University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Chemical & Biomolecular Engineering Theses, Chemical and Biomolecular Engineering, Dissertations, & Student Research Department of Summer 2010 Recombinant Factors for Hemostasis Jennifer Calcaterra University of Nebraska at Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/chemengtheses Part of the Biochemical and Biomolecular Engineering Commons Calcaterra, Jennifer, "Recombinant Factors for Hemostasis" (2010). Chemical & Biomolecular Engineering Theses, Dissertations, & Student Research. 5. https://digitalcommons.unl.edu/chemengtheses/5 This Article is brought to you for free and open access by the Chemical and Biomolecular Engineering, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Chemical & Biomolecular Engineering Theses, Dissertations, & Student Research by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Recombinant Factors for Hemostasis by Jennifer Calcaterra A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Interdepartmental Area of Engineering (Chemical & Biomolecular Engineering) Under the Supervision of Professor William H. Velander Lincoln, Nebraska August, 2010 Recombinant Factors for Hemostasis Jennifer Calcaterra, Ph.D. University of Nebraska, 2010 Adviser: William H. Velander Trauma deaths are a result of hemorrhage in 37% of civilians and 47% military personnel and are the primary cause of death for individuals under 44 years of age. Current techniques used to treat hemorrhage are inadequate for severe bleeding. Preliminary research indicates that fibrin sealants (FS) alone or in combination with a dressing may be more effective; however, it has not been economically feasible for widespread use because of prohibitive costs related to procuring the proteins. To meet future demands for hemostatic therapies, FS will likely include recombinant human fibrinogen (rFI) and recombinant human Factor XIII (rFXIII). The underlying hypothesis of the research presented in this dissertation is that a liquid fibrin sealant (LFS) composed of recombinant FI, FXIII and FIIa in optimized proportions can assist hemostasis in the presence and absence of a bioresorbable bandage while using considerably fewer biologics than commercial products currently available. This dissertation characterized rFI produced in the milk of transgenic cows, plasma-derived thrombin (pdFIIa) activated by sodium citrate and rFXIIIa expressed in genetically engineered Pichia pastoris with respect to their capacity to serve as components in a LFS. The ratios of these factors were optimized to yield a LFS with a rapid clot formation rate and high viscoelastic strength. This optimized LFS was preliminarily tested ex vivo and in vivo. The clotting kinetics and viscoelastic strength of our optimized LFS was equivalent to those of a commercially available LFS; however, it uses approximately 75% less fibrinogen and thrombin. Our optimal LFS successfully achieved hemostasis in a significant number of the wounds that included extensive tissue and vascular damage. LFS applied without the assistance of a dressing was able to stop bleeding of oozing wounds or those with small vessels; however, a scaffold was needed when wounds contained large vasculature. iv Copyright 2010 Jennifer Calcaterra v Dedicated to my parents Kathryn and Robert Calcaterra vi Author’s Acknowledgements I want to thank Dr. Velander for providing me with the opportunity to participate in his groundbreaking research and for his mentorship, guidance and moral support. I appreciate the generous advice of Dr. Anuradha Subramanian. I would also like to thank Mostafa Fatemi for introducing me to the analytical techniques used in blood chemistry. Leonard Akert’s assistance was crucial in overcoming numerous technical hurdles. I will be forever grateful to my family for their support, advice, encouragement, inspiration and proofreading. vii Table of Contents Page List of Tables ……………….………………………………………………………………… ix List of Figures ………………………………………………………………………………… x Chapter 1. Introduction ………………………………………………………………………. 1 Hemostasis ………………………………………………………………………... 2 Fibrinogen …………………………………………………………………………. 4 Hemorrhage treatment ……………………………………………………………... 8 Fibrin sealants ………………………………………………………………………. 9 Recobinant alternatives for fibrin sealants ………………………………………. 10 Hemophilia A ………………………………………………………………………. 11 Dissertation objectives ……………………………………………………………... 12 References …………………………………………………………………………... 14 Chapter 2. Sequence of Activation of Prothrombin by Sodium Citrate ……………………... 20 Abstract ……………………………………………………………………………… 21 Introduction ………………………………………………………………………..... 23 Materials and Methods …………………………………………………………. 29 Results ……………………………………………………………………………….. 36 rFXIIIa characterization ………………………………………………. 36 Prothrombin activation by ecarin, rFIIa, FXa and sodium citrate …………. 36 N-terminal sequencing of activated samples ……………………………… 44 Thrombin activity by chromogenic assay …………………………………. 46 Thrombin activity by SDS-PAGE ………………………………………. 47 Thrombin activity by thromboelastography ………………………………. 48 Discussion ………………………………………………………………………. 49 Acknowledgements ………………………………………………………………….. 57 References …………………………………………………………………………... 58 Chapter 3. Characterization of Recombinant Factor XIII Expressed in Pichia pastoris. ….... 61 Abstract ……………………………………………………………………………… 62 Introduction ………………………………………………………………………… 63 Materials and Methods …………………………………………………………. 69 Results ……………………………………………………………………………….. 78 rFI and pdFIIa characterization …………………………………….. 78 Expression and purification of rFXIII ……………………………………. 78 Comparison of pdFXIII and rFXIII by SDS-PAGE and Western blot ….... 79 Molecular weight estimates of expressed protein ……………………......... 80 Aggregation/molecular weight analysis (SDS-PAGE, DLS, AUC, SEC) .... 81 Activation and inactivation of rFXIII by thrombin ……………………….. 83 Chromogenic activity assay ……………………………………………...... 85 γ-γ crosslinking of fibrin(ogen) by FXIIIa ……………………………...... 86 Clot formation by thromboelastography …………………………………… 88 Discussion ……………………………………………………………………………... 89 Acknowledgements ………………………………………………………………….. 93 References …………………………………………………………………………… 95 Chapter 4. Optimization of a Liquid Fibrin Sealant to Achieve its Maximal Strength and … 103 Activation Speed Abstract ………………………………………………………………………………... 104 Introduction …………………………………………………………………………. 106 Materials and Methods ……………………………………………………………....... 110 Results ……………………………………………………………………………....... 116 rFXIIIa and pdFIIa characterization ……………………………………..... 116 γ-γ crosslinking of FI by FXIIIa ……………………………………………. 116 Thromboelastography analysis of fibrin clot formation …………………... 117 viii Clot lysis by thromboelastography ………………….……………………… 125 Discussion ………………………………………………………………………….… 126 Acknowledgements …………………………………………………………………… 130 References …………………………………………………………………………...… 131 Chapter 5. Ex vivo Evaluation of the Liquid Fibrin Sealant Components ………………… 135 Abstract ………………………………………………………………………………... 136 Introduction ……………………………………………………………………………. 138 Materials and Methods ………………………………………………………………. 139 Results ………………………………………………………………………………… 143 rFXIIIa and pdFIIa characterization …………………………………… 143 Donor data ………………………………………………………………… 144 TEG analysis of haemodilution ………………………………….………. 144 TEG analysis of protein doping into NHB, PRP and PPP ………..………... 149 TEG analysis of time to clot initiation ……………………………...……….. 150 TEG analysis of coagulation time …………………………………………... 153 TEG analysis of maximal clot strength …………………………………….. 155 TEG analysis of lysis ……………………………………………………… 158 Discussion ……………………………………………………………………………. 160 Acknowledgements …………………………………………………………………….. 164 References ……………………………………………………………………………. 165 Chapter 6. Preliminary in vivo Testing of the Optimized Liquid Fibrin Sealant ……………… 167 Abstract ………………………………………………………………………………… 168 Introduction …………………………………………………………………………... 169 Materials and Methods ………………………………………………………………… 172 Results ………………………………………………………………………………… 184 Evaluation of swine clotting factors ……………………………………… 184 LFS on hepatic wedge resections …………………………………………… 186 rLFS on hepatic stelate laceration …………………………………………... 189 rLFS dressing on femoral arteriotomy model ……………………………… 190 rLFS dressing on hepatic resections ………………………………………... 192 Discussion ……………………………………………………………………………. 195 Acknowledgements …………………………………………………………………… 200 References ……………………………………………………………………………. 202 Chapter 7. Biologically Active Single Chain Recombinant Human FVIII:von Willebrand …… 205 Factor Complexes Made Abundantly in Transgenic Milk Abstract ……………………………………………………………………………… 206 Introduction …………………………………………………………………………. 207 Materials and Methods ……………………………………………………………….. 209 Results ………………………………………………………………………………… 214 Identification and expansion of transgenic lineages ……………………….. 214 Coexpression of 226/N6-tg, vWF-tg and AAT-tg in trigenic mouse milk … 217 Presence of FVIII-vWF complexes in trigenic mouse milk ………………… 219 Coexpression of AAT ……………………………………………………….. 221 Quantitative analysis of rFVIII biological activity …………………………. 222 In vivo tail clip assay in the hemophilia A mouse ………………………....... 224 Discussion …………………………………………………………………………… 226 Authorship …………………………………………………………………………… 232 Acknowledgements ………………………………………………………………….