Philadelphia College of Osteopathic Medicine DigitalCommons@PCOM PCOM Biomedical Studies Student Scholarship Student Dissertations, Theses and Papers 7-2014 Characterization of Lipid-Anchored Inhibitor Rulers as a Measure of Enzyme Topography in Coagulation Enzyme Factor Xa Robin C. Conley Philadelphia College of Osteopathic Medicine, [email protected] Follow this and additional works at:http://digitalcommons.pcom.edu/biomed Part of theBiochemistry, Biophysics, and Structural Biology Commons, and theMedicine and Health Sciences Commons Recommended Citation Conley, Robin C., "Characterization of Lipid-Anchored Inhibitor Rulers as a Measure of Enzyme Topography in Coagulation Enzyme Factor Xa" (2014).PCOM Biomedical Studies Student Scholarship.Paper 76. This Thesis is brought to you for free and open access by the Student Dissertations, Theses and Papers at DigitalCommons@PCOM. It has been accepted for inclusion in PCOM Biomedical Studies Student Scholarship by an authorized administrator of DigitalCommons@PCOM. For more information, please [email protected]. Characterization of Lipid-Anchored Inhibitor Rulers as a Measure of Enzyme Topography in Coagulation Enzyme Factor X . a By: Robin C. Conley Department of Basic Sciences Philadelphia College of Osteopathic Medicine, Georgia Campus Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Biomedical Science May 2013 I PCOM Biomedical Sciences Degree Programs Signatory Page for Master's Thesis We approve the thesis of Robin Christina Conley. Date of Signature Assistant Professor of Biochemistry Thesis Advisor Date of Signature Associate Professor of Biochemistry Date of Signature Professor of Neuroscience, Physiology, and Pharmacology 7/17 po!1- Brian M. Matayoshi, PhD Date of Signature Graduate Program Director and Professor of Physiology II Table of Contents I. List of Figures II. List of Tables III. Abstract IV. Introduction i. Hemostasis ii. Platelets iii. Vasculature Walls iv. Coagulation Factors v. The Blood Coagulation Cascade vi. Extrinsic Pathway vii. Intrinsic Pathway viii. Cell-Based Model of Coagulation ix. Factor Structure x. Cascade Regulation xi. Traditional Therapies and Treatments xii. The Role of Kunitz Inhibitors xiii. The Importance of Factor X xiv. fX Topography a V. Research Design i. Current Information and Fluorescence Studies ii. Preliminary Studies and Results a) Proof-of-concept using lipid-tethered inhibitors b) Construction of a base vector c) Expression and purification of an active inhibitor d) Relipidation of recombinant proteins VI. Specific Aims i. Specific Aim 1 ii. Specific Aim 2 VII. Materials, Methods, and Results VIII. Discussion IX. Acknowledgements X. Appendices i. Appendix 1: Truncated DNA of pET11d vector system with HindIII, NcoI, and BamHI sites ii. Appendix 2: DNA and protein sequence of BPTI iii. Appendix 3: Primers iv. Appendix 4: Plasmids v. Appendix 5: Sequencing Data XI. References III List of Figures I. GPI Complex II. TF Interaction on the Cell Surface during Coagulation III. Factor Complexes IV. General Overview of Hemostasis V. TF/fVII Complex on Membrane Surface a VI. Overview of Cell-Based Coagulation—Initiation, Propagation, and Amplification VII. FVII Ribbon Diagram a VIII. General fX structure a IX. FX Catalytic Head Ribbon Diagram a X. BPTI Protein Structure XI. N-acyl, s-diacylglyceryl moiety group XII. EA K Linker Structure 3 XIII. Projected Graphic Map of p-LAGC-EAK10-BPTI XIV. Graphic DNA Map of pET11d Vector System XV. Verification of pET11d DNA Purification XVI. Restriction Digest with NcoI XVII. A and B. Alkaline Phosphatase-treated DNA XVIII. Oligonucleotide Synthesis XIX. Annealed LAGC and LINKER Oligonucleotides XX. Observation of Transformation Plates XXI. Ligation 1 Plate 1 XXII. Vector Map of Predicted Recombinant DNA including BamHI site XXIII. Pre-screening of Ligation Colonies IV XXIV. Data Analysis of Recombinant DNA XXV. Graphic Map of p-LAGC-EAK10 XXVI. Annealed BPTI Product XXVII. 9:1 Insert-Vector Ratio Plate XXVIII. Ligation Colonies digested with XhoI XXIX. Ligation Plates G, H, and p-LAGC-EAK10 without insert XXX. Restriction Digest with XhoI V List of Tables I. Serine Proteases and their Function II. Non-enzyme Coagulation Factors and their Function III. Cascade Regulation IV. Restriction Digestion with NcoI V. Dephosphorylation of pET11d + NcoI + HindIII VI. Annealing Components of Oligonucleotides VII. Ligation of pET11d Backbone with LAGC and EA K Linker 3 VIII. Transformation Plates IX. Ligation Colony Count X. Restriction Digest with BamHI XI. Pre-screening Colonies with BamHI XII. p-LAGC-EAK10 Restriction Digest with XbaI XIII. p-LAGC-EAK10/XbaI Restriction Digest with NcoI XIV. p-LAGC-EAK10/XbaI/NcoI Restriction Digest with AP-CIP XV. Ligation of BPTI into p-LAGC-EAK10/XbaI/NcoI XVI. Ligation Attempt 1 XVII. Ligation Attempt 2 XVIII. Ligation Attempt 3 XIX. Ligation Attempt 4 XX. Ligation Attempt 5 XXI. Ligation Attempt 6 1 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 pro- coagulant 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 (FX ). FX is a very important part of the blood coagulation cascade because a a its activation is primarily responsible for thrombin generation. Activation of FX alone is, a however, insufficient to produce a fully active thrombin. For this to occur, FX must form a a complex with its protein cofactor Factor V and its protein substrate prothrombin on a 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 FX a 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 FX . These small protein inhibitors fit just inside the a active site of the enzyme, making them ideal candidates with which to study the active site of FX . The goal of this project is to gain understanding of the topography of fully a active FX by using lipid-anchored BPTI with linkers that act as molecular rulers to a measure the range of reactive heights of the active site of FX in its pro-coagulant a complex on a phospholipid surface. 2 Introduction Hemostasis Hemostasis is the process responsible for initiating and terminating the blood clotting mechanism upon injury (1) . It is a tightly regulated and dynamic process by which bleeding as a result of vessel damage is stopped and, ultimately, returned to normal (1-3) . The hemostasis process works to maintain blood in a fluid, clot-free state under normal conditions, form a solid clot to block bleeding upon injury, and then return blood to a fluid state upon repair of that injury. This process involves a subset of processes including blood clotting, platelet activation, and vascular damage repair (3). Coagulation is the first integral part of this process as it is the formation of blood clots which assist with sealing the injury and returning blood flow to normal (2-4) . The coagulation cascade includes three major components some of which circulate in the blood in a deactivated state and become activated upon vascular injury. These components are platelets, vessel endothelium, and coagulation factors. Platelet activation is important in hemostasis for sealing vessel damage and accelerating the coagulation system overall. Vessel repair is a result of the hemostatic plug composed of fibrin and platelets that seals the damage as well as endothelial components which repair the tissue (3). To understand more about the blood coagulation cascade, it is important to investigate the blood components involved more closely. Platelets Platelets are specialized blood cells that play an important role in hemostasis (2, 5, 6). The structure of both the un-stimulated and stimulated platelet is important in its 3 functioning. In a deactivated state, they are small, disk-like, non-nucleated cell fragments of megakaryocyte cytoplasm in the blood (6). When activated, platelets structure morphs from smooth and discoid to rough cells with pseudopodia. As the term suggests, these pseudopodia are feet-like projections which serve as puzzle-like sites of attachment for other platelets and fibrin. The cytoplasm is surrounded by a plasma membrane constructed in an open canalicular system (6). This system transports nutrients in and out of the cell. The plasma membrane is composed of over 30% phospholipids and nearly 60% proteins. The phospholipids are organized as a bilayer with polar head groups on both the external and internal layers in addition to a hydrophobic center. Neutral phospholipids like phosphatidylcholine are oriented on the external layer while anionic phospholipids are oriented internally. These phospholipids include phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine (7) . Tasks assigned to the phospholipids include solubilizing cholesterol such that cholesterol can maintain the fluidity of the phospholipid bilayer. A small percentage of carbohydrates are present in the membrane in the form of glycoproteins and glycolipids. The outer layer of the bilayer contains a glycoprotein complex known as the GPI complex. There are subgroups of glycoproteins involved in this complex: GPI , GPI , and a b GPII /GPIII . These proteins work together to attach platelets to collagen and bind von b a Willebrand factor. This step-wise process involves the platelet exposure to coagulation- inducing proteins, adhesion, and cellular reorganization. A well-characterized coagulation factor named von Willebrand factor (vWf) attaches to a receptor on the GPI