Date of Award

Spring 5-16-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemical and Biomolecular Engineering

First Advisor

Scott L. Diamond

Abstract

Blood clotting is a highly conserved physiological response that prevents excessive blood loss following vessel injury. It involves a sequence of plasma reactions leading to the formation of thromin (the coagulation cascade) as well as tightly controlled intracellular reactions mediating platelet activation. These two events are inextricably coupled, with the active platelet surface serving as a cofactor for coagulation factor assembly and thrombin serving as a potent platelet agonist. Using the technologies of automated liquid handling, high throughput experimental systems were developed that allowed individual exploration of these two components of the thrombotic response under diverse initial conditions. Based on this high dimensional experimental exploration, a “bottom-up” mechanism based Ordinary Differential Equation (ODE) description of thrombin generation kinetics and a “top-down” data driven Neural Network model of platelet activation were developed. In the first study, “contact activation” (and not “blood-borne TF” alone) despite the best available inhibitor to prevent it, was found build up enough autocatalytic strength to trigger coagulation in the absence of exogenous tissue factor, particularly upon activated platelets. Further, the “Platelet-Plasma model” successfully predicted the stability of blood under multiple perturbations with active enzymes at various physiologically realizable conditions. In the second study, “Pairwise Agonist Scanning” (PAS), a strategy that trains a Neural Network model based on measurements of cellular responses to individual and all pairwise combinations of input signals is described. PAS was used to predict calcium signaling responses of human platelets in EDTA-treated plasma to six different agonists (ADP, Convulxin, U46619, SFLLRN, AYPGKF and PGE2). The model predicted responses to sequentially added agonists, to ternary combinations of agonists and to 45 different combinations of four to six agonists (R=0.88). Furthermore, PAS was used to distinguish between the phenotypic responses of platelets from healthy human donors. Taken together, these two studies lay the groundwork for integration of coagulation reaction kinetics and donor specific descriptions of platelet function with models of convection and diffusion to simulate thrombosis under flow.


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