Date of Award

Spring 5-17-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemical and Biomolecular Engineering

First Advisor

Scott L. Diamond

Abstract

Current ex vivo models of thrombosis either fail to accurately recreate the hemodynamics and focal nature of injuries commonly found throughout the vasculature or require excess amounts of material. Development of a novel microfluidic thrombosis model has allowed for the measurement of thrombus stability of platelet aggregates formed on collagen from a single non-lethal murine blood draw. It was observed that platelet accumulation exhibits a biphasic behavior, with maximum accumulation at an average wall shear rate of 400 1/sec, with engagement of alpha2beta1 integrin a mandatory factor due to complete loss of adhesion in blood from a knockout animal. It was found that PAR4 stimulation of pre-formed thrombi increased aggregate stability by 50%. Subsequent device designs were made to simultaneously measure platelet accumulation on collagen of eight samples under independent bulk pharmacological intervention. This provided a platform for rapidly and repeatable assessing the potency of receptor antagonists under relevant flow conditions with half-maximal inhibitor concentrations (P2Y1 inhibitor MRS 2179 IC50=0.23 uM, P2Y12 inhibitor 2-MeSAMP IC50=2.56 uM) comparable to published data, while an enzyme commonly used failed to have any effect due to transport limitations. Finally, investigation into the effects of tissue factor initiated coagulation in addition to collagen-induced platelet accumulation and activation revealed a number of technical hurdles required for further experimentation. The use of custom functional liposomes as linkers for surface immobilization provides a platform for careful titration of active tissue factor onto a surface at high densities (up to 35 active tissue factor molecules per um^2) for illumination into the role and extent of thrombin and fibrin formation during hemostatic injury. Additionally, geometries akin to late stage atherosclerotic vessels open the possibility of shear history affecting the growth and stability of platelet thrombi formed on collagen. Investigation of bulk phase shear-mediated von Willebrand Factor conformational changes could illuminate both mechanistic processes involved in pathological thrombosis and provide a basis for new biomarkers for patients at risk for a catastrophic thrombotic event.

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