Date of Award

7-2019

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Kimberly Baker, PhD

Second Advisor

Shu Zhu, MD, PhD

Third Advisor

Richard E. White, PhD, FAHA

Abstract

Blood coagulation, an important hemostatic property, is directed through activation of proteolytic enzymes known as serine proteases to generate a fibrin plug. Using multiple proteases allows for localized and precise regulation of blood coagulation within the circulatory system. Factor IX, a serine protease, is a key activator in the coagulation cascade by activating Factor X. For activation of FX to occur, FIXa must be in proper conformation on the platelet surface with cofactor FVIIIa to increase enzymatic abilities of FIXa a billion-fold. Current research looks at structure and function of FIXa yet neglects structural plasticity on the membrane, with function of FIX clearly being related to conformation on the platelet. The role of this investigation was to study the optimal reactive topography of FIXa by generating AMRI, Adjustable height Molecular Ruler with attached Inhibitor. AMRI composed of three primary regions contains an Anchor region, Linker region, and Inhibitor. AMRI was generated using the protein anchoring sequence LAGC to fixate to a lipid surface. A height adjustable linker area was then required between the anchor and inhibitor regions of AMRI to ensure proper vertical height of inhibitor. The linker sequence EAAAK forms a rigid structure with a vertical height of 7.5A above the membrane surface, thus we repeated this sequence to achieve a specific desired height. Completing the protein’s linker region is a flexible linker at end of the rigid set. That flexible linker ensures the inhibitor will fit in FIXa active site. Finishing the AMRI complex is a PN2-KPI inhibitor, which has been demonstrated to fit and inhibit the catalytic site of FIXa. Therefore, AMRI should allow for determination of optimal reactive height of the FIXa active site without affecting structural conformation. Through the generation of AMRI, a greater insight into blood coagulation will be gained, along with potential for site specific anticoagulant clinical applications.

Download
COinS