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Liposomal-encapsulated enzymes can be delivered to and modify platelet function ex vivo Chan, Vivienne Wai Tung 2018

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LIPOSOMAL-ENCAPSULATED ENZYMES CAN BE DELIVERED TO AND MODIFY PLATELET FUNCTION EX VIVO by  Vivienne Wai Tung Chan  B.Sc., The University of British Columbia, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2018  © Vivienne Wai Tung Chan, 2018 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Liposomal-encapsulated enzymes can be delivered to and modify platelet function ex vivo.  submitted by Vivienne Wai Tung Chan  in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology  Examining Committee: Dr. Christian J. Kastrup, Biochemistry and Molecular Biology Supervisor  Dr. Pieter R. Cullis, Biochemistry and Molecular Biology Supervisory Committee Member  Dr. Edward Conway, Experimental Medicine University Examiner Dr. Shyh-Dar Li, Pharmaceutical Sciences University Examiner  Additional Supervisory Committee Members: Dr. Nobuhiko Tokuriki, Biochemistry and Molecular Biology Supervisory Committee Member  iii Abstract Platelets are small, anucleate blood cells that are important mediators of many physiological and pathological processes. These include hemostasis, thrombosis, wound healing, inflammation, immunity, and malignancy. There are currently several uses for platelet therapy in the clinic, such as to increase platelet counts for the prevention of spontaneous bleeding, and to stop uncontrolled bleeding during trauma and surgery. Although platelet transfusions are an efficacious component in preventing and stopping bleeding in most cases, they are still insufficient to stop the most severe cases of surgical and traumatic bleeding. Traumatic bleeding is further complicated by trauma-induced coagulopathy, which often presents with platelet dysfunction and is not corrected by transfusions of normal platelets. Strategies to enhance the endogenous function of platelets to increase the efficacy of platelet transfusions has not been rigorously explored, especially during active bleeding in trauma-induced coagulopathy.  When activated by specific stimuli, platelets locally secrete a variety of biologically active molecules in order to contribute to many physiological and pathophysiological processes. For example, platelets can recognize areas of vascular damage and respond by locally adhering, aggregating, and activating to initiate primary hemostasis. Platelets also release procoagulant molecules and mediate the formation of active coagulation factor complexes to ultimately form an insoluble fibrin clot and seal the wound. Taken together, developing strategies to modify the endogenous function of platelets may be a first step towards a platform system that could target many diseases. Moreover, strategies to load platelets with biomolecules could allow for the local delivery of therapeutics to disease sites using endogenous platelet machinery. This provides iv significant motivation to test our overarching hypothesis, that the endogenous function of platelets can be modified ex vivo through the delivery of liposome-encapsulated enzymes.  The objectives of this thesis were to: i) develop a platform approach to deliver biomolecules to platelets, ii) engineer anucleate platelets to transcribe RNA, and iii) increase the coagulability of transfusable platelets. The results shown here demonstrate proof-of-concept that endogenous platelet function can be extended through the delivery of lipid-encapsulated enzymes, and provides new approaches to potentially enhancing current platelet transfusions. v Lay Summary Platelets are small cells that circulate in blood to help stop bleeding and aid in wound healing. Because of their ability to clot blood, platelets are often transfused to prevent spontaneous bleeding or stop active bleeding. However, transfusions of platelets and other blood components may not be sufficient to stopping bleeding in all cases, such as uncontrolled bleeding during trauma and surgery. Uncontrolled bleeding is one of the leading causes of preventable death following trauma, and enhancing the ability for platelets to clot blood could improve the effectiveness of platelet transfusions during traumatic bleeding. This thesis describes the development of a platform to load platelets with potential therapeutics, such as clotting factors, to enhance their natural function. Because platelets are also important in many other diseases, such as arthritis, atherosclerosis, and cancer, developing ways to change platelet function could also potentially lead to new therapies to treat these diseases. vi Preface Approvals for the study were given by the research ethics boards of the University of British Columbia. The UBC Ethics Certificate for human blood collection is H12-01516.  A modified version of Figure 2.3, Figure 2.4, and parts of chapter 3 have been published in Angewandte Chemie: V. Chan, S.K. Novakowski, S. Law, C. Klein-Bosgoed, C.J. Kastrup (2015). Controlled transcription of exogenous mRNA in platelets using protocells. V.C. and S.K.N. were co-first authors on the paper, designed and performed all experiments, analyzed and interpreted data, and wrote the paper. S.K.N. obtained data for Figure 2.3A, Figure 2.4B, Figure 3.8A and 3.8B. S.L. helped develop methods for testing uptake of liposomes by platelets and performed experiments for Figure 2.4A. C.K.-B. helped develop methods for platelet isolation. C.J.K. helped design experiments, interpret data and write the paper.  A modified version of chapter 4 has been accepted for publication in Journal of Thrombosis and Haemostasis: V. Chan, M. Sarkari, R. Sunderland, A.E. St. John, N.J. White, C.J. Kastrup (2018). Platelets loaded with thrombin-encapsulated liposomes have increased coagulability. V.C. designed and performed all of the experiments, analyzed and interpreted data and wrote the paper. M.S. and R.S. helped perform obtain data for Figures 4.9B and Figure 4.10B, and analyzed and interpreted corresponding data. A.E.S.J. and N.J.W. helped design TIC experiments, provided TIC patient samples, and helped write the paper. C.J.K. helped design experiments, interpret data and write the paper.  vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements .................................................................................................................. xviii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 1.1 Motivation and significance ............................................................................................ 1 1.2 Specific thesis objectives ................................................................................................ 5 1.2.1 Develop a platform approach to deliver biomolecules to platelets ............................. 5 1.2.2 Engineer anucleate platelets to transcribe RNA ......................................................... 5 1.2.3 Increase the coagulability of transfusable platelets .................................................... 7 1.3 Background and literature review ................................................................................... 9 1.3.1 Manipulating platelets has the potential to impact a variety of physiological and pathophysiological processes .................................................................................................. 9 Platelets play a central role in hemostasis and thrombosis ............................................... 11 Platelets contribute to wound healing ............................................................................... 12 Platelets contribute to inflammation and host defense ..................................................... 13 Platelets contribute to cancer progression......................................................................... 14 viii 1.3.2 Platelets are natural local delivery vehicles .............................................................. 15 Methods to locally deliver therapeutics are needed .......................................................... 15 Platelets endocytose natural and synthetic materials ........................................................ 16 Platelets can release biomolecules in response to specific biological stimuli .................. 17 1.3.3 Current applications and future opportunities for modified platelets in the clinic ... 18 Platelets are transfused to stop severe, traumatic bleeding ............................................... 18 Platelets are transfused to prevent bleeding in thrombocytopenic patients ...................... 19 Platelets lose function during storage ............................................................................... 20 PRP is topically injected for wound healing ..................................................................... 20 Platelets may be used to co-deliver other therapeutics ..................................................... 21 1.3.4 Previous approaches to modifying platelet function ex vivo .................................... 23 Engineered platelets can be derived from modified megakaryocytes .............................. 23 Platelets can be directly transfected with biomolecules ................................................... 24 1.3.5 Lipid-based nanoparticles for intracellular delivery of biomolecules to platelets .... 25 Chapter 2: Developing new strategies to deliver biomolecules to platelets ex vivo ................29 2.1 Rationale ....................................................................................................................... 29 2.2 Methods......................................................................................................................... 31 2.2.1 Isolating platelets from whole blood......................................................................... 31 2.2.2 Preparing liposomes .................................................................................................. 31 2.2.3 Measuring liposome uptake in isolated platelets. ..................................................... 32 2.2.4 Quantifying and imaging the uptake of liposomes by platelets. ............................... 32 2.2.5 Measuring platelet activation by flow cytometry ..................................................... 33 2.3 Results ........................................................................................................................... 34 ix 2.3.1 Optimized conditions for delivering liposomes to platelets ..................................... 34 2.3.2 Endocytosis of phosphatidylcholine-based liposomes by platelets .......................... 37 2.4 Discussion ..................................................................................................................... 41 Chapter 3: Transcription of exogenous mRNA in anucleate platelets....................................44 3.1 Rationale ....................................................................................................................... 44 3.2 Methods......................................................................................................................... 47 3.2.1 Plasmids and primers. ............................................................................................... 47 3.2.2 Loading platelets with RNA-synthesizing liposomes. .............................................. 47 3.2.3 Measuring protein expression from synthesized RNA. ............................................ 48 3.3 Results ........................................................................................................................... 50 3.3.1 Transcription can occur within nanoliposomes ........................................................ 50 3.3.2 Controlled RNA Transcription Using Light. ............................................................ 58 3.3.3 Controlled transcription of RNA in platelets. ........................................................... 61 3.4 Discussion ..................................................................................................................... 63 Chapter 4: Platelets loaded with liposomal thrombin have increased coagulability .............67 4.1 Rationale ....................................................................................................................... 67 4.2 Methods......................................................................................................................... 69 4.2.1 Preparing and characterizing liposomal thrombin. ................................................... 69 4.2.2 Loading platelets with liposomal thrombin. ............................................................. 69 4.2.3 Measuring clot retraction in PRP. ............................................................................. 70 4.2.4 Measuring thrombin generation in PRP. ................................................................... 70 4.2.5 Isolating plasma from patients with trauma-induced coagulopathy. ........................ 71 4.2.6 Determining clot initiation and clot strength by thromboelastography. ................... 71 x 4.2.7 Determining clot initiation and lysis by western blot. .............................................. 72 4.2.8 Determining release of endogenous platelet FXIII. .................................................. 72 4.2.9 Detecting fibrin formation and crosslinking. ............................................................ 72 4.2.10 Preparing fluorescent thrombin to measure uptake and release. ........................... 73 4.3 Results ........................................................................................................................... 74 4.3.1 Purified liposomal thrombin minimally activates platelets ...................................... 74 4.3.2 Platelet activation and clot characteristics of LT-PLTs ............................................ 78 4.3.3 Platelet response and clot formation during acidosis. ............................................... 83 4.3.4 Clot formation in the presence of antiplatelet drugs. ................................................ 85 4.3.5 Clot formation in plasma from patients with coagulopathies. .................................. 86 4.3.6 Platelet uptake of liposomal thrombin and assessing interactions with substrates of thrombin. ............................................................................................................................... 88 4.4 Discussion ..................................................................................................................... 92 Chapter 5: Conclusions ...............................................................................................................97 5.1 A range of functional biomolecules can be delivered to platelets ................................ 97 5.2 Platelet function can be enhanced ................................................................................. 98 5.2.1 Engineered anucleate platelets to transcribe RNA (Chapter 3) ................................ 98 5.2.2 Modified platelets have enhanced coagulability (Chapter 4) ................................... 99 Chapter 6: Future directions ....................................................................................................101 6.1 Exploring the safety and efficacy of LT-PLT transfusions in vivo ............................. 101 6.2 Optimizing the delivery of cargo to platelets .............................................................. 102 6.3 Other clinical opportunities for modified platelets ..................................................... 104 Bibliography ...............................................................................................................................107 xi List of Tables Table 1.1. Platelets are involved in many physiological and pathophysiological processes. ....... 10 Table 1.2. The roles of thrombin in hemostasis. ........................................................................... 23 Table 2.3. Summary of incubation conditions tested to optimize uptake of liposomes by platelets........................................................................................................................................................ 35  xii List of Figures Figure 1.1. Schematic of cargo encapsulated into liposomes which can be delivered to platelets........................................................................................................................................................ 28 Figure 2.1. Platelets take up liposomes more effciently in Tyrode's buffer than in CGS. ............ 36 Figure 2.2. Incubation of platelets with liposomes did not cause platelet activation. .................. 36 Figure 2.3. Nanoliposomes are internalized by platelets. ............................................................. 38 Figure 2.4. Liposomal uptake can be inhibited with several inhibitors of endocytosis. ............... 40 Figure 3.1. Transcription in nanoliposomes allows exoggenous RNA to be synthesized in anucleate cells such as platelets. ................................................................................................... 46 Figure 3.2. Extrusion inactivates components of the transcription reaction. ................................ 51 Figure 3.3. Incorporation a PEGylated lipid is required for efficient RNA synthesis in nanoliposomes............................................................................................................................... 52 Figure 3.4. Comparison of RNA yield in nanoliposomes before and after optimization of the concentrations of transcriptional components. ............................................................................. 54 Figure 3.5. Unencapsulated transcriptional components are removed from liposomes using an anionic exchange column. ............................................................................................................. 56 Figure 3.6. RNA is transcribed in liposomes of varoius sizes. ..................................................... 57 Figure 3.7. Incorporation of photo-caged ATP allows for controlled transcription initiation using light. .............................................................................................................................................. 59 Figure 3.8. Transcription of GFP and FLuc mRNA in nanoliposomes is controlled by light. ..... 60 Figure 3.9. Liposomes internalized by intact platelets synthesized RNA. ................................... 62 Figure 4.1. Thrombin binds to and is removed by a cationic exchange column. ......................... 75 xiii Figure 4.2. Unencapsulated thrombin is efficiently removed from the purified liposomal preparation. ................................................................................................................................... 77 Figure 4.3. LT-PLTS respond to collagen activation by increased P-selectin expression and clot contraction..................................................................................................................................... 79 Figure 4.4. Clots form faster with LT-PLTs than PLTs and are more resistant to fibrinolysis. ... 81 Figure 4.5. Several inhibitory effects of acidosis on platelets and clot formation can be reversed by LT-PLTs. .................................................................................................................................. 85 Figure 4.6. LT-PLTs can correct for delayed thrombin generation time in the presence of antiplatelet drugs. .......................................................................................................................... 86 Figure 4.7. LT-PLTs improve aspects of clotting in plasma from hemophilia A patients. .......... 87 Figure 4.8. Clotting of PRP from patients with TIC is faster with LT-PLTs. .............................. 88 Figure 4.9. Liposomal thrombin is actively taken up by platelets but not released upon activation........................................................................................................................................................ 89 Figure 4.10. Effects of inhibiting thrombin activity and PAR activity on LT-PLTs. ................... 91 Figure 4.11. Enhancing platelet coagulability with liposomal thrombin. ..................................... 93  xiv List of Abbreviations ADP  adenosine diphosphate ASA  acetylsalicylic acid ATP  adenosine triphosphate B.D.  below detection CD62P P-selectin CGS  citrate glucose saline buffer Col  collagen DNA  deoxyribonucleic acid DFP  diisopropylfluorophosphate DFP-PLT platelets loaded with liposomal, inactivated thrombin DHPE  1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine DMPC  1,2-dimyristoyl-sn-glycero-3-phosphorylcholine DMSO  dimethylsulfoxide DOPC  1,2-dioleoyl-sn-glycero-3-phosphocholine DOPE  1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DSPE  1,2-distearoyl-sn-glycero-3-phosphoethanolamine DTT  1,4-dithiothreitol EDTA  ethylenediaminetetraacetic acid EL-PLT platelets loaded with empty liposomes ePC  egg phosphatidylcholine FLuc  firefly luciferase FIX  coagulation factor IX xv FV  coagulation factor V FVIIa  activated coagulation factor VII FVIII  coagulation factor FVIII FX(a)  (activated)coagulation factor X FXIa  activated coagulation factor XI FXIII(A) (activated) coagulation factor FXIII GAPDH glyceraldehyde 3-phosphate dehydrogenase GFP  green fluorescent protein  GPS  gray platelet syndrome HBS  HEPES buffered saline HCl  hydrochloric acid LNP  lipid nanoparticle LT-PLT platelets loaded with liposomal thrombin MCS  multiple cloning site MFI  mean fluorescence intensity miRNA micro ribonucleic acid mRNA  messenger ribonucleic acid NET  neutrophil extracellular trap n.s.  not significant OCS  open canalicular system OG  Oregon Green PAR-1  protease activated receptor 1 PAR-4  protease activated receptor 4 xvi PBS  phosphate-buffered saline PDGF  platelet-derived growth factor PEG  polyethylene glycol PF4  platelet factor 4 PLT  normal, unmodified platelets PPP  platelet-poor plasma PRP  platelet-rich plasma PS  phosphatidylserine PSL  platelet storage lesion qPCR  quantitative polymerase chain reaction rFVIIa  recombinant activated coagulation factor FVII RNA  ribonucleic acid RNAi  ribonucleic acid interference rNTP  ribonucleotides RRL  rabbit reticulocyte lysate s.d.  standard deviation SDS-PAGE denaturing polyacrylamide gel electrophoresis SEM  standard error of mean siRNA  silencing ribonucleic acid T7RNAP T7 RNA polymerase TAFI  thrombin-activatable fibrinolysis inhibitor TBS  tris-buffered saline TEG  thromboelastography xvii TF  tissue factor TIC  trauma-induced coagulopathy Thr  thrombin tPA  tissue plasminogen activator TXA  tranexamic acid TXA2  thromboxane A2 VAMP3 vesicle associated membrane protein 3 v/v  volume to volume ratio vWF  von Willebrand factor w/v  weight to volume ratio w/w  weight to weight ratio WB  whole blood   xviii Acknowledgements  I would like to thank all past and present members of Dr. Christian Kastrup’s lab. In particular, the following members have contributed to or provided support for data collection for this dissertation: S. Novakowski, W.S. Hur, N. Ashok, S. Law, M. Sarkari, R. Sunderland, and E. Long. I thank my supervisor, Dr. Christian Kastrup, and my supervisory committee, Dr. Pieter Cullis and Dr. Nobuhiko Tokuriki, for their guidance and valuable research advice. I would also like to thank our collaborators and the members of their labs, including Dr. Pieter Cullis, Dr. Dana Devine, and Dr. Nathan White, for their contributions and research support.  xix Dedication  To everyone who has supported me throughout my Ph.D. career. 1 Chapter 1: Introduction  1.1 Motivation and significance Developing strategies to enhance or modify endogenous platelet function ex vivo could have the potential to enhance the current efficacy of platelet therapies in a range of clinical scenarios. There are currently several therapeutic applications for platelets isolated from whole blood; allogenic platelet units are often transfused during cancer treatment, haematopoetic stem cell transplants, or active bleeding scenarios to prevent or stop bleeding, and topical applications of autologous platelet-rich plasma (PRP) gels are increasingly being used to facilitate healing of various types of wounds. Although these platelet therapies are clinically approved and have been shown to be efficacious in a number of treatments, opportunities for improving current platelet therapies as well as exploring new therapeutic platelet applications remain.  Platelets are small, anucleate blood cells which are central in initiating hemostasis and thrombosis, and they are also important players in wound healing, inflammation, immunity, and malignancy. Currently, the main clinical application for platelet transfusions is to prevent or stop bleeding, and millions of platelet units are transfused each year worldwide for thrombocytopenic patients and patients who are actively bleeding. Platelet transfusions are usually effective at increasing total platelet count and reducing mortality associated with bleeding. However, transfusions of platelets and other blood components, along with other hemorrhage management strategies such as fluid resuscitation, wound dressings, and coagulation factor infusion, are still insufficient to control the most severe cases of hemorrhage. Controlling bleeding is particularly difficult when hemorrhage is complicated by coagulopathy, in which the ability for blood to clot 2 is impaired. Uncontrolled hemorrhage is the leading cause of preventable death resulting from trauma and remains a major cause of mortality worldwide, especially in young, healthy adults. Platelet dysfunction, which may involve decreased platelet activation and aggregation in response to agonists, is a major contributor to trauma-induced coagulopathy (TIC). TIC is also characterized by uncontrolled hemorrhage resulting from the consumption of coagulation factors, decreased clot stability, as well as increased clot degradation and clearance. In particular, decreased platelet sensitivity to agonists is strongly correlated with mortality during TIC, and platelet dysfunction cannot be reversed by simply transfusing normal, functional platelets back into the body. This suggests that the transfused platelets may become inactivated following transfusion, and thus are insufficient to overcoming coagulopathy. Strategies to improve the endogenous function of transfused platelets, such as enabling them to better contribute to clot initiation and clot strength, could have the potential to enhance the efficacy of platelet transfusions and hemorrhage management during TIC.  Platelets circulate in the body while containing a plethora of bioactive molecules, which could cause systemic thrombosis and inflammation if released in an unregulated manner. However, platelets can selectively secrete certain molecules by recognizing biological stimuli and specifically activating in response to them. This allows platelets to contribute and controllably regulate many physiological and pathophysiological processes, including coagulation, inflammation, immunity, and malignancy. Because they are able to naturally localize to and release their contents at disease sites, approaches to load platelets with biomolecules or therapeutics could potentially transform platelets into a biocompatible, targeted local drug delivery platform. This approach would be particularly useful in conjunction with 3 platelet therapies that are already used in the clinic. For example, cancer patients undergoing chemotherapy often develop thrombocytopenia, and require platelet transfusions to normalize their platelet count and prevent spontaneous bleeds. Since platelets naturally localize to and release their contents at tumor sites, methods to load platelets with anticancer drugs may allow targeted localization of chemotherapeutics to tumors. This may allow platelet transfusions to simultaneously increase platelet count and locally release chemotherapeutic drugs on a single platform, with the potential to enhance the therapeutic efficacy of the entire treatment regimen. Indeed, this idea of using platelets to deliver anticancer drugs has been recently explored in the literature, demonstrating significant interest in taking advantage of natural platelet secretion for therapeutic purposes. Platelet therapy is also used in the context of wound healing, such as following orthopedic or dental procedures. Although the efficacy of PRP gels in a number of wound healing applications is still being investigated, methods to load biomolecules into platelets may allow for the co-delivery of endogenous platelet growth factors and additional therapeutics to aid the healing process.  Platelets with modified functions could also be used to study platelet biology. Because platelets are anucleate and are prone to activation and aggregation during handling ex vivo, there are limited reports of reliable methods to manipulate the genomic or proteomic content of platelets while keeping them in a resting state. Developing such methods would provide tremendous mechanistic insight into the roles of platelets in various diseases. A deeper understanding of platelet biology during disease could also potentially lead to new therapies, such as enhancing or inhibiting certain platelet functions during disease, as well as uncovering new clinical scenarios where platelet therapies may be useful. Strategies to reliably study platelet 4 function through protein upregulation and knockdown could also allow for a better understanding of how platelets lose their endogenous function during storage, which is a major limiting factor for effectively maintaining sufficient platelet units in blood banking inventories. Furthermore, this could lead to the development of better storage conditions to prolong the shelf life of transfused platelets, which may address current blood banking challenges and potentially enhance the efficacy of current platelet transfusions.  Taken together, this provides significant motivation to develop approaches to reliably modify or enhance platelet function ex vivo. Currently, there are limited strategies to specifically tune platelet function because of premature activation and aggregation during handling ex vivo. We hypothesized that biologically relevant enzymes and other molecules can be encapsulated into liposomes and internalized by platelets to specifically modulate platelet function. Although there are many potential biological and clinical applications for modified platelets, the scope of this thesis is focused on developing the approaches to modify platelet function ex vivo, and using these approaches to explore the potential clinical relevance of modified platelets by enhancing their coagulability in vitro. The studies described here represents a platform system to deliver functional biomolecules to platelets, and is an important first step towards generating platelets with modified functions for therapeutic applications.    5 1.2 Specific thesis objectives  1.2.1 Develop a platform approach to deliver biomolecules to platelets The objective was to use lipid nanoparticle formulations which can encapsulate and deliver biomolecules to platelets ex vivo. Formulations found in previous studies were used as a starting point to determine lipid compositions which have been shown to encapsulate biomolecules with minimal cell toxicity. For example, neutral lipids that are normally found in biological membranes were used to make nanoparticles to ensure that the nanoparticles were biocompatible.  In order to maximize uptake efficiency while minimizing premature platelet activation, several variables were tested, including nanoparticle size and incubation conditions, such as varying incubation time, temperature, buffer, and nanoparticle concentrations. Following optimization of the nanoparticle formulation and incubation conditions, the mechanisms by which platelets took up liposomes were determined using small molecule inhibitors for specific pathways of endocytosis. This not only confirmed the internalization of liposomes by platelets, but also allowed for further characterization of the modified platelet system, which may potentially allow us to develop strategies to enhance liposomal uptake in the future.  1.2.2 Engineer anucleate platelets to transcribe RNA Platelets are involved in a multitude of physiological and pathophysiological processes, and developing methods to modify the genomic and proteomic contents of platelets could have significant implications in targeting platelets to treat disease as well as using platelets to localize the delivery of therapeutics. Platelets contain an abundance of mRNA and miRNA which are necessary for endogenous platelet function, and the machinery required for protein translation 6 and RNAi. However, the absence of components necessary for RNA transcription make platelets not amenable to traditional transfection reagents and techniques. This objective was focused on developing an approach to genetically manipulate platelets and generate platelets with new functions by enabling them to transcribe RNA. Although this was only a step towards modifying the RNA content of platelets, the strategies developed here may have important applications in genetically upregulating or downregulating specific targets within platelets.  The objective was first to build on the approaches developed in 1.2.1 to efficiently co-encapsulate the components of transcription, which included a DNA template, ribonucleotides, T7 RNA polymerase, and small molecules such as MgCl2, into liposomes which can be internalized by platelets ex vivo. In this system, ATP was replaced with a photo-caged ATP so that transcription is only initiated upon stimulation with light. This allowed transcription in liposomes and in platelets to be controllably initiated by an external stimulus. Several lipid compositions, as well as nanoparticle formulation conditions were tested in order to minimize the inactivation of transcriptional components and to maximize the RNA yield in liposomes. The entire transcriptionally-functional liposomal unit was then delivered to platelets ex vivo using the approaches developed in 1.2.1 and platelets were stimulated with light for transcription to occur. The system was optimized to maximize transcription of full length green fluorescent protein (GFP) mRNA, but other templates such as firefly luciferase (FLuc) mRNA was also tested to ensure that the system can be extended to express a number of different transcripts within platelets.  7 1.2.3 Increase the coagulability of transfusable platelets Building on the strategies developed in 1.2.1 and 1.2.2, the objective was to deliver thrombin to transfusable platelets to increase their coagulability. Platelet therapy is most commonly used in the clinic as allogeneic platelet transfusions to prevent and stop bleeding, but platelet transfusions are not always sufficient to stop bleeding, especially in the most severe cases of hemorrhage. Platelets can become dysfunctional during coagulopathy, and reduced sensitivity to agonists is strongly correlated with mortality. Furthermore, platelets undergo deleterious changes in structure and function during storage, correlating to reduced recovery, platelet survival and hemostatic potential after transfusion. This provides support for developing approaches to enhance the endogenous coagulability of platelets, which has the potential to reduce the transfusion requirement to stop bleeding, and may have important implications in enhancing the efficacy of platelet transfusions during severe hemorrhage.  The objective was first to build on the strategies developed in 1.2.1 to efficiently encapsulate thrombin and temporarily shield its prothrombotic activity from platelets. Thrombin is a coagulation factor and potent platelet agonist, and its hemostatic activity may be highly beneficial during uncontrolled bleeding. However, clinical use of thrombin is currently limited to topical applications due to its high thrombotic risk if used systemically, and exposure of platelets to thrombin would cause intense premature platelet activation. Therefore, liposomal thrombin was delivered to platelets ex vivo based on the approaches developed in 1.2.1 and 1.2.2 and its effect on platelets was determined. Platelet coagulability was measured by monitoring platelet activation upon stimulation with specific agonists, as well as the contribution of modified platelets to clotting characteristics such as clot initiation, clot strength, clot contraction, and clot 8 degradation. Platelet coagulability was also tested under conditions where either platelet function or overall coagulation is compromised, in order to determine whether enhancing platelet coagulability will be a useful approach to restoring hemostasis under these conditions. This included acidosis, antiplatelet drugs, hemophilia A, and TIC. In addition to the in vitro clotting experiments, we also attempted to determine the intracellular substrates with which the delivered thrombin interacts. These characterization studies allowed us to better understand the mechanism of enhanced coagulability following modification with liposomal thrombin, and could potentially allow for further optimization in increasing the coagulability of transfusable platelets. These studies are a first step towards determining whether transfusing modified platelets could enhance the efficacy of platelet transfusions during coagulopathy.    9 1.3 Background and literature review  1.3.1 Manipulating platelets has the potential to impact a variety of physiological and pathophysiological processes Platelets are small, anucleate blood cells that are generated by megakaryocytes in the bone marrow. Although platelets are generally thought of as merely contributing to blood clotting, they are actually involved in a multitude of physiological and pathophysiological processes, making them attractive therapeutic targets. These processes are summarized in Table 1.1, which is not an extensive list, but is representative of the major categories of the diverse physiological and pathophysiological processes to which platelets contribute. Developing strategies to reliably manipulate platelet function could have the potential to impact many of these pathological processes and effectively treat many diseases. The following subsections are more detailed summaries of several of the major processes in which platelets play a major role.   10 Process Platelet Contribution Mechanism Hemostasis and Thrombosis Adhesion  Platelet GPIb binds soluble vWF anchored on to collagen from subendothelium in a shear dependent manner [1, 2]  Aggregation  GPIb-IX-V and GPIIbIIIa bridged by fibrinogen or vWF [3]  Platelets aggregate on top of layer of adhered platelets to form hemostatic plug [4, 5]  Activation  Platelets activated by thrombin, collagen, complement, ADP, epinephrine, TXA2 [6]   Release of alpha and dense granules [7] Activation and propagation of coagulation cascade  Exposure of platelet PS [8]   Platelet-bound FXIa activates FIX [9]   Assembly of tenase complex [10] and prothrombinase complex [11] Inflammation Development of atherosclerosis  Recruitment of monocytes and lymphocytes to atherosclerotic plaques [12, 13]  Thrombotic vessel occlusion following plaque rupture [14, 15]  Activation of endothelial cells to release cytokines [16, 17]  Progression of arthritis  Platelet-derived microparticles amplify inflammation in arthritic joints [18]  Release of cytokines  Platelets contain and release CD62P, CD40L, PF4, CCL3, CCL5, IL-1 to promote inflammation and recruit leukocytes [19]  Wound healing Recruiting cells and revascularization  Release of growth factors such as PDGF and VEGF to promote the recruitment, proliferation, and differentiation of smooth muscle cells and endothelial progenitor cells [20, 21]  Malignancy Tumor growth  Platelet growth factors promote tumor cell proliferation and vascular permeability [22-24]  Metastasis  Platelet TGF-β causes tumor cells to transition to invasive phenotype [25]   Aggregate and protect embolic tumor cells [26]   Promotes egress of micrometastases and adhesion to endothelium [27]  Immunity Activation of neutrophils  Platelet TLR4 promotes formation of NETs to capture bacteria in blood [28-30]  Direct interaction with bacteria  Release of antimicrobial molecules [31]   Internalize pathogens [32, 33]  Table 1.1. Platelets are involved in many physiological and pathophysiological processes.  11 Platelets play a central role in hemostasis and thrombosis One of the most important physiological roles of platelets is in clot initiation and primary hemostasis, which includes the adhesion, aggregation, and activation of platelets to form a platelet plug in response to damage in the vasculature.[8, 34] This is a highly regulated process, involving the recognition of damaged blood vessels and local recruitment of platelets and other cells. In the secondary wave of hemostasis, platelets further contribute to blood clotting by activating enzymes in the coagulation cascade, which is primarily achieved through specifically releasing procoagulant molecules, providing negatively-charged surfaces for coagulation factor assembly, and contributing to cell-based thrombin generation to ultimately form an insoluble fibrin clot and stop bleeding.  This procoagulant role of platelets is necessary for maintaining hemostasis, because thrombocytopenia (low platelet count) or inherited thrombocytopathies (defective platelet function) are associated with prolonged bleeding time, defective clot formation, and spontaneous, possibly life-threatening hemorrhage. [35] For example, gray platelet syndrome (GPS) is a storage pool disease characterized by thrombocytopenia and abnormally large platelets that lack α-granules, where procoagulant proteins such as vWF, fibrinogen, and FV are stored. [7, 35] Patients with GPS have a mild to moderate bleeding tendency; although the exact mechanism is unclear, the bleeding diathesis may be attributed to defects in platelet aggregation and intracellular signaling within platelets, highlighting the importance of platelets to maintaining hemostasis. [36] Platelets further promote clot formation by exposing phosphatidylserine (PS) on the cell surface to promote assembly of coagulation factor complexes such as the prothrombinase complex and the tenase complex to activate thrombin and FX, 12 respectively. [10, 11] In addition to initiating and sustaining coagulation, platelets contribute to the hemostatic response by releasing antifibrinolytic proteins such as α2-antiplasmin, plasminogen activator inhibitor-1 (PAI-1), and thrombin-activatable fibrinolysis inhibitor (TAFI) to inhibit fibrinolysis to delay clot degradation and clearance. [37-39]   On the other hand, because of the high hemostatic potential of platelets, dysregulation of platelet activation could cause thrombotic events. For example, erosion or rupture of atherosclerotic plaques results in the exposure of thrombogenic material to blood and platelets, causing excessive platelet activation and clot formation, which could potentially lead to vessel occlusion, resulting in myocardial infarction or ischemic stroke. [14, 15] In deep vein thrombosis and the associated venous thromboembolism, unstable clots form in the areas of low blood flow and may embolize to other areas of vasculature. The interaction of platelets with endothelial vWF in areas of restricted blood flow have been shown to play a key role in initiating thrombus formation in animal models. [40] This demonstrates that platelets play an important role in both physiological and pathological clot formation, and strategies to target the dysregulation of platelet function may be useful in treating thrombosis in several clinical scenarios.  Platelets contribute to wound healing Following clot formation and stabilization, the final step to repairing damaged vasculature involves the formation of new tissue and revascularization, which is in part facilitated by platelets. Wound healing occurs as a result of a complex series of cell-cell and cell-matrix interactions, and platelets can contribute to these processes directly by binding and recruiting various cells, as well as indirectly by releasing numerous growth factors and cytokines 13 to activate and initiate interactions of specific cells to the wound. [21] The release of PDGF and TGF-β by platelets facilitates in initiating an inflammatory response, in addition to promoting the migration of neutrophils and macrophages to the wound site. [20] Platelets have been shown to contribute to several stages of the wound healing process, including matrix deposition, collagen production, and epithelialization. [41] The ability to modulate platelet function may have an enormous impact in facilitating wound healing in many clinical applications.  Platelets contribute to inflammation and host defense Many vascular pathologies, such as atherosclerosis and sepsis, operate at the interface of coagulation and inflammation, and immunity. While platelets can readily respond to thrombotic and proinflammatory stimuli, they can also express and secrete chemokines such as CCL5 and CXCL, which activate and recruit leukocytes and endothelial cells to initiate and promote an inflammatory response. [42, 43] During infection, neutrophils are recruited to engulf and kill bacteria, as well as produce neutrophil extracellular traps (NETs) to trap microbes and induce a proinflammatory response. [44, 45] NETs consist of scaffolds of extracellular DNA and antimicrobial proteins, and have been shown to interact with platelets and contribute to thrombus formation. [46] On the other hand, platelets bind to and are activated by NETs, and stimulation of platelets with lipopolysaccharide or collagen can trigger NET formation, leading to a vicious cycle of further NET formation and platelet activation. [30, 47] In addition to facilitating NET formation and binding, platelets also play an active role in innate and adaptive immunity by recognizing pathogens through toll-like receptors, [48] releasing microbicidal molecules, and engulfing pathogens such as Staphylococcus aureus and HIV. [33, 49] In some cases, pathogens have taken advantage of being internalized by platelets to evade the host response and promote 14 bacterial dissemination. [32] These studies demonstrate that platelets play an active role in inflammatory disease and during infection, and platelet function may be an attractive therapeutic target in treating these diseases.  Platelets contribute to cancer progression The role of platelets in cancer, particularly in tumor growth and metastasis, has been well characterized. Cancer cells induce platelet activation by various mechanisms, including secretion of soluble platelet agonists such as ADP and TXA2, direct engagement of platelet surface receptors, or activation of the coagulation cascade through expression of tissue factor. [50] Activated platelets can then secrete a number of molecules which can promote tumor cell proliferation, angiogenesis, and assist cancer cells to evade apoptosis. [51] Malignant tumor cells can cause platelets to aggregate and form a protective outer coating to evade the body’s immune system and to withstand high shear forces in circulation. [52] Furthermore, platelets have been shown to contribute to metastasis by facilitating the migration, invasion, and adhesion of tumor cells to the endothelium, as well as promoting tumor growth and vascularization through the release of growth factors. [22, 53-55] These studies suggest that targeting certain platelet functions may be useful in treating cancer, particularly for inhibiting tumor growth and metastasis.  Taken together, the importance of platelets in all of these processes provides significant motivation to develop a platform approach to target platelet function for treating many different diseases.  15 1.3.2 Platelets are natural local delivery vehicles In the previous section, the roles of platelets in a diverse set of physiological and pathophysiological processes were discussed. Platelets can mediate such processes primarily through their ability to recognize and activate in response to biological stimuli, resulting in the local release of bioactive molecules. [56] There are over 300 molecules that can be secreted by platelets, which could potentially cause systemic thrombosis and inflammation if they were all released in an uncontrolled manner. Therefore, the release of these bioactive molecules must be tightly regulated spatiotemporally in order to coordinate the function of normal platelets in a diverse range of physiological processes. [21]   Methods to locally deliver therapeutics are needed Localizing the delivery of therapeutics is an important goal in medicine, particularly for drugs with adverse systemic side effects. For example, current procoagulant or anticoagulant therapies are commonly associated with risks of complications such as thrombosis or hemorrhage; [57-63] effective localization and targeting of these drugs to areas of vascular damage or clots could greatly enhance their efficacy while minimizing risks of off target effects. Many strategies have been developed to localize drugs to their intended sites of action. This includes local direct injections, as well as the encapsulation of therapeutics into natural or synthetic carriers to shield therapeutic activity, prolong circulation times, and enhance drug accumulation at disease sites. [64-67] For example, long-circulating nanoparticles containing chemotherapeutic drugs that evade clearance by the host can be passively targeted to tumors by exploiting the leaky vasculature in the local tumor microenvironment. [68] This allows nanoparticles to preferentially accumulate at tumors and effect cytotoxicity in a local manner. 16 This strategy has been used to deliver doxorubicin and daunorubicin for treating various cancers. [68-70] Furthermore, nanoparticles can also be functionalized with various moieties that can recognize and bind to specific ligands at the intended site to target therapeutics to specific cells. [71, 72]  Platelets endocytose natural and synthetic materials  Platelets take up small molecules and large particles through distinct mechanisms. The uptake of small molecules was determined to occur primarily through random collisions and non-specific adsorption to the plasma membrane. [73-75] This process is not energy dependent and ultimately leads to the internalization of small molecules through the open canalicular system (OCS), which is a platelet-specific invagination of the plasma membrane. [76]  Platelets also actively take up and sequester larger particles and plasma proteins such as vWF, albumin, fibrinogen, FV, and latex particles though energy dependent processes. [73, 77, 78] The mechanism of uptake was similar to phagocytosis by leukocytes, and involved plasma membrane invaginations that were not related to the OCS. The endocytosis of fibrinogen is dependent on GPIIbIIIa, which is the fibrinogen membrane surface receptor on platelets, but the internalization of fibrinogen by platelets did not cause significant activation. [79] Although the exact mechanisms of platelet endocytosis remain to be completely elucidated, platelet endocytosis involves coated membranes and vesicles, such as clathrin and VAMP3, in order to traffick endocytosed materials into specific granules for platelet function and secretion. [80, 81]  17 Platelets can release biomolecules in response to specific biological stimuli In nature, the challenge of locally delivering biomolecules has in part been addressed by platelets. Platelets are natural local delivery vehicles because they can preferentially accumulate at sites of vascular damage; they possess a diverse set of membrane surface receptors that can recognize and activate in response to specific biological stimuli. [8, 34, 82] During vascular damage, sub-endothelial proteins such as collagens are exposed to blood components and platelets. Platelets can bind collagen directly via the GPVI and α2β1 receptors present on the plasma membrane, as well as indirectly via vWF, a circulating glycoprotein, through the GPIb-IX-V receptor, ultimately leading to platelet adhesion and initiation of the first wave of hemostasis. [83] Vascular injury also results in the exposure of tissue factor to blood, and triggers activation of the coagulation system, eventually leading to the generation of thrombin, which is the most potent activator of platelets. [84]   Collagen and thrombin stimulates platelet degranulation, leading to the release of more platelet adhesion molecules such as vWF and fibrinogen, as well as molecules that further contribute to the activation of platelets and the coagulation cascade, such as ADP and Ca2+. [7, 82, 85-88] In addition to procogulant and hemostatic molecules, several reports have demonstrated the release and delivery of platelet contents such as proteins and nucleic acids to target cells to modulate their function. [89] For example, platelets have been shown to package miRNA and RNAi machinery into small membrane-enclosed microparticles, which are released upon platelet activation and internalized by endothelial cells to regulate gene expression. [89-91] Furthermore, there is evidence that platelets are differentially activated by specific stimuli, during which subsets of granule contents are secreted based on the specific activation conditions. 18 [21] While the mechanisms of differential platelet activation remain to be completely elucidated, [21, 92, 93] these studies demonstrate that the release of platelet contents is highly regulated, and suggests that strategies to manipulate the genomic or proteomic contents of platelets could potentially allow for a way to deliver biomolecules in a controlled and specific manner.  1.3.3 Current applications and future opportunities for modified platelets in the clinic Because of the central role of platelets in maintaining hemostasis and their contribution to wound healing, there are currently several clinical applications for platelet therapy. In the following paragraphs, current platelet therapies, along with some of their limitations and challenges that remain to be addressed, as well as the future opportunities for potential clinical applications for modified platelets will be discussed.  Platelets are transfused to stop severe, traumatic bleeding One important indication for platelet transfusions is in massive transfusion protocols for patients with severe, active hemorrhage. [94, 95] During traumatic hemorrhage, total blood volume, blood cells, and coagulation protein levels can become extremely low due to uncontrolled bleeding and excessive blood loss. Recent clinical studies have demonstrated the advantages of early platelet transfusions, as well as the benefits to transfusing a higher ratio of platelets relative to plasma and red blood cells compared to past protocols. [96, 97] However, despite transfusions of blood components and aggressive resuscitation protocols, trauma remains to be the major cause of mortality in young, healthy adults worldwide. [98] Uncontrolled bleeding during trauma is further complicated by TIC, which is characterized by acidosis, decreased clot strength, increased fibrinolysis, and platelet dysfunction, where platelets no longer 19 respond to agonists such as ADP and collagen. [98-100] Platelet dysfunction is strongly correlated with mortality, and is not easily corrected by transfusions of normal platelets. Developing strategies to enhance endogenous platelet function, such as increasing their sensitivity to agonists, could have the potential to greatly increase the efficacy of platelet transfusions during TIC.  Platelets are transfused to prevent bleeding in thrombocytopenic patients Platelet transfusions were first shown to reduce mortality from hemorrhage in leukemia patients and have since been integrated into several clinical applications, including cancer treatment, haematopoietic stem cell transplantation, and traumatic hemorrhage. [96, 101] Millions of platelet units are transfused worldwide each year, with the goal of preventing or stopping bleeding. [101] Traditionally, prophylactic platelet transfusions were administered when platelet counts fall below a certain number, conventionally set at 10 000 platelets per µL for stable patients in most clinical practices. [102] However, more recent studies support the use of platelet transfusions only during active bleeding even for patients with low platelet counts, and this was reported to be safe for patients undergoing peripheral blood stem-cell transplants. [103] Nevertheless, platelet transfusions remain an integral aspect of treatment regimens for patients with decreased platelet counts. In these cases, developing strategies to enhance the endogenous coagulability of platelets could potentially decrease the number of platelet transfusions required to prevent bleeding. This could decrease the burden on blood banking inventories as well as mitigate the inherent immunological risks associated with allogeneic transfusions. [104]   20 Platelets lose function during storage Platelet concentrates used for transfusions are stored for up to five or seven days at room temperature with agitation.[105] The short shelf life is primarily due to risks of contamination with pathogens, as well the development of platelet storage lesion (PSL), where platelet quality deteriorates after storage. [106] PSL is correlated with deleterious effects in platelet structure and function, and has been shown to reduce recovery, survival, and hemostatic potential following transfusion. PSL has been associated with premature platelet activation and degranulation during storage, progressive energy consumption, and apoptosis and necrosis. [106] Although the exact mechanisms by which PSL occurs have not been well defined, the degradation of platelet function following transfusion suggests that the efficacy of platelet transfusions could be enhanced by transfusing higher-quality platelet products. Developing ways to enhance platelet function and increase their coagulability ex vivo could be a first step in generating higher-quality platelet units for transfusion purposes.  PRP is topically injected for wound healing Platelet-rich plasma (PRP) gels are used in a variety of clinical application for healing acute and chronic wounds, such as cutaneous ulcers, traumatic wounds, and surgical wounds, in many fields, including orthopedics, dentistry, and sports medicine. [107-111] PRP gels are loosely defined as a portion of the autologous plasma fraction enriched with platelets, and are used to stimulate wound healing because they are rich in growth factors, cytokines, and chemokines. [112] These gels contain a fibrin scaffold derived from the patient’s blood, and function as both a sealant and a local drug delivery system. A multitude of growth factors are released from PRP gels, such as platelet factor 4, epidermal growth factor, and vascular 21 endothelial growth factor; they contribute to the healing process by recruiting undifferentiated cells and triggering cell division. [41, 113] PRP gels may also modulate inflammation and interact with macrophages to stimulate tissue regeneration, promote angiogenesis and accelerate epithelialization. [114-117] Although PRP gels can localize the release of growth factors to wound sites and show promise for certain wound healing scenarios, their efficacy and application for broad clinical use remains controversial. Strategies to manipulate platelet function may allow for tailoring of the cocktail of growth factors that are released by PRP gels and potentially enhance their efficacy for wound healing applications.  Platelets may be used to co-deliver other therapeutics Platelet transfusions in the clinical situations described above are often accompanied by therapeutic strategies, such as chemotherapeutics in cancer patients, and procoagulant and hemostatic molecules for traumatic bleeding. Strategies to combine therapies or co-deliver therapeutics on a single platform could not only enhance their efficacy, but also has the potential to simultaneously address different challenges in complex clinical scenarios. For example, in severe traumatic hemorrhage, transfusions of blood components is supplemented with other management strategies, such as fluid resuscitation, [118, 119] rFVIIa to promote clot formation, [120] and tranexamic acid (TXA) to inhibit fibrinolysis. [121, 122] Since platelets naturally localize to sites of vascular damage and clot formation, developing tools to co-deliver other procoagulant or antifibrinolytic molecules with platelets could localize these therapeutics to clots and synergistically enhance the efficacy of these therapies.  22 In this thesis, thrombin was loaded into platelets as a strategy to increase the endogenous coagulability of platelets. Thrombin is a coagulation factor and potently contributes to several stages of the hemostatic response, which is summarized in Table 1.2. Following vascular injury, thrombin is first generated inefficiently through FXa activation of prothrombin. [123-125] Through positive-feedback activation of the coagulation cascade, assembly of the prothrombinase complex leads to acceleration of thrombin generation to quickly initiate a hemostatic response. Thrombin is also responsible for the conversion of fibrinogen to fibrin, which then spontaneously assembles and aggregates to from the basic meshwork of a blood clot. [126, 127] Activation of FXIII by thrombin results in the covalent crosslinking of the fibrin mesh to form a stable clot that is more resistant to degradation. [127, 128] Thrombin is also a potent platelet agonist; it binds and cleaves the protease-activated receptors, PAR-1 and PAR-4, on the surface of platelets, resulting in a cascade of signaling events that lead to platelet activation, aggregation, and degranulation. [129, 130] Thrombin further participates in the hemostatic response through inhibiting anticoagulation. Thrombin can bind thrombomodulin that is present on the endothelial surface and subsequently activate TAFI, which removes binding sites for fibrinolytic proteins in the fibrin meshwork and effectively decreases the rate of clot degradation. [131, 132] On the other hand, the binding of thrombin to thrombomodulin can also activate protein C, which then acts as a potent anticoagulant by inhibiting by inactivating FVa and FVIIIa. [133] Nevertheless, due to thrombin being a potent activator of coagulation, its application during severe bleeding may have many potential benefits. However, thrombin is currently only approved for topical applications due to high risks of thrombosis associated with systemic administration. Developing strategies to localize thrombin activity may be highly beneficial for use in severe bleeding scenarios. 23 Primary hemostasis  Activation of platelets through cleavage of PAR-1 and PAR-4 [129, 130] Activation of coagulation cascade  Activation of FV, FVIII, and FXI [124]  Positive feedback to generate more thrombin [124]  Conversion of fibrinogen to fibrin [126] Clot stabilization  Activation of FXIIIa to covalently crosslink fibrin mesh [127, 128]  Activation of TAFI to inhibit fibrinolysis [131, 134] Table 1.2. The procoagulant roles of thrombin. 1.3.4 Previous approaches to modifying platelet function ex vivo To our knowledge, there are limited methods in the literature to deliver biomolecules to platelets with the intent of modifying or enhancing platelet function. The following subsections will discuss some of the strategies that were described in previous reports of manipulating platelet function, including deriving platelets from modified megakaryocytes, and the direct transfection of platelets ex vivo.  Engineered platelets can be derived from modified megakaryocytes Targeting expression of FVIII to platelets has been demonstrated to be a promising gene therapy approach for the treatment of hemophilia A. By inserting a lineage-specific FVIII gene into megakaryocytes, FVIII expression was directed to platelets and was found to be stored in the α-granules of the differentiated platelets. [135] Strategies to express FVIII in platelets effectively restored hemostasis in FVIIInull murine and canine models, even in the presence of high-titer inhibitory antibodies. [136-138] Although the manipulation of megakaryocyte function is laborious and requires bone marrow transplantation for clinical use, this strategy was effective for hemophilia A treatment because a permanent, sustained expression of FVIII is needed to 24 maintain consistent FVIII levels in the blood and prevent spontaneous bleeding. However, this would be inappropriate for applications such as traumatic bleeding, wound healing, or anti-cancer therapy, where a temporary and reversible modification of platelet function is desired. Currently, there are also challenges in deriving enough platelets from megakaryocytes at a clinically and commercially viable scale, [139, 140] limiting the technology to transplantation of modified precursor cells. Therefore, strategies to manipulate the function of transfusable platelets ex vivo may be a simpler and more clinically relevant approach for acute clinical scenarios.  Platelets can be directly transfected with biomolecules There is one report where platelets have been directly transfected ex vivo with siRNA using lipofectamine and electroporation, resulting in downregulation of platelet GAPDH transcript. [141] Although this work provides proof-of-concept evidence that platelets can be directly transfected with exogenous biomolecules, the levels of protein knockdown was not measured and further applications of this strategy to generate modified platelets for clinical use have not been described. Since platelet function and reactivity following transfection was not tested in this study, it remains unclear whether these are viable methods to modify platelets for clinical purposes. A more recent study described a new potential therapy using platelets loaded with doxorubicin for treating lymphoma. [142] In this case, platelets were loaded with doxorubicin ex vivo through the open canalicular system, which protected the drug from immune surveillance and targeted delivery of the chemotherapeutics to tumors. Loading platelets with doxorubicin did not induce significant morphological or functional changes. The system had enhanced chemotherapeutic efficacy while attenuating off-target side effects, further highlighting the potential for using platelets as therapeutic carriers. These studies suggest platelet function can 25 be modified or enhanced ex vivo, and provides motivation to further characterize the use of modified platelets for an extended range of clinical applications.  1.3.5 Lipid-based nanoparticles for intracellular delivery of biomolecules to platelets Although there is potential to use platelets as localized therapeutic delivery vehicles, significant challenges remain in effectively modifying platelet function. As discussed in 1.3.4, there are limited strategies to delivering biomolecules to platelets ex vivo. Even though ex vivo platelets can be directly transfected with biomolecules, this approach would be limited to inert molecules that do not cause platelet activation or cytotoxicity. For example, thrombin is a coagulation factor and potent platelet agonist which may be useful in conjunction with platelet therapy to stop active bleeding. [143] However, its activity must be shielded from platelets during the loading process in order to minimize platelet activation. One major class of local delivery vehicles is lipid-based nanoparticles (LNPs). Because many lipids and membranes are naturally found in the bloodstream and remain relatively inert while in contact with platelets, LNPs have the potential to be used to encapsulate and deliver biomolecules to platelets without causing significant premature platelet activation.  LNPs are one of the most extensively characterized classes of nanotherapeutics, with liposomal doxorubicin being one of the first nanoparticle formulations to be approved for clinical use. [144-146] Since then, at least 15 more liposomal formulations have been clinically approved and many are in preclinical and clinical trials, for applications ranging from drug delivery to imaging. [147] Because of their amphipathic nature, lipids will self-assemble into nanoparticles in solution in order to exclude the hydrophobic tails from the aqueous environment. [148] 26 Depending on the composition of the various lipids in the mixture and the structures of each individual lipid, lipid nanoparticles may take the form of micelles, liposomes, or solid-core nanoparticles. [149-151] Each of these different structures have advantages in encapsulating different cargo; hydrophobic cargo will associate with the hydrophobic tails of the lipids, whereas hydrophilic drugs may be passively encapsulated into the aqueous core of liposomes or bind the hydrophilic heads of lipids. [68, 152, 153] Nanoparticle size can be controlled by extrusion, or high-efficiency mixing using microfluidic devices, resulting in a homogenous population of drug-encapsulated nanoparticles. [154-156]   In addition to localized delivery, encapsulating biomolecules such as nucleic acids and proteins into lipid-based carriers offers several advantages. First, encapsulation effectively shields cargo from the outside environment, reducing inactivation of the loaded cargo by external degrading enzymes. [68, 157] Second, encapsulation of nucleic acids and proteins facilitates intracellular delivery; these charged macromolecules cannot efficiently cross the plasma membranes of cells and are generally not internalized on their own. [150] Lipid nanoparticles have been shown to be endocytosed by various cell types through multiple mechanisms, [150, 158, 159] and specificity and internalization can be increased by incorporating ligands for cell surface receptors into the liposome formulation. [160-163] For example, negatively-charged liposomes containing weakly acidic molecules were endocytosed through coated vesicle structures. [164, 165] In order to encapsulate different cargo, the lipid compositions and formulation methods can be easily modified to enhance drug loading efficiency and cellular uptake. [150, 166] Moreover, modification of the lipid composition can also facilitate endosomal escape and intracellular targeting following internalization. [167] This is especially useful in 27 certain applications such as the intracellular delivery of siRNA, which requires the RNA to localize to the cell cytoplasm and RNAi machinery to exert its gene silencing effects. [168, 169] One approach is to incorporate pH-sensitive lipids into the nanoparticle formulation. [170] These liposome formulations are generally composed of a neutral, cone-shaped lipid such as DOPE, which forms a hexagonal structure, and rapidly destabilizes in acidic conditions. This allows the liposomes to fuse with the endosomal membrane during endosomal maturation and acidification, allowing the liposomal contents to be released into the cytoplasm of the cell. [171-174]   The use of lipid-based carriers to deliver molecules to platelets has not been well characterized prior to the work in this dissertation. To our knowledge, platelets have been transfected with siRNA using lipofectamine,[141] but there have not been reports of using lipid nanoparticles to facilitate the uptake of molecules in platelets. Manipulation of platelet contents and function is challenging because they are anucleate and are prone to premature activation and aggregation during handling. Because of the diverse role of platelets and the potential to use platelets as drug delivery vehicles as outlined in 1.3.1 and 1.3.2, there is significant motivation to develop and test the use of lipid nanoparticles for delivering biologically and clinically relevant molecules to platelets (Figure 1.1).    28  Figure 1.1. Schematic of cargo encapsulated into liposomes which can be delivered to platelets. The delivered cargo may be used to modify or enhance platelet function, or may be released from platelets during platelet activation. 29 Chapter 2: Developing new strategies to deliver biomolecules to platelets ex vivo  2.1 Rationale Local delivery of therapeutics to sites of damage within blood vessels remains a major challenge in managing hemorrhage and cardiovascular disease. For example, most coagulation factors remain inactive in circulation as zymogens, and are only specifically activated at wounds so that coagulation is not initiated systemically. [175] Nature has in part overcome this challenge of local delivery by using platelets as delivery vehicles for small molecules and biological macromolecules that localize coagulation and inflammation to specific areas of vasculature. [56] This makes platelets attractive therapeutic targets due to their ability to locally modulate pathological diseases. However, modifying the genomic or proteomic contents of platelets is not a trivial matter, since ex vivo platelets are easily activated during handling and storage, resulting in platelets that do not function normally following transfusion back into the bloodstream. The idea of using platelets as drug delivery vehicles has been explored, primarily through the manipulation of megakaryocytes. [135] However, reliable approaches to enhance or modify the natural function of transfusable platelets have not been reported.  Platelets are routinely transfused to prevent and treat bleeding. One application is for patients who develop thrombocytopenia following chemotherapy or hematopoietic progenitor cell transplantation. [101] Another clinical scenario is during severe, active hemorrhage. [96] In all cases, the ability to co-deliver small molecule or macromolecular drugs with platelets could greatly enhance the local delivery and efficacy of therapeutics and transfused platelets at sites of 30 platelet activation. This could include chemotherapeutic drugs for cancer treatment, or procoagulant and antifibrinolytic drugs in cases of severe bleeding. Therefore, an approach to directly modify the contents of transfusable platelets could allow direct targeting of platelets, as well as allow therapeutics to be co-delivered during platelet transfusions.  Lipid-based nanoparticles have been used extensively to encapsulate and deliver biomolecules to cells. Their advantages include spontaneous formation, low immunogenicity, the ability to encapsulate a wide variety of cargo within the hydrophobic bilayer or in the aqueous core of a liposome. [176] Encapsulation of drugs into nanoparticles has allowed for intravenous injection of drugs with undesired side effects, such as chemotherapeutics, by efficiently shielding the cargo and enhancing localization and delivery of drugs into target cells. [177] To our knowledge, there are limited reports on developing lipid-based systems to enhance or modify platelet function. One example is the in vitro transfection of platelets with siRNA targeted to GAPDH using lipofectamine. [141] Although this is the first report of directly introducing nucleic acids to platelets, the system has not been well characterized and the potential clinical or biological relevance of these transfected platelets have not been explored further. We therefore hypothesized that lipid-based nanoparticles, which have a more defined structure and mechanism of internalization compared to lipofectamine, can be delivered to and internalized by platelets ex vivo with minimal premature platelet activation. These nanoparticles will then be further optimized and used in chapters 3 and 4 to encapsulate and deliver potentially therapeutically relevant biomolecules to platelets.  31 2.2 Methods  2.2.1 Isolating platelets from whole blood Approvals for the study were given by the research ethics boards of the University of British Columbia. Informed, signed consent was obtained from healthy volunteers prior to collecting whole blood from donors. Whole blood was collected into tubes containing sodium citrate (105 mM). Platelets were isolated from citrated whole blood by centrifuging at 100 × g for 20 min, and collecting the platelet-rich plasma (PRP), which was the top 75% (v/v) of the upper layer. To obtain platelet-poor plasma (PPP), the remaining whole blood was centrifuged at 1000 × g for 10 min. To obtain purified platelets, PRP was centrifuged at 250 × g for 20 min and the plasma was removed. The platelets were washed once in a buffer containing citrate and glucose (CGS; 120 mM NaCl, 30 mM D-glucose, 11 mM trisodium citrate, pH 6.5) and once in Tyrode’s-HEPES buffer (1.8 mM CaCl2, 1.1 mM MgCl2, 2.7 mM KCl, 137 mM NaCl, 0.4 mM NaH2PO4, 10 mM HEPES, 5.6 mM D-glucose, pH 6.5) at 250 × g for 10 min. Washed platelets were resuspended in Tyrode’s-HEPES at a final concentration of 200 × 109 platelets L-1.  2.2.2 Preparing liposomes Liposomes were prepared using a thin-film hydration technique. Lipids (1 mg DOPC or DMPC) were dissolved in chloroform and coated in a thin film on a centrifuge tube. The lipid films were dried overnight in a vacuum to remove all residual solvents. Tris buffered saline (TBS; 50 mM Tris-Cl, 150 mM NaCl, pH 7.5; 100 µL) was used to rehydrate lipid films and form a heterogeneous mixture of lipid particles. To prepare unilamellar liposomes of specific sizes, the rehydrated lipid mixture was extruded 10 times through a LIPEX extruder containing a 32 filter with specific pore sizes. Liposome sizing was determined using a dynamic light scattering instrument (Malvern Zetasizer Nano).  2.2.3 Measuring liposome uptake in isolated platelets. Liposomes (10% v/v) were added to 200 µL washed platelets, and the mixture was incubated for 30 min at 37°C while uptake occurred. To remove excess liposomes, platelets and liposomes were centrifuged at 250 × g for 5 min and the supernatant was removed. Platelets were then washed twice in Tyrode’s-HEPES buffer at 250 × g for 5 minutes. To inhibit uptake of liposomes, platelets were pre-incubated for 30 minutes with nystatin (0.45 mM), chlorpromazine (8.4 mM), dynasore (Sigma), sodium azide (NaN3; Sigma), cytochalasin D (Calbiochem), PAO (Sigma), amiloride (Sigma), or dimethylsulfoxide (0.5% v/v; Sigma) before addition of liposomes. When inhibitors were used, they were present in platelet samples at all times.  2.2.4 Quantifying and imaging the uptake of liposomes by platelets. Uptake of liposomes was analyzed by flow cytometry and confocal microscopy. In flow cytometry experiments, fluorescent Oregon Green 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (OG-DHPE; 0.2 – 0.5% w/w of total lipids; Invitrogen) was added to the liposomes. Lipids were resuspended in TBS, extruded into nanoliposomes, and incubated with platelets. After removal of excess liposomes, platelets were stained with anti-human CD42b-APC antibody (5 ng μL-1; eBiosciences) for 30 min and analyzed by flow cytometry (Calibur, BD Biosciences). For flow cytometry, platelets were identified using forward and side scatter, as well as APC staining. Single color controls were used to determine the background fluorescence of each stain and to draw quadrants.  For confocal microscopy experiments, fluorescent Texas 33 Red-DHPE (1% w/w of total lipids, Invitrogen) was added to the liposomes. Incubation of liposomes and platelets was performed as described for flow cytometry experiments. Platelets were fixed with 4% paraformaldehyde (w/v) and adhered onto cover slips. Cover slips were washed twice with phosphate buffered saline (PBS; LifeTechnologies), blocked with 5% goat serum, and stained with anti-human CD42b-FITC antibody (5 ng μL-1; eBiosciences) overnight. Images were acquired using a 60 x objective on a Nikon Eclipse TI microscope and NIS-Elements AR software.  2.2.5 Measuring platelet activation by flow cytometry After removing excess liposomes, platelets were resuspended in Tyrode’s-HEPES buffer (pH 7.4, unless otherwise indicated). To determine the level of activation, platelets were stained with 10 ng μL-1 anti-human CD42b-FITC and 2 ng μL-1 anti-human CD62P-APC antibodies (eBiosciences) and incubated at 37°C for 30 min. For flow cytometry analysis (FACSCalibur, BD Biosciences), platelets were identified using forward and side scatter, as well as positive FITC staining. Single colour controls were used to determine the background fluorescence of each stain and to define quadrants.    34 2.3 Results  2.3.1 Optimized conditions for delivering liposomes to platelets The platelet and liposome incubation conditions were optimized to maximize uptake and minimize platelet activation. Several conditions were tested, including buffer, temperature, incubation time, liposome sizes, platelet to liposome ratio, as well as the number of washes to efficiently removed liposomes that were not internalized. Liposome uptake was measured by incorporating a fluorescently tagged lipid into the liposome, and measuring the liposomal fluorescence intensity associated with platelets. A summary of the tested conditions is listed in Table 1.1. The optimum conditions were determined to be 10% liposomes (v/v) in Tyrode’s buffer (pH 6.5), at room temperature for one hour (Figure 2.1). Smaller liposomes were internalized more efficiently by platelets, but may be limited to encapsulating smaller molecules. Neutral liposomes were used in order to minimize platelet activation during uptake. A minimum of three washes at 250 × g for 5 minutes were necessary to remove excess liposomes.  Platelet activation measured by flow cytometry confirmed that the liposome preparations do not cause significant platelet activation (Figure 2.2) In contrast, incubating platelets with thrombin, a potent platelet agonist and positive control, caused significant platelet activation.    35 Condition Variables Tested Buffer Plasma CGS (pH 6.5) Tyrode’s (pH 6.5)* Tyrode’s (pH 7.4) Temperature 4°C 20°C* 37°C Time 15 min 30 min 60 min* Liposome diameter 100 nm 200 nm* 400 nm 1µm Liposome to platelet ratio (v/v) 1:5 1:10* 1:20 1:100 Washes to remove excess liposomes 1 2 3* Table 2.1. Summary of incubation conditions tested to optimize uptake of liposomes by platelets. Conditions where liposomal uptake occurred are bolded. Optimized conditions that were used for experiments in this chapter are indicated with *.  36  Figure 2.1. Platelets take up liposomes more effciently in Tyrode's buffer than in CGS. Liposomes containing DHPE-Oregon Green were incubated with platelets in the indicated buffers over three hours. The mean fluorescence intensity (MFI) was recorded for a population gated by forward scatter, side scatter, and CD42b, a platelet marker.   Figure 2.2. Incubation of platelets with liposomes did not cause platelet activation. Platelets were incubated with liposomes according to the optimized conditions, stained with CD42b-FITC and CD62P-APC, and analyzed by flow cytometry to determine the level of platelet activation. * P < 0.05  37 2.3.2 Endocytosis of phosphatidylcholine-based liposomes by platelets The ability of platelets to take up liposomes was first assessed using flow cytometry and confocal microscopy. Empty, fluorescently-tagged liposomes were incubated with isolated platelets in a buffered solution. Excess liposomes were removed, and platelets were stained with a fluorescent antibody against CD42b, a cell-surface marker expressed only on platelets and megakaryocytes. [178] Liposomes co-localized with 35 to 65% of platelets, with variation between platelet donors (Figure 2.3A). Confocal microscopy of liposome-treated platelets confirmed that the majority of co-localization corresponded to internalized liposomes, where the liposomes tagged with a red fluorophore is consistently observed inside of the platelet membrane, which is stained with a green fluorophore (Figure 2.3B).  38   Figure 2.3. Nanoliposomes are internalized by platelets. (A) Representative histograms of platelets (shaded grey curve) from flow cytometry depict shifts in fluorescence from liposomes, corresponding to liposome internalization (black curve). (B) Confocal images of platelets (green) and liposomes (red) showing internalization of liposomes by platelets. Scale bar is 10 µm.   To determine the mechanism by which platelets were taking up liposomes, platelets were pre-incubated with several inhibitors of endocytosis prior to incubation with liposomes. The 39 reduction in uptake that was observed in the presence of these inhibitors further confirmed that liposomes were being endocytosed, as opposed to only binding the platelet surface. Using microscopy, a reduction in liposome uptake was observed in the presence of nystatin (0.45 mM) or chlorpromazine (8.4 mM), which inhibit caveolae- and clathrin-mediated endocytosis, respectively (Figure 2.4A). In the chlorpromazine-inhibited samples, liposomes were observed to bind the platelet surface, but not be internalized. Flow cytometry showed that the co-localization of platelets and liposomes decreased by over 60% by cytochalasin D (4 μM), an inhibitor of actin polymerization, as well as by sodium azide (NaN3; 50 mM), a general metabolic inhibitor (Figure 2.5B). Using inhibitors specific to different endocytotic pathways, uptake was reduced by over 60% by dynasore (25 µM) and 35% by phenylarsine oxide (PAO; 10 µM), while amiloride (1 mM) did not significantly reduce uptake (Figure 2.4B). Dynasore inhibits dynamin-dependent endocytosis pathways, including caveolae- and clathrin-mediated endocytosis; PAO is an inhibitor of clathrin-mediated endocytosis, and amiloride is an inhibitor of phagocytosis and micropinocytosis. [179] DMSO was used as a solvent for the inhibitors, and therefore served as a control. Pre-incubation of platelets with DMSO had no effect on the amount of liposomes that were taken up by platelets.  40  Figure 2.4. Liposomal uptake can be inhibited with several inhibitors of endocytosis. (A) Platelets were loaded with liposomes with DHPE-Oregon Green (green) and then stained with CD42b-APC (red) for visualization. Platelets were pre-treated with 0.45 mM nystatin or 8.4 mM chlorpromazine for 30 min at room temperature prior to incubation with liposomes. (B) Uptake of liposomes by platelets was reduced when platelets were pre-treated with a metabolic inhibitor and inhibitors of endocytosis, quantified by flow cytometry. Error bars represent SEM (n = 3). * P < 0.05, ** P < 0.001, n.s. indicates it is not significant compared to no inhibitor.    41 2.4 Discussion The encapsulation of small molecules, proteins, and nucleic acids into lipid nanoparticles has been well documented and explored in the literature. Lipid-based systems have been used extensively because they spontaneously self-assemble, are biocompatible, and have the ability to encapsulate a variety of cargo due to their amphipathic nature. [176] For the purposes of this dissertation, we identified liposome formulations and incubation conditions which allowed liposomes to be taken up by platelets in vitro, while minimizing premature platelet activation. A range of conditions allowed for the uptake of liposomes, such as temperatures ranging from room temperature to 37°C, liposome sizes of 100 nm to 200 nm in diameter, and liposome to platelet ratios of 1 to 20% (v/v). However, some of these incubations promoted premature platelet activation, including incubation at 37°C and increasing liposome to platelet ratios. Furthermore, smaller liposomes may be less efficient in encapsulating larger molecules such as nucleic acids and proteins. Therefore, the conditions identified here will only be used as a starting point for delivering transcriptional machinery (chapter 3) and thrombin (chapter 4) to platelets, and some optimization for encapsulation and delivery will be needed for the specific cargo.  For the purposes of characterizing liposome endocytosis for platelets, optimized conditions were determined to be 200 nm liposomes, incubated with platelets in a 1:10 ratio in Tyrode’s buffer (pH 6.5) at 20°C for 1 hour. Excess liposomes were removed by washing the platelets for 5 min at 250 × g three times. Using a combination of microscopy and flow cytometry, fluorescently tagged liposomes were observed to accumulate within platelets. By pre-treating platelets with different inhibitors of endocytotic pathways, it appears that multiple 42 mechanisms contribute to the overall uptake of liposomes by platelets. The flow cytometry results suggest platelets primarily take up liposomes through multiple dynamin-dependent endocytotic pathways, since uptake is inhibited by dynasore.[180, 181] These results are further confirmed by microscopy, which shows that uptake is reduced following incubation with nystatin and chlorpromazine, which inhibit caveolae- and clathrin-mediated endocytosis, respectively. [182, 183] Interestingly, in the presence of chlorpromazine, liposomes are observed to be bound to the surface of platelets but not internalized. Chlorpromazine inhibits clathrin-mediated endocytosis by disrupting LDL-receptor trafficking, leading to the loss of clathrin-coated pits on the plasma membrane and thus inhibiting the endocytotic pathway. [183] Both caveolae- and clathrin-mediated endocytosis are dynamin-dependent, further confirming the flow cytometry results with dynasore. Previously, platelets have been reported to internalize nanoparticles through the open canalicular system (OCS), a surface-connected system of channels within the core of the platelet, as well as by cell engulfment and trafficking to storage vacuoles. [142, 184] This is consistent with the results observed here, including the inability of any single inhibitor to completely abrogate uptake, and suggests that multiple mechanisms are involved in the internalization of liposomes.   Taken together, the data suggests that lipid-based nanoparticles can be used to encapsulate and deliver various cargo to ex vivo platelets. The liposomes can be modified to encapsulate proteins and nucleic acids. Furthermore, platelets actively endocytose liposomes, given the proper incubation conditions. The approaches developed in this chapter will serve as a basis to encapsulate and deliver biologically relevant cargo to platelets in chapters 3 and 4. Although some modifications will be needed to optimize the formulation for specific cargo, the 43 parameters determined here will be useful as a starting point for delivering a variety of molecules to platelets. In the next two chapters, liposomes will be used to deliver synthetic nuclei (chapter 3) and a coagulation factor (chapter 4) in order to modify or enhance the endogenous function of platelets. Although these systems will require much more development and testing before translation to the clinic, these studies provide proof-of-concept that endogenous platelet function can be modified ex vivo, and is a first step towards developing engineered platelets for therapeutic applications. 44 Chapter 3: Transcription of exogenous mRNA in anucleate platelets  3.1 Rationale Platelets have an abundance of mRNA and miRNA which regulate platelet function using endogenous platelet translation and RNAi machinery. [185-187] The ability to genetically modulate platelet function could potentially have implications in treating many pathologies, since platelets are important contributors to processes such as coagulation and inflammation. However, platelets are not amenable to traditional transfection techniques due to their anucleate nature, and ex vivo platelets have a tendency to activate and aggregate during handling and storage. Manipulation of platelets at the genetic level requires altering cultured megakaryocytes, which is laborious and its clinical use requires bone marrow transfections and results in permanent alteration of the platelet genome. Moreover, significant hurdles remain before platelets cultured from megakaryocytes can be used clinically, such as developing techniques to generate enough platelets in vitro for transfusion purposes. [139, 140] This provides significant motivation to directly transfect platelets ex vivo, and these strategies will allow for a temporary modification of the platelet genome, since platelets only have a life span of approximately one week. Additionally, engineering transcription in platelets provides a level of control, where transcription can be regulated with specific stimuli.  Cell- and nuclei-free systems have been developed for synthesizing RNA and proteins using a phage RNA polymerase and translational machinery extracted from cells. Encapsulating cell extracts within synthetic lipid bilayers has led to the development of “protocells”, liposomes capable of protein expression. [188] Protocells have been used to model early cellular life, [189-45 195] and significant advances have been made in studying and maximizing protein expression within nano- [196] and micron-sized liposomes. [197-199] Several applications for protocells in synthetic biology and drug delivery have been explored. These include the use of protein-synthesizing vesicles as synthetic vaccines, [200] as reactors for directed evolution, [201] as stimuli-responsive vehicles for modifying bacterial cell behavior, [202] and for applications toward in vivo drug delivery. [203] Can existing protocell technology be adapted to function within platelets to enable transcription of exogenous RNA and modification of natural platelet function? In a step towards answering this question, we hypothesized that RNA-synthesizing nanoliposomes could be delivered to and function within platelets (Figure 3.1).  To test this hypothesis, we used liposomes capable of light-induced RNA synthesis, [203] in order to initiate transcription only after liposomes have been internalized by platelets. Although light-initiated transcription in protocells have been used previously to remotely control protein synthesis in vivo, the incorporation of photo-caged components into our system was not meant for in vivo use, but only to minimize transcription until the liposomes have been internalized by platelets. Components of a transcription reaction, consisting of T7 RNA polymerase (T7RNAP), a linear DNA template, and ribonucleotide triphosphates (rNTPs), including a photo-caged adenosine triphosphate (caged-ATP), were encapsulated into nanoliposomes. Although protocells typically contain coupled RNA and protein synthesis, we focused only on transcription to bypass the difficulties in co-encapsulating components of translation, [204] in order to demonstrate as a proof-of-concept that a variety of functional biomolecules can be co-loaded into platelets ex vivo, and that platelet function can be manipulated through the delivery of exogenous cargo. 46    Figure 3.1. Transcription in nanoliposomes allows exoggenous RNA to be synthesized in anucleate cells such as platelets. (A) Transcriptional components (dark green), including T7RNAP, uncaged ribonucleotides, DNA and photo-caged ATP (light green) were encapsulated into nanoliposomes (blue and orange), which synthesized RNA (red lines) following irradiation. (B) Transcription of RNA is a basic function of nuclei (dark purple and yellow), but anucleate cells, such as platelets, are incapable of de novo RNA synthesis. RNA-synthesizing nanoliposomes allow transcription to occur inside of anucleate cells. 47 3.2 Methods 3.2.1 Plasmids and primers. The GFP coding sequence from pRSET-EmGFP (Invitrogen) was cloned into the multiple cloning site (MCS) of pT7CFE1-CHis (ThermoScientific) using EcoRI and XhoI (Fermentas) to create the GFP plasmid template. The FLuc coding sequence from pEJ3 (a gift from E. Jan, UBC) was cloned into the MCS of pT7CFE1-CHis using Pst1 and PvuII (Fermentas) to create the FLuc plasmid template. PCR using primers forward 5’-GGC CTC TTC GCT ATT ACG C-3’ and reverse 5’-CGA GGA AGC CCG GAT ATA GT-3’ were used to amplify the sequences for EmGFP and FLuc transcription in liposomes. Resulting PCR amplicons contained a T7 promoter for transcription initiation, an EMCV internal ribosome entry site (IRES) for mRNA translation, the protein coding sequence, and a polyA tail for stability. Primers forward 5’-GAT GAC GGA AAA AGA GAT CG-3’ and reverse 5’-TTT TTC TTG CGT CGA GTT TT-3’ were used for quantification of FLuc. Primers forward 5’-CAA CAG CCA CAA GGT CTA T-3’ and reverse 5’-GGT GTT CTG CTG GTA GTG-3’ were used for quantification of EmGFP. Primers forward 5’-CAA GGC TGT GGG CAA GGT-3’ and reverse 5’-GGA AGG CCA TGC CAG TGA-3’ were used for quantification of GAPDH.  3.2.2 Loading platelets with RNA-synthesizing liposomes. Liposomes were prepared using a protocol previously described, with minor modifications. [205] Briefly, a thin film of 12 µmol of lipids, consisting of egg-phosphotidylcholine:cholesterol:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]  (ePC:chol:DSPE-PEG2000; 58.9:39.6:1.5 molar ratio; Avanti Polar Lipids, cholesterol from Sigma) was rehydrated with deionized water, extruded ten 48 times through a 200 nm filter using a LIPEX extruder (Northern Lipids Inc.), and lyophilized. A small percentage of a PEG lipid was incorporated into the formulation because it has previously been reported to enhance protein translation within liposomes in a buffered solution. [197] The lyophilized lipid was rehydrated with deionized water to 24 mM and extruded through a 200 nm filter using a Mini-extruder (Avanti) eleven times.  While on ice, a transcription mixture containing the supplier’s transcription buffer, 10 ng µL–1 of DNA template, 0.75 mM each of GTP, CTP, UTP, and photo-caged ATP (Invitrogen), and 0.07 unit µL–1 of T7RNAP (Toyobo), was added to the formed liposomes and the entire liposome mixture was freeze-thawed once. The linear DNA templates were created through amplification by PCR of plasmid templates using specific primers. Liposomes were purified by passing undiluted liposomes through an anionic exchange column (Vivapure Q Mini H, Sartorius) to remove unencapsulated material. Liposomes were irradiated with white light for 30 sec using a fluorescence microscope (Leica DMI6000B). After incubating the sample for 1 h at 37ᵒC, RNA was extracted using Trizol reagent (Invitrogen), digested with TURBO DNA-free (Invitrogen), reverse transcribed into cDNA using oligo-DT primers and MMLV-reverse transcriptase (Invitrogen), and quantified using qPCR with gene-specific primers and SYBR green reagents (Applied Biosystems). RNA was quantified using 2-ΔΔCt, with GAPDH as an internal control when applicable. Statistical significance was determined using two-tailed Student’s t-test.  3.2.3 Measuring protein expression from synthesized RNA. Liposomes were prepared as described above using the FLuc template (10 ng uL-1). RNA was extracted from 50 uL of liposomes using Trizol reagent, with modifications from the manufacturer’s protocol. During isopropanol precipitation, 100 µg glycogen (Invitrogen) was 49 added, and the precipitation was allowed to proceed for 30 min before being spun down at 21,000 × g for 10 min and washed with 70% (v/v) ethanol. The final pellet was re-suspended in 25 µL of nuclease-free water (Promega). A rabbit reticulocyte lysate (RRL) system (Promega) was used for cell-free protein expression. RNA (6.5 µL) isolated from liposomes was used in a 25 µL reaction of the RRL system to express the protein. After incubating the reaction mixture at 30ᵒC for 90 min, 50 µL of D-luciferin reagent (6 M; Thermo Scientific) was added to the reaction and luminescence was measured in a microplate reader.   50 3.3 Results  3.3.1 Transcription can occur within nanoliposomes In order to formulate liposomes with efficient transcriptional activity that can also be delivered to platelets, some modifications to the composition and preparation of liposomes were made to a previously published method. [203] Even though cationic lipids are generally used to increase the encapsulation of negatively-charged nucleic acids, [150] neutral lipids were used in our system in order for nucleic acids to remain free in solution to allow for polymerase access. Moreover, optimized formulations of LNPs containing cationic lipids for siRNA encapsulation have been shown to adopt solid core structures, [206] but the liposomes needed to have an aqueous core for transcription components to freely diffuse and for RNA synthesis to occur. Using the formulation and extrusion methods from chapter 2, we found that passing the transcription reaction through a LIPEX extruder (200 nm filter) inactivated the transcription components, resulting in no RNA being synthesized after extrusion, as observed by the loss of all bands following five or ten passes through the extruder (Figure 3.2).  The method to encapsulate transcription components were then modified such that only the lipids, without the transcription components, were extruded to the appropriate size. Transcriptional components were then added to the extruded, sized liposomes and freeze-thawed once for encapsulation. At the same time, a lipid functionalized with polyethylene glycol (PEG) was incorporated, which has previously been shown to enhance coupled transcription and translation within nanoliposomes. [197] Incorporation of the PEGylated lipid, along with the new liposome preparation method, increased the RNA yield in liposomes by 10-fold (Figure 3.3). 51    Figure 3.2. Extrusion inactivates components of the transcription reaction. Transcription reactions were passed through the LIPEX extruder for 5 or 10 passes, then incubated at 37°C for 1 or 2 hours. Reactions were run on a 1% agarose gel and visualized with ethidium bromide to detect transcribed RNA.   52  Figure 3.3. Incorporation a PEGylated lipid is required for efficient RNA synthesis in nanoliposomes. An initial formulation consisted of ePC:chol (59.8:40.2 mol ratio) liposomes was optimized by adding DSPE-PEG2000, which led to a 10-fold increase in the amount of GFP mRNA synthesized, measured by qPCR. Error bars represent SEM (n = 3).    53 The specific concentrations of the various components of the transcription reaction, including the concentrations of rNTPs, MgCl2, DNA template, and T7RNAP were varied to determine the conditions with the highest RNA yield. The optimized concentrations were determined to be 10 ng µL–1 of DNA template, 0.75 mM each of GTP, CTP, UTP, and photo-caged ATP, and 0.07 unit µL–1 of T7RNAP, in comparison to the control concentrations, which consisted of 1 ng µL–1 of DNA template, 0.375 mM each of GFP, CTP, UTP, and photo-caged ATP, and 0.7 unit µL–1 of T7RNAP. Combined with the modified liposomal preparation methods and lipid formulation, these optimized concentrations resulted in a 50-fold increase in the amount of RNA synthesized in nanoliposomes (Figure 3.4).  Unencapsulated material was removed from the liposomal preparation in order to inhibit transcription occurring outside of liposomes, and to remove possibly immunostimulatory reagents prior to incubation with platelets. The purification of liposomes was achieved by using an anionic exchange column to remove unencapsulated T7RNAP and DNA, as indicated by the loss of bands on the respective gels, and subsequent incubation of the eluate at 37°C did not produce RNA as seen on an agarose gel (Figure 3.5A and 3.5B). In purified liposomes, encapsulated reagents were protected from the anionic exchange column and transcription was able to occur inside of liposomes (Figure 3.5C).    54  Figure 3.4. Comparison of RNA yield in nanoliposomes before and after optimization of the concentrations of transcriptional components. Nanoliposomes were prepared with different concentrations of transcriptional components and incubated at 37 °C for 1 hour. RNA was quantified using qPCR. Error bars represent SEM. (n = 3)  55   56 Figure 3.5. Unencapsulated transcriptional components are removed from liposomes using an anionic exchange column. (A) SDS-PAGE showing that unencapsulated T7 is removed when the transcription components are passed through an anionic exchange column. (B) Agarose gel showing that unencapsulated DNA, and possibly other transcription components, are removed after passing through the column, resulting in loss of transcriptional activity. (C) In liposomes, transcriptional components are encapsulated and protected from the anionic exchange column, which allows for transcription of GFP mRNA as measured by qPCR. Error bars represent SEM (n = 3).  Transcription was also tested in two batches of liposomes with average diameters of 270 ± 50 nm and 430 ± 120 nm (mean ± s.d.). Since the amount of RNA synthesized in these two populations were not significantly different (Figure 3.6), the smaller liposomes were used to maximize delivery efficiency to platelets, which are only 2-5 µm in diameter. 57  Figure 3.6. RNA is transcribed in liposomes of various sizes. (A) Two groups of liposomes with average diameters of 270 ± 50 nm and 430 ± 120 nm (mean ± s.d.) were prepared. The average diameters were measured using dynamic light scattering. (B) These two groups of liposomes had transcriptional activity that was not significantly different (p > 0.95), measured using qPCR. Error bars represent SEM (n = 3). 58 3.3.2 Controlled RNA Transcription Using Light. In order to control the initiation of RNA transcription in nanoliposomes, ATP in the transcription reaction was replaced by a photo-caged ATP (Amax = 360 nm). In solution, the samples required 5 seconds of irradiation with white light (λ > 300 nm) to release active ATP and allow for transcription to occur, whereas samples with normal ATP was not affected by irradiation with light (Figure 3.7).  Purified nanoliposomes (220 ± 110 nm) containing all the components necessary for transcription were irradiated for 30 s with white light to release ATP from caged ATP and incubated for one hour at 37°C for transcription to occur. A 2300-fold increase in GFP mRNA was detected in purified liposomes following irradiation and incubation, using quantitative polymerase chain reaction (qPCR), whereas only a 6-fold increase was observed in samples that were not irradiated, indicating that transcription initiation could be controlled by light (Figure 3.8A). Another DNA template, for firefly luciferase (FLuc) mRNA, was also transcribed in liposomes, resulting in a 12-fold increase in mRNA in irradiated samples and no significant difference in samples that were not irradiated (Figure 3.8B). To confirm that mRNA transcribed within liposomes was function, a cell-free expression system was used to translate the FLuc mRNA into protein. A 6-fold increase in luminescence was observed for RNA samples isolated from irradiated liposomes, indicating that functional FLuc mRNA was synthesized inside of the nanoliposomes (Figure 3.8C). 59  Figure 3.7. Incorporation of photo-caged ATP allows for controlled transcription initiation using light. Transcription reactions were set up with normal ATP (not caged) or photo-caged ATP (caged) and irradiated with light for 0-10 seconds. Reactions were incubated at 37°C for 1 hour, run on a 1% agarose gel, and visualized with ethidium bromide.  60  Figure 3.8. Transcription of GFP and FLuc mRNA in nanoliposomes is controlled by light. (A-B) The amount of GFP (n = 6) and FLuc mRNA (n = 3) increased in liposomes only when irradiated with light, measured using qPCR. (C) FLuc mRNA extracted from liposomes produces functional enzyme that activates a luminescent substrate in a separate cell-free translation system (n = 3). Error bars represent SEM, * P < 0.05, ** P < 0.01, n.s. indicates it is not significant compared to 0 h. 61 3.3.3 Controlled transcription of RNA in platelets. To test if protocells could function in platelets and enable transcription to occur inside of platelets, light-inducible RNA-synthesizing liposomes were incubated with platelets in the dark. Following incubation at 37°C, a 59-fold increase in mRNA was detected in irradiated platelets, compared to only a 2-fold increase when platelets were first treated with dynasore, which inhibits uptake of liposomes into platelets. To confirm that the increase in mRNA was not due to excess liposomes that did not get removed during washes, two control samples, consisting of liposomes without platelets or liposomes incubated with platelets that were first lysed by freeze-thawing, were purified in the same manner as samples containing intact platelets. In both of these control samples, no significant increases in mRNA occurred, indicating efficient removal of all liposomes not internalized by platelets (Figure 3.9).   62 Figure 3.9. Liposomes internalized by intact platelets synthesized RNA. A significant increase in RNA occurred one hour after irradiation only in uninhibited, intact platelets. When platelets were lysed or absent, no RNA was detected. In dynasore-inhibited platelets, no significant increase in RNA was detected. Error bars represent SEM (n = 3), * P < 0.01, n.s. indicates it is not significant compared to the respective 0 h sample, B.D. indicates it is below detection by qPCR.  63 3.4 Discussion To encapsulate a functional transcription reaction within liposomes that can be delivered to platelets, the liposomes must possess several inherent features. First, the liposomes need to maintain a stable, aqueous core for the transcription reaction to take place. Second, the required transcriptional components must be co-encapsulated in concentrations that allow transcription to occur. Lastly, the lipid composition and size of the liposome must allow for platelet endocytosis. In order to meet these requirements, several modifications were made to the approaches described in chapter 2.  By extruding the empty liposomes alone, transcriptional components were protected from inactivation caused by extrusion at high pressures. Since the transcriptional components need to be at specific relative concentrations in order for transcription to occur, extrusion may inactivate the reaction by simply depleting certain necessary reagents for transcription. Extrusion at high pressures may also denature the DNA or T7 RNA polymerase, resulting in no RNA being synthesized. In order to encapsulate the transcriptional components into the pre-formed, extruded liposomes, the transcription reaction was added to the outside of empty liposomes and subjected to a single freeze-thaw cycle. Freezing and thawing the liposomes resulted in a transient opening of lipid bilayer, allowing the reagents to be encapsulated into the aqueous core of the liposomes. [207-209] In our experiments, this single freeze thaw cycle did not decrease the RNA yield of the transcription reaction.  This modified formulation method, along with incorporating a PEGylated lipid and optimizing the concentrations of the transcription reaction, allowed for a 50-fold increase in the 64 amount of RNA that could be synthesized within nanoliposomes. Although PEGylated lipids are known to inhibit interaction and uptake of LNPs by cells, a small percentage was incorporated to increase the RNA yield in liposomes, which is consistent with previous literature on optimizing protein translation in liposomes. [197] By running the liposomes through an anionic exchange column, unencapsulated components were removed. Removal of the transcriptional components was necessary because unencapsulated nucleic acids and proteins could potentially be immunostimulatory, and could cause premature platelet activation. This also allowed us to measure only the RNA that was synthesized within the platelets, and excluded any RNA that may be made in the supernatant and was subsequently internalized by platelets. This single-step method for purification has been used with nanoparticles containing nucleic acids. [210] The use of these columns is faster than dialysis and is an alternative to previously published methods of adding ethylenediaminetetraacetic acid (EDTA) or nucleases to inhibit transcription outside of liposomes, [195, 205] which are likely unsuitable for subsequent cellular delivery since EDTA causes irreversible structural and functional damage to platelets. [211, 212]   By replacing the ATP in the transcription reaction with a photo-caged ATP, transcription could be controlled using light. The incorporation of light-induced transcription into our system was to ensure that RNA synthesis occurred after internalization by platelets. Using this system, a GFP mRNA and a FLuc mRNA was controllably synthesized in liposomes following irradiation. The lower transcriptional activity observed with the FLuc template (2 kb) compared to the GFP template (1 kb) suggests some optimization is needed to maximize RNA synthesis of larger templates. However, this data shows that this system can be easily extended to other DNA templates, and provides a basic approach with room for optimization, such as varying the 65 concentration of the transcription reaction and potentially liposome size for encapsulation of larger substrates. Nevertheless, this data demonstrates that RNA transcription can be suppressed and specifically initiated using light in nanoliposomes.  This work shows, for the first time, that transcription of exogenous RNA can occur in a controllable manner within an anucleate cell, such as platelets ex vivo. This proof-of-concept study demonstrated that multiple biomolecules can be co-encapsulated into liposomes, delivered to platelets, and function intracellularly. Moreover, loading platelets with transcriptional machinery rather than just purified RNA could potentially allow for an additional level of regulation, where RNA synthesis is only initiated upon light irradiation. The methods described here has the potential to be adapted to transcribe RNA from other templates, including siRNA and miRNA for RNAi. Although the scope of the experiments described here did not include testing for protein expression from the newly synthesized RNA, it is an important next step toward using this technology to genetically alter platelet function. To answer this, it will be important to determine the intracellular localization of the newly synthesized RNA and possibly include signals to target the RNA for protein expression if necessary, since protein expression in platelets is highly regulated. [213] For example, platelets have been found to translate Bcl-3 and IL-1β following activation, [214-216] suggesting specific signaling pathways exist to translate certain RNA transcripts.  Another area of exploration is to test whether modified platelets to release the newly synthesized RNA. Since platelets specifically release RNA and other biomolecules to other cells such as endothelial cells and macrophages during platelet activation, [89-91] there is potential to 66 engineer platelets to deliver exogenous RNA to their target cells and modulate pathological processes such as inflammation, thrombosis, and malignancy. The release and delivery of platelet contents is also highly regulated and requires packaging into certain platelet compartments such as α-granules and dense granules; engineering platelets to deliver exogenous RNA would likely require developing strategies to target liposomes and their contents to specific platelet compartments.  67 Chapter 4: Platelets loaded with liposomal thrombin have increased coagulability  4.1 Rationale Platelet transfusions are used clinically to reduce bleeding, but transfusions of these and other blood components are not always effective during severe hemorrhage. [217, 218] A major component of coagulopathy is platelet dysfunction, where platelets no longer respond to agonists such as ADP and collagen, and platelet dysfunction is strongly correlated with mortality during traumatic bleeding. [99, 219, 220] Furthermore, transfusions of normal platelets are insufficient to reverse platelet dysfunction; therefore, strategies to enhance the efficacy of current platelet transfusions in the clinic are needed for the management of severe bleeding.  Several approaches are being developed to enhance or replace platelet transfusions. These include using cold-stored platelets, lyophilized platelets, modified platelets, as well as artificial platelets. In particular, several promising nanoparticle-based systems of artificial platelets, or platelet mimics, have been developed toward augmenting or replacing platelet transfusions in acute severe bleeding. [221-224] Although these systems have the advantage of long shelf lives and low immunogenicity,[225, 226] they cannot adequately mimic all the important and diverse aspects of platelet structure and function on a single platform, including the ability to deliver a wide range of biomolecules that collectively contribute to clot formation, stabilization, contraction, and longer term wound healing. [224, 227] Alternatively, developing a strategy that improves the natural function of transfused platelets, specifically during platelet activation, 68 would be an important step towards increasing the efficacy of platelet transfusions during severe bleeding.  Previous studies on modified platelets demonstrated that enhancing platelet coagulability could improve overall hemostasis in vivo; [135, 136] however, clinical use of this technology would currently require bone marrow transplantation, which is not appropriate for cases of acute hemorrhage. Nanoparticles have been used extensively to encapsulate enzymes and shield their enzymatic activity until they are delivered to the intended cells or sites of action, where the enzymes can be released into the cytoplasm of the cell or extracellularly. [203, 228, 229] Thrombin, a potent hemostatic enzyme, has also been encapsulated into polymeric and aptamer-based nanoparticles for controlled local delivery and release. [230, 231] Although the hemostatic potential of thrombin could be useful during severe hemorrhage, particularly during coagulopathy, it is currently only approved for topical administration because high concentrations of active thrombin in circulation causes systemic thrombosis. [232] Thus, we hypothesized that thrombin can be encapsulated into nanoliposomes and be internalized platelets ex vivo to be temporarily shielded, resulting in enhanced platelet coagulability during platelet activation.    69 4.2 Methods 4.2.1 Preparing and characterizing liposomal thrombin. Washed platelets were loaded with liposomes as previously described, with minor modifications. [233] Briefly, 4.3 μM of human alpha thrombin or DFP active site blocked human alpha thrombin (Haematologic Technologies Inc.) in Tris-buffered saline (TBS) was added to a 5 mg lipid film, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine:cholesterol:1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPC:chol:DOPE; 23:23:4; Avanti Polar Lipids, cholesterol from Sigma) and extruded five times through a 200 nm filter using a LIPEX extruder (Northern Lipids Inc.). Liposomes were then passed through a cationic exchange column (Vivapure S Mini H, Sartorius) to remove unencapsulated thrombin. Efficient removal of unencapsulated thrombin was confirmed by measuring thrombin activity of the column eluate using a fluorogenic substrate (Boc-Asp(OBzl)-Pro-Arg-MCA; Peptide Institute Inc.), and incubating the column eluate with platelets (1:20 v/v) to determine platelet activation by monitoring CD62P expression using flow cytometry. Fibrin formation time in PPP following incubation with purified liposomal thrombin (1:100 v/v) was measured in the absence of additional calcium, with fibrin formation monitored visually. Encapsulation efficiency of thrombin was calculated by lysing purified liposomes with 0.1% Triton-X and measuring the thrombin activity using a fluorogenic substrate.  4.2.2 Loading platelets with liposomal thrombin. Purified liposomes were added to isolated platelets (1:100 v/v) and incubated at room temperature for 1 h with gentle agitation for uptake to occur. The uptake of liposomes into platelets was confirmed as previously described. To remove excess liposomes, platelets and 70 liposomes were centrifuged at 250 × g for 5 min and the supernatant was removed. Platelets were then washed twice in Tyrode’s-HEPES buffer at 250 × g for 5 min and resuspended either in Tyrode’s-HEPES buffer or PPP as indicated.  4.2.3 Measuring clot retraction in PRP. After removing excess liposomes, platelets were resuspended in PPP to make PRP. PRP (50 μL) was mixed into a total of 200 μL pH 7.4 Tyrode’s-HEPES buffer with a final concentration of 3.75 mM CaCl2, and 10 μg mL-1 rat tail collagen or 120 pM recombinant tissue factor (Innovin, Dade Behring) as indicated. Samples were incubated at 37°C and images of the clots were obtained every 10 min. The total areas of the clot and the sample were measured using Quorum Analysis software.  4.2.4 Measuring thrombin generation in PRP. After removing excess liposomes, platelets were resuspended in PPP with 10 μM of the fluorogenic substrate for thrombin. In the platelet inhibitor experiments, platelets were preincubated with either acetylsalicylic acid (ASA, 5 µg mL-1) or naproxen (5 µg mL-1) for 15 min at room temperature. In the trauma patient plasma experiments, platelets were resuspended in PPP obtained from TIC patients. Reconstituted PRP containing 10 μM of the fluorogenic substrate for thrombin was mixed 3:1 with 40 mM CaCl2, with a final concentration of 10 μM adenosine diphosphate (ADP; Sigma) where specified. Samples were incubated at 37ᵒC and fluorescence (excitation: 360 nm; emission: 465 nm) was measured every 30 s using a plate reader (TECAN GENios Plus). The lag time for thrombin generation was calculated by plotting the first derivative of the fluorescence readings over time. 71 4.2.5 Isolating plasma from patients with trauma-induced coagulopathy. For trauma patient samples, human subjects (≥ 18 years of age) who were not pregnant or incarcerated, presented directly from the place of injury, and received fewer than three units of transfused blood products were enrolled as part of a single-center observational study taking place at the Harborview Medical Center Emergency Department in Seattle, WA, USA. Delayed, written consent was obtained for study participation from all study subjects in accordance with the Declaration of Helsinki in a protocol approved by the University of Washington Institutional Review Board. Blood was collected from a peripheral intravenous line at Emergency Department arrival and 0.6 mL of plasma was stored at -80°C until used for this study.  4.2.6 Determining clot initiation and clot strength by thromboelastography. After removing excess liposomes, platelets were resuspended in PPP. In whole blood experiments, washed platelets wasere resuspended in PPP and mixed 1:1 with RBCs washed in PBS. In the acidosis experiments, reconstituted PRP was preincubated with HCl (10 mM) for 15 min at room temperature, resulting in a plasma pH ~ 7.2 to mimic mild acidosis. The pH of acidified plasma was determined using a plasma spectrophotometric assay with phenol red. [234] In the hemophilia A plasma experiments, platelets were resuspended in congenital FVIII deficient PPP (HA-PRP; George King Bio-Medical, Inc.) and clotting was initiated with 2 pM recombinant tissue factor. Reconstituted PRP or whole blood was mixed 3:1 with 40 mM CaCl2 in a TEG cup and R time was measured at 37°C using a thromboelastography (TEG) Hemostasis Analyzer System 5000 (Haemonetics Corporation). 72 4.2.7 Determining clot initiation and lysis by western blot. After removing excess liposomes, platelets were resuspended in PPP. In the hemophilia A patient experiments, platelets were resuspended in congenital FVIII deficient PPP. Reconstituted PRP (20 μL) was mixed into a total of 40 μL HBS, with a final concentration of 10 mM CaCl2, 0.6 pM tissue factor, and 1 nM tissue plasminogen activator (tPA), and incubated at 37°C. At specific time points, the reaction was stopped with 100 μL of quench buffer (1 M DTT, 0.5 M EDTA, 8 M urea) and incubated at 60°C for 1 h to dissolve the clot. Samples were run on a 10% SDS-PAGE gel, transferred on to a nitrocellulose membrane, and probed using a fibrinogen antibody (1:50,000; Dako) and a goat anti-rabbit secondary antibody (1:15,000; Abcam).  4.2.8 Determining release of endogenous platelet FXIII. After removing excess liposomes, platelets were resuspended in pH 7.4 Tyrode’s-HEPES buffer and activated with 10 μg mL-1 rat tail collagen at 37°C for 30 minutes. Platelets were spun down at 250 × g for 5 minutes to separate the platelet pellet from the releasate in the supernatant. Both pellet and supernatant samples were run on a 10% SDS-PAGE gel, transferred on to a nitrocellulose membrane, and probed using a FXIII antibody (1:1,000; Affinity Biologicals) and a rabbit anti-sheep antibody (1:10,000; Abcam).  4.2.9 Detecting fibrin formation and crosslinking. After removing excess liposomes, platelets were resuspended in PPP and activated with 10 μg mL-1 rat tail collagen or 0.35 μM thrombin at 37°C for 30 minutes. Western blotting for fibrin(ogen) was done as described above. 73  4.2.10 Preparing fluorescent thrombin to measure uptake and release. Human alpha thrombin (1 mg) was fluorescently labeled using an Alexa Fluor 488 Protein Labeling Kit (Molecular Probes), purified using a centrifugal filter with a 3 KDa cut-off (Millipore), and resuspended in 50% glycerol (v/v). Fluorescent thrombin was loaded into liposomes and platelets as described above, visualized by running both pellet and supernatant samples on a 10% SDS-PAGE gel, and imaged with an Alexa 488 filter on a ChemiDoc Imaging System (Biorad) using Image Lab software.    74 4.3 Results  4.3.1 Purified liposomal thrombin minimally activates platelets To confirm that unencapsulated thrombin was removed from the liposomal thrombin preparation, a thrombin solution was passed through the cationic exchange column and thrombin activity was measured using a fluorogenic substrate. No thrombin was activity was detected in the solution that passed through the column, compared to a 1:100 thrombin solution that was not passed through the column (Figure 4.1A). The column eluate of the thrombin solution also did not induce platelet activation, measured by CD62P exposure with flow cytometry (Figure 4.1B).  Following purification, liposomal thrombin did not initiate fibrin formation in plasma in the absence of calcium, further confirming the removal of unencapsulated thrombin from the liposomal preparation (Figure 4.2A). Purified liposomal thrombin did not change clot initiation time in recalcified plasma, measured by R time with TEG (Figure 4.2B). Without platelet agonists, LT-PLTs were 20 (± 4) % activated, which is higher than the 8.6 (± 0.8) % activation for PLTs, or the 7 (± 0.03) % for platelets loaded with empty liposomes (EL-PLTs), but much lower than the 89 (± 4) % activation for PLTs stimulated with unencapsulated thrombin (Figure 4.2C). Using the fluorogenic substrate to measure thrombin activity following lysis of the liposomes, the encapsulation efficiency of thrombin into nanoliposomes was determined to be 1.6%, producing a stock solution of approximately 70 nM liposomal thrombin, which was approximately 0.7 nM when added to platelets. After loading into platelets, the bulk concentration of thrombin in LT-PLTs was around 7 pM, which is very low compared to 75 circulating prothrombin, which is approximately 3 µM, or the local concentration of thrombin during clotting, spanning concentrations at the picomolar to low micromolar range. [235, 236]  Figure 4.1. Thrombin binds to and is removed by a cationic exchange column. (A) Thrombin activity was measured for samples that did not pass through the column (before column; diluted 1/100) and the column eluate (after column) using a fluorogenic substrate. (B) Activation of platelets by before column and after column samples as measured by CD62P expression on flow cytometry. (n = 3) * P < 0.01 compared to control. N.S. is not statistically significant compared to control. Data represent SEM.  76    77 Figure 4.2. Unencapsulated thrombin is efficiently removed from the purified liposomal preparation. (A) Fibrin formation time in PPP after incubation with liposomal thrombin that did not pass through the column (before column) and samples that passed through the column (after column) samples. (B) Clot initiation time (R time) of platelet-poor plasma in the presence of liposomal thrombin (LT), measured by TEG. (n = 3). (C) Basal activation without treating platelets with collagen, comparing PLTs, platelets loaded with empty liposomes (EL-PLT), and LT-PLTs to unencapsulated thrombin (85 nM).    78 4.3.2 Platelet activation and clot characteristics of LT-PLTs To determine the effect of delivering liposomal thrombin to platelets, we first tested the sensitivity of normal (PLTs) and modified platelets (LT-PLTs) to activation by collagen, a subendothelial protein and platelet agonist that is exposed at sites of damaged vasculature. [237] PLTs were handled and washed in the exact manner as LT-PLTs, except without the addition of liposomes. Platelet activation was determined by measuring CD62P exposure using flow cytometry.  Compared to PLTs, a higher percentage of LT-PLTs became activated when exposed to collagen (Figure 4.3A). Collagen stimulation of LT-PLTs resulted in an additional 28 (± 5) % activation compared to no agonist, whereas PLTs were only 11 (± 3) % more activated after collagen stimulation. Clot contraction was also accelerated with LT-PLTs (Figure 4.3B). Collagen-stimulated clot contraction in PRP with LT-PLTs was approximately 2.2-fold faster than PLTs, and the final contracted clot was similar in size to those made using recombinant tissue factor (TF), a strong activator of clot formation and contraction. [238]  The lag time of thrombin generation in PRP was shorter with LT-PLTs, measured using a fluorogenic substrate, occurring at 5.1 (± 0.1) min, compared to PLTs at 7.0 (± 0.4) min (Figure 4.4A). Platelets loaded with an inactivated form of thrombin (DFP-PLTs) that contained an irreversible active site inhibitor (diisopropylfluorophosphate-thrombin, DFP) had an endogenous thrombin generation lag time of 6.0 (± 0.2) min. The enzymatic activity of DFP-inhibited thrombin was measured using a fluorogenic substrate of thrombin, and had approximately 0.01% of the activity of uninhibited thrombin. 79   Figure 4.3. LT-PLTS respond to collagen activation by increased P-selectin expression and clot contraction. (A) Activation of LT-PLTs and unmodified platelets (PLTs) by collagen (0.5 µg mL-1), measuring CD62P surface expression using flow cytometry. (B) Clot contraction in response to collagen (10 μg mL-1) and tissue factor (120 pM) stimulation. (n = 4) * P < 0.05. Data represent SEM.    80 TEG was used to further characterize the clots formed by LT-PLTs compared to PLTs. On average, the clot initiation time (R time) for PRP containing LT-PLTs (8.8 ± 1.7 min) was approximately 26% faster compared to PLTs (11.9 ± 2.5 min), as measured by the clot initiation time (R time) using TEG (Figure 4.4B). In whole blood, the R time for LT-PLTs (11.2 ± 1.2 min) was approximately 13% faster compared to PLTs (12.9 ± 1.2 min). The clot strength, as measured by G on TEG, was 16% higher in LT-PLTs (13.4 ± 1.7 kdyne cm-2) compared to PLTs (11.5 ± 1.6 kdyne cm-2) in PRP, and 22% higher (5.0 ± 0.6 kdyne cm-2 vs 4.1 ± 0.4 kdyne cm-2) in whole blood (Figure 4.4C). To determine characterize the effect of LT-PLTs on fibrin crosslinking and the susceptibility of fibrin to degradation, PRP clots with either PLTs or LT-PLTs were made in the presence of tPA. A western blot for fibrin(ogen) was used to monitor the α, β, and γ chains, as well as higher molecular weight bands corresponding to crosslinked fibrin, and lower molecular weight bands corresponding to fibrin degradation products. The formation and degradation of high molecular weight bands were similar, but the β chain (~56 kDa) was stable for several hours longer in clots made with LT-PLTs compared to PLTs (Figure 4.4D).   81  Figure 4.4. Clots form faster with LT-PLTs than PLTs and are more resistant to fibrinolysis. 82 (A) Lag time for thrombin generation in platelet-rich plasma (PRP) was measured using a fluorogenic substrate of thrombin. LT-PLTs were compared to both PLTs and to platelets loaded with inactivated thrombin (DFP-PLTs). (n = 5) * P < 0.05 compared to LT-PLTs. (B-C) Clot initiation and clot strength was measured using TEG (R time and G, respectively) in recalcified PRP and whole blood (WB) with normal platelets or modified platelets. (n = 5) * P < 0.05. Data represent SEM. (D) Western blot for fibrin(ogen) of clots formed with tissue factor (0.6 pM) in the presence of tPA (1 nM).   83 4.3.3 Platelet response and clot formation during acidosis. More LT-PLTs responded to collagen activation compared to PLTs at pH levels ranging from 6.8 to 7.4 (Figure 4.5A), the range of blood pH that occurs during mild to severe acidosis. [239] To determine the effect of LT-PLTs on clotting during acidosis, thrombin generation time was measured in acidified PRP (pH ~ 7.2). On average, normal PRP (pH ~ 7.4) had a thrombin generation time of 8.1 (± 1.0) min. Acidifying PRP (pH ~ 7.2) that contained PLTs slowed the generation of endogenous thrombin by 1.0 (± 0.4) min (Figure 4.5B). In contrast, LT-PLTs sped up thrombin generation time by 1.6 (± 0.2) min in acidified PRP. Using TEG to measure clot initiation, normal PRP at physiological pH had a R time of 8.5 (± 1.4) min. Acidified PRP containing PLTs clotted approximately 10% slower compared to normal PRP, but acidified PRP containing LT-PLTs clotted approximately 10% faster compared to normal PRP (Figure 4.5C).   84   85 Figure 4.5. Several inhibitory effects of acidosis on platelets and clot formation can be reversed by LT-PLTs. (A) Activation of platelets with collagen (0.5 µg mL-1) at pH 6.8 to 7.4, measuring CD62P surface expression using flow cytometry. (n = 4) * P < 0.01, compared to PLTs at the corresponding pH. (B) Lag time for thrombin generation of acidified PRPs relative to PLTs in PRP with a neutral pH. (n = 3) * P < 0.05 compared to PLTs in acidified PRP. (C) Clot initiation (R time) was measured using TEG. (n = 3) * P < 0.05 compared to acidified platelets. Data represent SEM.  4.3.4 Clot formation in the presence of antiplatelet drugs. Normal PRP had an ADP-stimulated thrombin generation time of 7.2 (± 0.7) min. This was 1.3 (± 0.5) min slower in the presence of ASA or naproxen (Figure 4.7). In contrast, PRP with LT-PLTs generated thrombin 0.4 (± 0.4) and 0.6 (± 0.4) min faster in the presence of ASA and naproxen compared to normal PRP, respectively.   86  Figure 4.6. LT-PLTs can correct for delayed thrombin generation time in the presence of antiplatelet drugs. Lag time for thrombin generation in the presence of ASA (5 µg mL-1) or naproxen (5 µg mL-1). (n = 4) * P < 0.05, ** P < 0.01, compared to the respective PLT group. Data represent SEM.  4.3.5 Clot formation in plasma from patients with coagulopathies. HA-PRP is deficient in FVIII and has compromised thrombin generation. [240, 241] On average, normal PRP had a R time of 5.3 (± 0.5) min, when clotting was initiated with TF and measured by TEG. R time for HA-PRP was 33 (± 5) % slower compared to normal PRP, whereas HA-PRP with LT-PLTs was only 17 (± 3) % slower compared to normal PRP (Figure 4.7A). In a western blot for fibrin(ogen), clots made with PLTs in the presence of tPA had faster β chain degradation when made with HA-PRP compared to normal plasma (Figure 4.7B). In contrast, the fibrin formed in clots of HA-PRP with LT-PLTs had a similar rate of degradation as clots made in normal PRP. 87  Figure 4.7. LT-PLTs improve aspects of clotting in plasma from hemophilia A patients. (A) Clot initiation (R time) of PRP with tissue factor (2 pM) from patients with hemophilia A (HA-PRP), measured by TEG, relative to normal PRP. (n = 4) * P < 0.05. Data represent SEM. (B) Western blot for fibrin(ogen) of clot formation and degradation. Clots were formed with tissue factor (0.6 pM) in the presence of tPA (1 nM).  The effects of modified platelets in the plasma of patients with TIC was assessed by measuring the endogenous thrombin generation time. In two TIC patient samples, thrombin generation times were 5.0 (± 0.1) and 6.7 (± 0.2) min for PLTs. In both cases, adding LT-PLTs resulted in thrombin generation times of 3.9 (± 0.1) and 5.9 (± 0.1) min, respectively (Figure 4.8).  88  Figure 4.8. Clotting of PRP from patients with TIC is faster with LT-PLTs. Lag time for thrombin generation in plasma collected from two patients. (n = 3 technical replicates for each patient) * P < 0.05, ** P < 0.01 compared to respective PLT group. Data represent SEM.  4.3.6 Platelet uptake of liposomal thrombin and assessing interactions with substrates of thrombin. Liposomal thrombin was internalized by 38 (± 9.2) % of platelets, which decreased to 13 (± 5.5) % of platelets that were pretreated with a general metabolic inhibitor (Figure 4.9A). Thrombin appeared to remain inside of platelets after they were activated by collagen, because fluorescent thrombin was detected inside of platelets, but not in the supernatant upon activation (Figure 4.9B). 89  Figure 4.9. Liposomal thrombin is actively taken up by platelets but not released upon activation. (A) Uptake of fluorescent liposomes by platelets was measured by flow cytometry, detecting CD42b. (n = 4) * P < 0.05. Data represent SEM. (B) Image of fluorescent thrombin on a SDS-PAGE gel, showing cell lysates (p) and supernatant fractions (s) of LT-PLTs that were non-activated (NA) or activated with collagen (Col; collagen 10 µg ml-1).  Cleavage of fibrinogen was not detected within the platelets or surrounding plasma when platelets were activated with collagen in the absence of calcium (Figure 4.10A). Compared to PLTs, EL-PLTs, and DFP-PLTs, only LT-PLTs released endogenous platelet FXIII upon activation by collagen (Figure 4.10B). Inhibiting the platelet surface thrombin receptors PAR-1 and PAR-4 of LT-PLTs resulted in a similar level of collagen activation compared to uninhibited LT-PLTs (43.7% versus 50.3%; Fig. 4.10C). 90    91 Figure 4.10. Effects of inhibiting thrombin activity and PAR activity on LT-PLTs. (A) Western blot for fibrin/ogen in PRP. In this experiment only, calcium was not added. Platelets were either not activated (NA) or activated with collagen (Col; 10 μg mL-1) or unencapsulated thrombin (Thr; 0.35 μM). (B) Western blot for coagulation factor XIII (FXIII) in the pellet (p) and supernatant (s) of platelets that were either not activated (NA) or activated with collagen (Col; 10 μg mL-1). (C) Platelet activation by collagen (0.5 μg mL-1) or thrombin (1 nM) in the presence of inhibitors of PAR1 and PAR4 (FR171113,1 µM, and ML354, 1 µM). * P < 0.05 Data represent SEM.    92 4.4 Discussion The results show that several aspects of coagulability of ex vivo, transfusable platelets can be enhanced by loading them with an exogenous coagulation factor (Fig 4.11). Thrombin was encapsulated by passive loading into liposomes consisting of natural lipids and delivered to platelets isolated from fresh whole blood. It was important to minimize the extent that platelets are activated prior to transfusion to minimize the risk of adverse events. Although the hemostatic potential of thrombin would be highly beneficial if transfused during bleeding, unencapsulated thrombin would cause intense platelet activation and lead to thrombosis. Therefore, excess, unencapsulated thrombin was removed from the liposome preparation using a cationic exchange column prior to incubation with platelets. Passing unencapsulated thrombin through the column resulted in no detectable thrombin activity in the eluate. Incubating the eluate with platelets did not result in platelet activation, and purified liposomal thrombin did not cause fibrin formation in plasma. Additionally, no enzymatically active thrombin was found outside of LT-PLTs before or after they were activated. Taken together, this suggests that unencapsulated thrombin is efficiently removed from the liposomal preparation, and is suitable for use with platelets. Nevertheless, incubating the platelets with liposomal thrombin increased the background activation from approximately 8.6% to 20%. However, this is well within the range of platelet activation considered safe for transfusions, [106] and LT-PLTs retained the ability to be further activated by conventional platelet agonists. In future studies, it will be important to determine whether LT-PLTs are associated with a greater risk of thromboses compared to normal platelets. 93  Figure 4.11. Enhancing platelet coagulability with liposomal thrombin. Platelets loaded with liposomes (orange) containing thrombin (red) have enhanced coagulability. LT-PLTs remain mostly in their resting state (green) until activated (blue) with an agonist, such as collagen.  The coagulability of LT-PLTs was improved in several ways. First, the ability of platelets to activate in response to agonists was tested. In buffer and in plasma, LT-PLTs were more sensitive and responsive to collagen activation compared to PLTs in several assays, including platelet activation and clot contraction. The ability of platelets to contribute to clot formation and clot strength were then assessed using TEG and by observing fibrin formation and degradation using western blots. LT-PLTs significantly decreased the clot initiation time, and increased clot firmness in both PRP and whole blood. The fibrin formed in the presence of LT-PLTs were also less susceptible to lysis by tPA. To our knowledge, this is the first report of directly modifying ex vivo platelets to enhance their coagulability. Direct modification of transfusable platelets may be useful in cases of acute bleeding, where a temporary increase in platelet coagulability may be desirable. Previous work in modifying precursor cells is time-consuming and would permanently alter platelet function, [135, 136] which would not be appropriate in an acute bleeding scenario. 94 Although the current approach would require much more development and testing before translation to clinical use, these proof-of-concept experiments suggest that increasing the endogenous coagulability of transfusable platelets without increasing their basal activation may have important implications for treating severe bleeding with platelet transfusions. This could be particularly important for cases of TIC, where damaged blood vessels cannot be efficiently sealed due to decreased platelet sensitivity to agonists, decreased clot strength, and hyperfibrinolysis. [98, 99]  In a step towards determining the efficacy of LT-PLTs during severe bleeding, we tested LT-PLTs in several scenarios where platelet function and/or thrombin generation are compromised, using various combinations of the above assays. In all cases, endogenous thrombin generation was used as an indirect measure of the activation of the coagulation cascade, supplemented by other assays such as TEG and western blotting for fibrin to further characterize clot initiation, and aspects of mechanical strength and stability when applicable. Severe hemorrhage is often complicated by acidosis, which contributes to coagulopathy by impairing the activity of coagulation complexes such as FXa/Va, ultimately leading to decreased thrombin generation and prolonged clotting times. [98, 242, 243] In previous studies with animal models of acidosis, these coagulation defects could not be reversed by correcting the pH back to physiological levels. [244, 245] Acetylsalicylic acid (ASA) and naproxen are antiplatelet drugs that can require platelet transfusions for emergency reversal during severe bleeding, though platelet transfusions are sometimes insufficient to restore hemostatic function. [63, 246, 247] Using LT-PLTs, some of the in vitro coagulation defects caused by acidosis and antiplatelet 95 drugs were reversed, such as normalizing the lag time to thrombin generation and enhancing platelet activation following collagen activation.  In order to determine the potential clinical relevance of platelets with enhanced coagulability, ex vivo clotting parameters, including thrombin generation time, clot initiation, and clot strength, were measured in two different patient plasmas. Hemophilia A is a genetically acquired bleeding disorder resulting from FVIII deficiency. [248] Previously, platelet precursors were genetically modified to produce FVIII-expressing platelets, which were effective at treating several animal models of hemophilia A. [135, 136] In our experiments, LT-PLTs improved the clotting parameters in HA-PRP, which is likely due to enhancing thrombin and fibrin generation. TIC is an example of acute, severe bleeding which may benefit from LT-PLT transfusions, since it often presents with impaired platelet function, and transfusions of platelets or other blood components do not always correct coagulopathy. [100, 217, 218] In plasma samples collected from patients with TIC, LT-PLTs sped up endogenous thrombin generation times. These experiments with patient plasma samples are proof-of-concept experiments suggesting that enhancing platelet coagulability through increased platelet sensitivity to agonists and faster thrombin generation may be an effective strategy to overcoming certain aspects of coagulopathies.  To identify the molecules that liposomal thrombin acted on, we analyzed several substrates of thrombin in platelets, including fibrinogen, FXIII, PAR-1 and PAR-4. Although we did not identify a specific substrate, the results provide several insights into the mechanism of action of LT-PLTs. Thrombin was internalized and appeared to remain inside the platelets and 96 was not released in easily detectable amounts. While the enzymatic activity of thrombin contributes to enhanced platelet coagulability, as demonstrated by several control experiments with DFP-inhibited thrombin, neither cleavage of FXIII nor fibrinogen was detected within platelets. This is especially interesting, because FXIII is present primarily in the platelet cytoplasm, [249] and fibrinogen is found primarily in alpha granules. [78] This suggests that the exogenously delivered thrombin remains separated from both of these compartments. Inhibition of platelet thrombin receptors, PAR-1 and PAR-4, did not abrogate the increased sensitivity of LT-PLTs to collagen activation, suggesting that liposomal thrombin did not activate the receptors on the outer surface of the platelet. Platelet FXIIIA is normally externalized on the surface of activated platelets and contributes to clot contraction and thrombus stabilization. [249] The release of platelet FXIIIA by LT-PLTs may explain the decreased fibrinolysis and enhanced clot contraction, though it could also be a result of enhanced platelet activity and thrombin generation. [128, 250, 251] Clots formed in the presence of higher concentrations of thrombin have been shown to be less susceptible to fibrinolysis. [252] This may explain why clots formed with LT-PLTs, which contributes to faster thrombin generation in plasma, have a higher mechanical strength and decreased fibrinolysis in the presence of tPA. Either way, the exact mechanism by which the delivered thrombin leads to increased agonist responsiveness and FXIIIA release remains to be elucidated. Future studies on the mechanism of enhanced coagulability of LT-PLTs may determine the primary targets of exogenously delivered thrombin, as well as the intracellular location of the delivered thrombin.97 Chapter 5: Conclusions This thesis describes approaches to encapsulate a variety of biomolecules in lipid nanoparticles and deliver them to platelets to modulate or enhance platelet function in vitro. First, lipid nanoparticles were formulated to encapsulate biomolecules such as nucleic acids, proteins, and small molecules. The conditions to deliver these liposomes to platelets were determined by maximizing internalization and minimizing premature platelet activation, and the mechanisms of liposome internalization by platelets were determined (Chapter 2). Based on the approaches developed in chapter 2, platelets were modified to: i) transcribe a reporter mRNA using delivered transcriptional machinery (Chapter 3), and ii) have increased coagulability under physiological and coagulopathic conditions (Chapter 4). The studies here showed promise in modifying platelet function in vitro, and future studies to determine their clinical relevance in vivo will be described in chapter 6.  5.1 A range of functional biomolecules can be delivered to platelets The incubation conditions for platelets to take up liposomes were determined, including buffer composition, temperature, time, liposome size, and platelet to liposome ratio. Using lipid nanoparticles approximately 200 nm in diameter, a range of biomolecules, including small molecules, nucleic acids, and enzymes were delivered to and internalized by platelets ex vivo. Lipid nanoparticles were composed mostly of neutral phospholipids and cholesterol, with molecules encapsulated into the aqueous core by passive loading using a thin-film rehydration method. The cargo remained functional following encapsulation and endocytosis, and were able to modulate endogenous platelet function, as demonstrated by the transcriptionally active platelets in chapter 3 and platelets with increased coagulability in chapter 4. Although 98 internalization of nanoparticles by platelets appears to occur through multiple pathways, receptor-mediated endocytosis, including caveolae- and clathrin-mediated endocytosis, were the predominant mechanisms of uptake, as inhibition of these processes significantly reduced liposome internalization. The current studies did not identify specific ligands or receptors to trigger the activation of these endocytotic pathways, since uptake of liposomes occurred in purified buffer systems and in the absence of normal proteins found in circulating blood. Previous studies have shown that platelets can take up unencapsulated small molecules (doxorubicin) through the open canalicular system. Platelets have also been shown to engulf bacteria and internalize siRNA, although the mechanisms of uptake in these systems have not been completely characterized. Previous studies have demonstrated that larger particles such as proteins and latex particles are internalized by platelets through receptor-mediated endocytosis in an energy-dependent manner.  Our studies confirm these results, and to our knowledge, this is one of the first reports of platelets taking up synthetic lipid nanoparticles through receptor-mediated endocytosis.  5.2 Platelet function can be enhanced  5.2.1 Engineered anucleate platelets to transcribe RNA (Chapter 3) Because platelets are anucleate and therefore do not have endogenous transcriptional machinery, many traditional genetic transfer techniques cannot be used to genetically modify platelets. To bypass these challenges, protocell technology was used to co-deliver DNA with ribonucleotides, T7 RNA polymerase, and small molecules to form a functional, synthetic “nuclei” capable of transcribing RNA. Following delivery of liposomes to platelets, transcription 99 within platelets was controllably initialized using light as an external stimulus, demonstrating that RNA synthesis could be regulated in vitro. This work not only confirms that a variety of biomolecules can be delivered to and function within platelets, but is also proof-of-concept that platelet function can be manipulated through the delivery of exogenous cargo. Although some optimization for transcription efficiency may be required to maximize RNA yield, the approaches described here can likely be extended to transcribe other DNA templates, potentially allowing for synthesis of mRNA, miRNA, and siRNA to be expressed in platelets. The experiments described here did not determine whether the newly synthesized RNA can be translated into proteins or if the system can downregulate proteins through RNA silencing mechanisms. However, these studies represent the first step in directly manipulating the genomic and potentially proteomic contents of platelets, which may have important applications for studying platelet biology or creating modified platelets for future therapeutic use.  5.2.2 Modified platelets have enhanced coagulability (Chapter 4) Encapsulating thrombin, a potent hemostatic enzyme and platelet activator, into liposomes resulted in efficient shielding of thrombin activity, such that thrombin-encapsulated liposomes only caused minimal platelet activation compared to unencapsulated thrombin. Encapsulation of thrombin also allowed for endocytosis by platelets ex vivo. Platelets loaded with liposomal thrombin became more sensitive to collagen activation, and resulted in clots that formed faster and stronger in vitro compared to normal platelets. This increase in platelet coagulability persisted in conditions where coagulation and/or platelet function is normally compromised, including acidosis, antiplatelet drugs, hemophilia A, and TIC. The in vitro data suggests that increasing platelet coagulability may be a viable approach to decreasing bleeding 100 during severe traumatic bleeding, which is often complicated by acidosis and TIC. Although the enzymatic activity of thrombin was necessary for the increased coagulability of LT-PLTs, the exact target of the internalized thrombin and its intracellular localization remains to be elucidated. While others have demonstrated that platelets derived from modified megakaryocytes can contribute to enhanced clotting in hemophilia, this is the first report of directly modifying transfusable platelets to increase their endogenous coagulability, which may have important implications for treating acute, severe hemorrhage with platelet transfusions. 101 Chapter 6: Future directions  6.1 Exploring the safety and efficacy of LT-PLT transfusions in vivo Based on the results presented in chapter 4, LT-PLTs enhanced clot formation and clot strength under physiological and coagulopathic conditions in several in vitro assays. LT-PLTs sped up thrombin generation in TIC plasma, suggesting that loading platelets with liposomal thrombin may be an effective strategy to potentially enhancing the efficacy of platelet transfusions during severe hemorrhage. In order to test this hypothesis, a porcine model of traumatic arterial hemorrhage may be used to assess the efficacy of transfusing LT-PLTs compared to PLTs. Similar models have been used to test the efficacy of infusing synthetic platelet mimetics and other hemorrhage management strategies during traumatic hemorrhage, in which animal survival, blood loss, and vitals are measured over the course of the experiment. [253-255] Blood can also be drawn from the animal throughout the experiment to measure clotting parameters such as clot initiation time, clot strength, and clot lysis times using TEG. At the same time, some aspects of the safety of transfusing LT-PLTs may be obtained by monitoring complement activation, biodistribution, and organ histology to determine whether thromboses occurred in the major organs. Because of the high hemostatic potential of activated platelets and thrombin, it will be important to determine whether transfusing LT-PLTs is associated with a greater risk of systemic thrombosis compared to normal platelet transfusions. These in vivo studies will allow us to determine whether platelet coagulability is a major limiting factor to stopping severe traumatic hemorrhage, and whether strategies to increase platelet coagulability can potentially increase the efficacy of platelet transfusions during severe bleeding.  102 6.2 Optimizing the delivery of cargo to platelets Incubation of platelets and the various liposomal formulations described in this thesis resulted in approximately 40 to 60% uptake, depending on the incubation conditions used. From the confocal microscopy experiments, it appears the amount of internalized liposomes differed between individual platelets. Studies to correlate platelet activation or other intrinsic platelet properties to the amount of internalized liposomes may identify a subpopulation of platelets which are more amenable to modification, and may be useful in increasing the loading efficiency of cargo into platelets. There are several ways to enhance the loading efficiency of potentially therapeutic cargo into platelets ex vivo. First, the encapsulation efficiency of cargo into liposomes may be enhanced by modifying the liposomal composition and formulation method. For example, the encapsulation efficiency of thrombin was relatively low at 1.6%. Using similar rationale from studies on encapsulating negatively-charged nucleic acids into positively-charged nanoparticles, [256, 257] the encapsulation efficiency of thrombin may be increased by incorporating negatively-charged lipids to bind the positively-charged exosites on thrombin through charge-charge interactions. Another strategy to enhance the loading efficiency of therapeutic cargo is to modify the lipid composition to facilitate endosomal escape of the delivered cargo. For example, this can be achieved by incorporating anionic, pH-sensitive lipids into the liposomal bilayer. These lipids will convert from a lamellar to an inverted micelle structure at low pH, such as during endosomal maturation, leading to the fusion of liposomal and endosomal membranes, ultimately allowing the encapsulated cargo to be released into the cell cytoplasm and escape degradation in the lysosomes. [164]  103 The studies performed in this thesis did not directly identify the exact fate of delivered cargo, including the lipids and the encapsulated molecules, following internalization by platelets. However, based on the experiments in chapter 4, there was no detectable thrombin activity in the platelet supernatant with or without platelet activation, suggesting that thrombin was not released by platelets after it was internalized. The accumulation of lipid fluorescence in platelets over time (chapter 2) also suggests that liposomes remain within platelets following endocytosis under the conditions that were tested. Based on results from chapter 3, liposomes remained intact shortly after delivery to platelets, which allowed for localization of the transcriptional components and for RNA synthesis to occur. Based on the results from chapter 4, thrombin activity was important for the increased coagulability of platelets following activation by agonists. This suggests that the delivered thrombin escapes the liposomes following platelet activation and exerts its activity on intracellular targets to modulate platelet function. However, the experiments conducted here did not determine the exact molecular target or the intracellular localization of the delivered thrombin.  One reason that the delivered contents may not be released by platelets is that the liposomes are not localizing to platelet granules. Because the release of platelet contents is a tightly regulated process, only certain activation conditions will trigger the release of molecules that are packaged in specific platelet compartments. Further characterization of the modified platelet system described here will be needed in order to use these modified platelets as drug delivery vehicles in the clinic. This includes determining the intracellular localization of the delivered contents, the stability of delivered cargo within platelets, and the fate of the contents following activation by different platelet agonists. These studies may be done by radiolabeling or 104 fluorescently labeling the delivered cargo and performing extensive microscopy studies to determine the co-localization of the delivered material with platelet granules and other compartments. These experiments should be performed for resting platelets as well as during activation with platelet agonists such as collagen, ADP, or thrombin.  6.3 Other clinical opportunities for modified platelets There are many short term and longer term clinical opportunities for platelets with modified or enhanced function, such as in drug delivery and in enhancing current platelet therapies. The ability of platelets to localize and specifically release their contents at disease sites provides motivation to continue developing modified platelets for drug delivery, such as to target sites of inflammation and infection. However, since platelets are not currently part of the treatment regimen for such diseases, and transfusing platelets into patients who are not at risk of bleeding may carry risks of thromboses, the most probable short term translation pathways for modified platelets in the clinic will likely involve applications where platelet therapy is already used. Although there is much more characterization and optimization that is needed to be able to use modified platelets effectively and safely in the clinic for localized drug delivery, the work described here will have important implications in modifying platelet function for current platelet therapies.  As a first step to increasing the efficacy of platelet transfusions during severe hemorrhage, we increased delivered liposomal thrombin to platelets ex vivo. The studies presented in this thesis demonstrated that internalization of liposomal thrombin enhanced the hemostatic potential of platelets in several in vitro assays. During severe hemorrhage, platelet 105 transfusions are only one part of a larger hemorrhage management strategy; other procoagulants and antifibrinolytics are also administered to promote clot formation and inhibit clot degradation. Loading platelets with molecules such as FVIIa, which binds tissue factor in wounds to initiate thrombin generation, FXIII, which crosslinks and stabilizes fibrin clots to increase clot strength and adhesion, as well as TXA, an antifibrinolytic which has been shown to be efficacious during traumatic bleeding, could potentially further enhance the efficacy of platelet transfusions in acute bleeding scenarios. The nanoparticle formulations and delivery approaches described here can serve as a basis for encapsulating and delivering these molecules to platelets, and platelet coagulability can be measured using similar clotting assays. For example, FVIIa and FXIII are larger proteins (approximately 52 kDa and 81 kDa, respectively) compared to thrombin (37 kDa), which may not be encapsulated efficiently in the liposomes described in our studies. Further optimization, such as modifying the lipid composition, may be required to efficiently deliver these proteins to platelets. TXA is a small, acidic molecule, which can likely be encapsulated efficiently using previous methods to encapsulate weakly acidic anticancer drugs. The small size of TXA (157 Da) may also allow efficient loading into platelets through the OCS, similar to the spontaneous uptake of serotonin (176 Da) by platelets.  Because platelets are transfused in cancer patients who develop thrombocytopenia, the liposomal approaches described here may also be used to localize chemotherapeutics to tumors to combine platelet therapy and chemotherapy in a single platform. Indeed, others have loaded doxorubicin into platelets and demonstrated an increase in antitumor efficacy. The strategies described in this thesis allows for the delivery of macromolecules such as nucleic acids and active enzymes to platelets. Macromolecules do not generally enter cells in their naked form, and 106 would likely be difficult to deliver without the use of nanoparticles. Our approaches could likely encapsulate and deliver anticancer siRNA therapeutics to platelets, which may have the potential to localize the siRNA to tumors following platelet transfusions. This may mitigate off-target effects and potentially enhance delivery of anticancer siRNA into tumor cells.  Another clinical application for modified platelets is enhancing the efficacy of PRP gels for wound healing. The exact molecular compositions of PRP gels are currently not well defined, which may be why their efficacy in various wound healing scenarios remains controversial. The strategies described in this thesis allow for modification of platelet contents, which might be useful in determining the key active components in PRP gels. The liposomal system can also be used to upregulate or downregulate certain factors such as PF4 and PDGF that are secreted by platelets in PRP gels, and have demonstrate efficacy in wound healing studies. This could allow for a deeper understanding of the key components of PRP gels and may allow for fine tuning of PRP gels for specific wound healing purposes.  107 Bibliography 1 Ruggeri ZM. Mechanisms initiating platelet thrombus formation. Thrombosis and Haemostasis. 1997; 78: 611-6. 2 Ni HY, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. 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