the endogenous feedback, a more uniform spatial distribution of adhesion was obtained.

Though these agonists, activator (ADP) and inhibitor (NO), act on different receptors through different signaling pathways, they all lead to increase or decrease in the intracellular Ca2+ concentration ([Ca2+]) depending on the agonist produced. Ca2+ is a key component and serves as second messenger in all cells regulatory processes. The increase in [Ca2+] leads to several steps of activation. The process leading Ca2+ to increase and decrease and its underlying mechanisms remained largely unknown.

To examine Ca2+ response, platelets were loaded with the Fluo-4 Ca2+ ion indicators and fluorescence waveform was monitored. We found that Ca2+ response increased dose dependently with addition of an activator (ADP) and addition of endogenous (L-Arginine) and exogenous (DPTA NONOate) NO donor showed decrease in Ca2+ response in ADP stimulated platelets.

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