Date of Award

7-2014

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Kimberly J Baker, PhD

Second Advisor

Francis E Jenney, Jr, PhD

Third Advisor

Harold Komiskey, PhD

Fourth Advisor

Brian M Matayoshi, PhD

Abstract

The blood coagulation cascade is activated under various circumstances such as an injury. This system involves a tightly regulated series of events. The enzymes involved assemble with their respective cofactors on lipid membranes to reach their full procoagulant complex potential. The coagulation cascade is divided into intrinsic and extrinsic portions, both of which converge into a common pathway with the activation of factor X (FXa). FXa is a very important part of the blood coagulation cascade because its activation is primarily responsible for thrombin generation. Activation of FXa alone is, however, insufficient to produce a fully active thrombin. For this to occur, FXa must form a complex with its protein cofactor Factor Va and its protein substrate prothrombin on a phospholipid surface. This suggests that the topography of the enzyme on the lipid surface changes when in a fully pro-coagulant state. However, the topography of FXa alone and in complex with these cofactors is poorly understood. Kunitz-type protein inhibitors, such as basic pancreatic trypsin inhibitor (BPTI), are globular proteins, which inhibit serine proteases such as FXa. These small protein inhibitors fit just inside the active site of the enzyme, making them ideal candidates with which to study the active site of FXa. The goal of this project is to gain understanding of the topography of fully active FXa by using lipid-anchored BPTI with linkers that act as molecular rulers to measure the range of reactive heights of the active site of FXa in its pro-coagulant complex on a phospholipid surface.

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