Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
DSpace
UBCV
Zhao, Han Qi
Doctor of Philosophy - PhD
University of British Columbia
Platelets are an integral part of transfusion medicine. In Canada, over 80% of the platelet units for transfusion are produced via the buffy coat method from whole blood and stored in plasma. The storage condition is at 22°C under constant agitation for a maximum of 7 days. These are known as room temperature stored platelets (RPs). There are several disadvantages associated with RPs. First, RPs experience a significant decrease in platelet quality during storage known as the platelet storage lesion. Second, RPs have increased risks of microorganism growth. Last, RPs have a very limited shelf life which contributes to product wastage. Platelets stored at 4°C or cold-stored platelets (CPs) have been gaining renewed interest due to their potential of overcoming the disadvantages of RPs. Preliminary studies have shown that CPs have preserved mitochondrial functions, superior hemostatic functions and stable pH compared to RPs. Additionally, CPs have been hypothesized to be a superior product especially for bleeding patients. In this thesis, we compared the in vitro storage characteristics of CPs versus RPs over 14 days of storage. We showed that CPs have better aggregation response to physiological agonists and formed stronger clots compared to RPs. We also examined the underlying biological changes of CPs and RPs during storage by performing an untargeted metabolomic study. We showed that CPs exhibited significantly less metabolomic stress during storage compared to RPs as exemplified by the stable reduced glutathione pools in the CPs but not RPs. Additionally, we explored the potential benefits of CPs in bleeding scenarios by using an in vitro trauma transfusion model. From this experiment, we showed that donor whole blood mixed with CPs is more resistant to hyperfibrinolysis that is commonly observed in trauma. Finally, we explored the importance of oxidative stress in platelet storage by inhibiting the activity of the glucose 6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway. We found that RPs with G6PD inhibitors seem to have higher P-selectin expression and lower maximum clot firmness, indicating worse quality. These results indicate that CPs should be further considered as a transfusion product specifically for bleeding patients.
THE EVALUATION OF COLD-STORED PLATELETS by Han Qi Zhao BSc (Hons), University of Toronto, 2014 MSc, University of British Columbia, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2022 ©Han Qi Zhao, 2022ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: The evaluation of cold-stored platelets submitted by Han Qi Zhao in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pathology and Laboratory Medicine Examining Committee: Dr. Dana Devine, Professor, Pathology and Laboratory Medicine, UBC Supervisor Dr. Suzanne Vercauteren, Clinical Professor, Pathology and Laboratory Medicine, UBC Supervisory Committee Member Dr. Hugh Kim, Associate Professor, Biochemistry and Molecular Biology, UBC University Examiner Dr. Cedric Carter, Associate Professor Emeritus, Pathology and Laboratory Medicine, UBC University Examiner Additional Supervisory Committee Members: Dr. Ed Conway, Professor, Pathology and Laboratory Medicine, UBC Supervisory Committee Member Dr. Christian Kastrup, Associate Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member iii Abstract Platelets are an integral part of transfusion medicine. In Canada, over 80% of the platelet units for transfusion are produced via the buffy coat method from whole blood and stored in plasma. The storage condition is at 22°C under constant agitation for a maximum of 7 days. These are known as room temperature stored platelets (RPs). There are several disadvantages associated with RPs. First, RPs experience a significant decrease in platelet quality during storage known as the platelet storage lesion. Second, RPs have increased risks of microorganism growth. Last, RPs have a very limited shelf life which contributes to product wastage. Platelets stored at 4°C or cold-stored platelets (CPs) have been gaining renewed interest due to their potential of overcoming the disadvantages of RPs. Preliminary studies have shown that CPs have preserved mitochondrial functions, superior hemostatic functions and stable pH compared to RPs. Additionally, CPs have been hypothesized to be a superior product especially for bleeding patients. In this thesis, we compared the in vitro storage characteristics of CPs versus RPs over 14 days of storage. We showed that CPs have better aggregation response to physiological agonists and formed stronger clots compared to RPs. We also examined the underlying biological changes of CPs and RPs during storage by performing an untargeted metabolomic study. We showed that CPs exhibited significantly less metabolomic stress during storage compared to RPs as exemplified by the stable reduced glutathione pools in the CPs but not RPs. Additionally, we explored the potential benefits of CPs in bleeding scenarios by using an in vitro trauma transfusion model. From this experiment, we showed that donor whole blood mixed with CPs is more resistant to hyperfibrinolysis that is commonly observed in trauma. Finally, we explored the importance of oxidative stress in platelet storage by inhibiting the activity of the glucose 6-iv phosphate dehydrogenase (G6PD) in the pentose phosphate pathway. We found that RPs with G6PD inhibitors seem to have higher P-selectin expression and lower maximum clot firmness, indicating worse quality. These results indicate that CPs should be further considered as a transfusion product specifically for bleeding patients. v Lay summary Platelet transfusions are given to patients who are bleeding or have chronic low platelet counts. Currently, our platelets that are donated are stored at room temperature (22°C) which unfortunately allows bacteria and other microorganisms to grow quickly. Previous research has shown that by storing platelets at a cooler temperature (4°C), the growth of microorganisms can be slowed, and the platelet shelf life can be extended. These platelets are known as cold-stored platelets. Since cold-stored platelets are not currently in use in Canada, we decided that an investigation of cold-stored platelets is needed prior to implementation in the clinics. Our investigation revealed that cold-stored platelets are more activated during storage compared to room-temperature stored platelets. We also found that cold-stored platelets are able to form stronger clots which are especially beneficial for bleeding patients. From our study we believe that cold-stored platelets are a promising product with numerous benefits for patients. vi Preface This research study has been approved by the University of British Columbia (UBC) Research Ethics Board under the project name “Refrigerated Platelets”. The ethics ID for this research is H17-02708. This research study has also been approved by the Canadian Blood Services Research Ethics Board. The ethics ID is 2018.003b4r. This thesis was conducted under the direct supervision of Dr. Dana Devine at the Centre for Blood Research of UBC. Dr. Devine initially proposed the idea of thoroughly investigating cold-stored platelets using modern analytical techniques. Chapter 1 contains the relevant literature review of platelets, platelet storage, and in particular, cold-stored platelets. A version of Chapter 1 has been published as a review paper in the International Journal of Molecular Sciences: Zhao H, Devine DV. The Missing Pieces to the Cold-Stored Platelet Puzzle. Int J Mol Sci. 2022 Jan 20;23(3):1100. doi: 10.3390/ijms23031100. PMID: 35163024; PMCID: PMC8835703. I was responsible for conducting the literature review, creating figures, compiling tables, and writing the first draft of the manuscript. Chapter 2 contains results on validating the analytic techniques, instruments and small storage bags of platelets known as non-destructive test containers (NDTCs). I designed this experiment under the supervision of Dr. Devine and Dr. Serrano. I conducted the experiments, analyzed the data and compiled all results into graphs. The metabolomic results in Chapter 3 came from a collaboration with Dr. Angelo D’Alessandro and Davide Stefanoni at the University of Colorado in Denver. A version of Chapter 3 has been published in the Journal of Proteome Research: Zhao HW, Serrano K, Stefanoni D, D'Alessandro A, Devine DV. In Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets. J vii Proteome Res. 2021 May 7;20(5):2251-2265. doi: 10.1021/acs.jproteome.0c00792. Epub 2021 Mar 29. PMID: 33780259. Dr. Devine initially proposed the idea of investigating the metabolomic profile of stored platelets. I designed the experiment under the supervision of Dr. Devine, Dr. Serrano and Dr. D’Alessandro. I performed the in vitro platelet characterization experiments with the help of research associate Dr. Zhongming Chen. I prepared the samples for metabolomic analysis and sent them to Dr.D’Alessandro and Davide for analysis. I flew to the University of Colorado and performed the metabolomic analysis (mass spectrometry and high-performance liquid chromatography) with Davide under the supervision of Dr. D’Alessandro. Davide created the metabolomic graphs (Figure 3-4 to 3-8). I wrote the first draft of the manuscript. Chapter 4 was inspired by work previously done by Dr. Ahmad Arbaeen and Dr. Cedric Carter. A version of Chapter 4 has been accepted for publishing in the Trauma, Hemostasis, Oxygenation, and Research (THOR) supplement in the Journal of Transfusion. The manuscript is titled “Cold-stored platelets are effective in an in vitro model of massive transfusion protocol assessed by rotational thromboelastometry”. I designed this experiment under the supervision of Dr. Devine and Dr. Serrano. I performed all experiments, analyzed the data and created graphs. I wrote the manuscript as well. Finally, I would like to emphasize that although I am the primary author for the above publications, my co-authors have contributed significantly to the work through countless hours of editing, discussion and advising. They all contributed to the work intellectually to produce the high-quality publications above. viii Publications: Zhao H, Devine DV. The Missing Pieces to the Cold-Stored Platelet Puzzle. Int J Mol Sci. 2022 Jan 20;23(3):1100. doi: 10.3390/ijms23031100. PMID: 35163024; PMCID: PMC8835703. Zhao HW, Serrano K, Stefanoni D, D'Alessandro A, Devine DV. In Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets. J Proteome Res. 2021 May 7;20(5):2251-2265. doi: 10.1021/acs.jproteome.0c00792. Epub 2021 Mar 29. PMID: 33780259. Zhao HW, Serrano K, Culibrk B, Chen ZM, and Devine DV. Cold-stored platelets are effective in an in vitro model of massive transfusion protocol assessed by rotational thromboelastometry. (Accepted for publication on February 9th 2022). National/International Conference Presentations: Oral Presentations: Norman Bethune Symposium 2019: Cold-Stored Platelets: an in vitro Analysis. Zhao HW, Serrano K and Devine DV. Canadian Blood Services Centre for Innovation Research Day May 29th, 2019: Cold-stored Platelets: An Old Product with New Hope. Zhao HW, Serrano K and Devine DV. Earl Davie Symposium 2020: Metabolomic Analysis of Cold-Stored Platelets Zhao HW, Serrano K, Stefanoni D, D'Alessandro A, Devine DV. International Society of Blood Transfusion (ISBT) 2022: The inhibition of Glucose-6-Phosphate dehydrogenase in room and cold-stored platelets. Zhao HW, Serrano K and Devine DV. Poster Presentations: ix American Association of Blood Bank Conference (AABB) 2020: In Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets Zhao HW, Serrano K, Stefanoni D, D'Alessandro A, Devine DV. American Association of Blood Bank Conference (AABB) 2021: An In Vitro Transfusion Model to Assess Cold-stored Platelets in Trauma Zhao HW, Serrano K and Devine DV. x Table of contents Abstract ......................................................................................................................................... iii Lay summary ................................................................................................................................. v Preface ........................................................................................................................................... vi Table of contents ........................................................................................................................... x List of tables................................................................................................................................. xv List of figures .............................................................................................................................. xvi List of abbreviations .................................................................................................................. xix Acknowledgements .................................................................................................................. xxiii Dedication .................................................................................................................................. xxv Chapter 1: Introduction ............................................................................................................... 1 1.1: Platelets ................................................................................................................................ 1 1.1.1: Platelets in transfusion medicine ................................................................................... 1 1.1.2: Problems with current platelet storage methodology .................................................... 2 1.2: Cold-stored platelets............................................................................................................. 3 1.2.1: Cold-stored platelet clearance ....................................................................................... 4 1.2.2: Physiological changes of platelets in the cold ............................................................... 5 1.2.3: Cold-stored platelet in vitro characterization ................................................................ 5 1.2.4: “Omics” and cold-stored platelets ................................................................................. 7 1.2.5: Reduction of CPs storage lesion .................................................................................... 8 xi 1.2.6: Pathogen inactivation technology and platelet additive solutions in cold-stored platelets .................................................................................................................................. 10 1.2.7: Changes in cold storage methodologies: delayed cold storage and temperature cycling ............................................................................................................................................... 14 1.2.8: CPs in the clinics: regulatory approval, application and where in vitro analyses fit in ............................................................................................................................................... 16 1.3: Research question, hypothesis, and specific aims .............................................................. 18 1.3.1: Research question ........................................................................................................ 18 1.3.2: Hypothesis ................................................................................................................... 18 1.3.3: Specific aims ............................................................................................................... 18 1.3.3.1: Aim 1: Instrument and test container validations ................................................ 18 1.3.3.2: Aim 2: In vitro characterization and metabolomic analysis of CPs .................... 19 1.3.3.3: Aim 3: Effectiveness of CPs in trauma ................................................................ 19 1.3.3.4: Aim 4: The role of pentose phosphate pathway in CPs ....................................... 19 Chapter 2: Instrument and non-destructive test container validation .................................. 21 2.1: Introduction ........................................................................................................................ 21 2.2: Materials and methods ....................................................................................................... 22 2.2.1: Instrument validation ................................................................................................... 22 2.2.1.1: Platelet handling, storage, and sampling ............................................................. 22 2.2.1.2: Instrument validation: In vitro assay variability assessment ............................... 23 xii 2.2.2: Non-destructive test container validation of CPs ........................................................ 25 2.2.2.1: Platelet handling, storage, sampling, and experimental setup ............................. 25 2.2.2.2: In vitro characterization of NDTCs versus MU ................................................... 25 2.2.3: Statistical analysis ....................................................................................................... 26 2.3: Results ................................................................................................................................ 27 2.3.1: Instrument validation ................................................................................................... 27 2.3.2: NDTCs validation of CPs ............................................................................................ 39 2.4: Discussion .......................................................................................................................... 42 Chapter 3: In vitro characterization and metabolomic analysis of cold-stored platelets .... 45 3.1: Introduction ........................................................................................................................ 45 3.2: Materials and methods ....................................................................................................... 46 3.2.1: Sample processing and experimental setups ............................................................... 46 3.2.2: Platelet in vitro assays ................................................................................................. 47 3.2.3: Metabolomic analysis of stored platelet concentrates ................................................. 48 3.2.4: Statistical analysis ....................................................................................................... 49 3.3: Results ................................................................................................................................ 50 3.3.1: Platelet in vitro storage characteristics ........................................................................ 50 3.3.2: Metabolomic analysis of stored platelets .................................................................... 56 3.3.2.1: Overview of the effect of storage temperature and time on the platelet metabolomic profile .......................................................................................................... 64 xiii 3.3.2.2: Changes in metabolites of platelets stored under different conditions ................ 64 3.3.2.3: Correlation of metabolites with platelet in vitro characteristics and function ..... 66 3.4: Discussion .......................................................................................................................... 68 Chapter 4: The functions of cold-stored platelets assessed by the in vitro transfusion model....................................................................................................................................................... 73 4.1: Introduction ........................................................................................................................ 73 4.2: Materials and methods ....................................................................................................... 74 4.2.1: Blood collection, storage, and research ethics............................................................. 74 4.2.2: In vitro transfusion model setup .................................................................................. 75 4.2.3: Rotational thromboelastometry (ROTEM) .................................................................. 77 4.2.4: Plasminogen activator inhibitor-1 (PAI-1) assay ........................................................ 77 4.2.5: Statistical analysis ....................................................................................................... 77 4.3: Results ................................................................................................................................ 78 4.4: Discussion .......................................................................................................................... 85 Chapter 5: The role of the pentose phosphate pathway in platelet storage .......................... 89 5.1: Introduction ........................................................................................................................ 89 5.2: Materials and methods ....................................................................................................... 90 5.2.1: Inhibitor preparation, platelet handling, and experimental setup ................................ 90 5.2.2: Platelet in vitro testing ................................................................................................. 92 5.2.3: Statistical analysis ....................................................................................................... 92 xiv 5.3: Results ................................................................................................................................ 93 5.3.1: G6PDi-1 in platelet storage ......................................................................................... 93 5.3.2: 6-aminonicotinamide in platelet storage ..................................................................... 96 5.4: Discussion .......................................................................................................................... 99 Chapter 6: Conclusion .............................................................................................................. 102 6.1: Significance of this thesis in the field of platelet storage................................................. 102 6.2: Future directions of cold-stored platelets ......................................................................... 105 6.2.1: Cold-stored platelet production and storage methodologies ..................................... 105 6.2.2: CPs and randomized clinical trials ............................................................................ 106 Bibliography .............................................................................................................................. 107 Appendices ................................................................................................................................. 120 Appendix A: Detailed method on sample processing, metabolite extraction, data processing, and metabolite assignment. ..................................................................................................... 120 Appendix B: PCA loadings plot with selective annotations for Figure 3-4a. ......................... 123 Appendix C: Correlation coefficient (r) and P value (P) of the Correlation analysis between in vitro parameters and metabolites............................................................................................. 124 xv List of tables Table 1-1: Different platelet additive solutions (PAS) used in recent investigations of cold-stored platelets (CPs) ............................................................................................................................... 13 Table 2-1: Major ROTEM parameters and their definition .......................................................... 24 Table 3-1: Flow cytometry analysis of buffy-coat derived platelets stored in different conditions........................................................................................................................................................ 51 Table 4-1: Changes in platelet count throughout storage in room and cold temperature. ............ 84 xvi List of figures Figure 1-1:Summary of known effects of cold storage on platelets and aspects of cold-stored platelets currently under investigations. ......................................................................................... 4 Figure 2-1: Examples of non-destructive test containers (NDTCs).............................................. 22 Figure 2-2: A typical ROTEM result graph .................................................................................. 24 Figure 2-3: Non-destructive test container experimental setup overview .................................... 26 Figure 2-4: Variability of platelet counts measured by the hematology analyzer ........................ 27 Figure 2-5: Variability of pH measured by the blood gas analyzer .............................................. 28 Figure 2-6: Variability of glucose concentration measured by the blood gas analyzer ................ 29 Figure 2-7: Variability of the lactate concentration measured by the blood gas analyzer ............ 30 Figure 2-8: Variability of P-selectin expression measured by flow cytometry ............................ 31 Figure 2-9: Variability of the increase in P-selectin expression of platelets in response to ADP stimulation..................................................................................................................................... 32 Figure 2-10: Variability of phosphatidylserine expression of platelets measured by flow cytometry ...................................................................................................................................... 33 Figure 2-11: Variability of platelet clotting time measured by ROTEM ...................................... 34 Figure 2-12: Variability of platelet clot formation time measured by ROTEM ........................... 35 Figure 2-13: Variability of maximum clot firmness measured by ROTEM ................................. 36 Figure 2-14: Count and biochemical properties of platelet stored in NDTCs versus MU ........... 39 Figure 2-15: Activation level and response to ADP of platelet stored in NDTCs versus MU measured by flow cytometry ......................................................................................................... 40 Figure 2-16: ROTEM parameters of platelets stored in NDTCs versus MU ............................... 41 xvii Figure 3-1: Changes in count and in vitro biochemical parameters of stored buffy coat platelet concentrates................................................................................................................................... 50 Figure 3-2: ROTEM analysis of stored buffy coat platelet concentrates...................................... 52 Figure 3-3: Aggregation responses of stored platelets stimulated with physiological agonists. .. 53 Figure 3-4: The impact of storage condition on metabolomic phenotypes of platelets. ............... 56 Figure 3-5: Changes of metabolites in platelets stored at different conditions............................. 59 Figure 3-6: Changes in amino acid level during platelet storage. ................................................. 60 Figure 3-7: Lipidomic analysis of stored platelets. ....................................................................... 62 Figure 3-8: Correlation between metabolites and in vitro platelet parameters. ............................ 63 Figure 4-1: Schematics of in vitro transfusion model. .................................................................. 76 Figure 4-2: ROTEM analysis activated with INTEM of hemodiluted donor whole blood (diluted with saline to 20% hematocrit) transfused with warm or cold package. ...................................... 78 Figure 4-3: ROTEM analysis activated with EXTEM of hemodiluted donor whole blood (diluted with saline to 20% hematocrit) transfused with warm or cold package. ...................................... 79 Figure 4-4: ROTEM analysis activated with INTEM of hyperfibrinolytic donor whole blood (mixed with recombinant tissue plasminogen activator to a final concentration of 8.8 µg/mL) transfused with warm or cold package.......................................................................................... 80 Figure 4-5: ROTEM analysis activated with EXTEM of hyperfibrinolytic donor whole blood (mixed with recombinant tissue plasminogen activator to a final concentration of 8.8 µg/mL) transfused with warm or cold package.......................................................................................... 81 Figure 4-6: The activity of plasminogen activator inhibitor 1 (PAI-1) in in vitro transfusion packages. ....................................................................................................................................... 82 Figure 5-1: Overview of the pentose phosphate pathway ............................................................. 90 xviii Figure 5-2: Inhibition of G6PD in platelet storage using NDTCs experimental setup ................. 91 Figure 5-3: Platelet count and blood gas analysis of stored platelets with G6PDi-1 inhibitor. .... 93 Figure 5-4: Surface marker expression and ROTEM function of stored platelets with G6PDi-1. 94 Figure 5-5:Platelet count and surface marker expression of stored platelets with 6-AN inhibitor........................................................................................................................................................ 96 Figure 5-6: Aggregation profile of stored platelets with 6-AN. ................................................... 97 Figure 5-7: Maximum clot firmness of stored platelets with 6-AN.............................................. 98 xix List of abbreviations 6-AN 6-Aminonicotinamide ADP Adenosine diphosphate AMP Adenosine monophosphate ATP Adenosine triphosphate CBS The Canadian Blood Services CPAs Cold-stored platelets with agitation CPs Cold-stored platelets DMSO Dimethyl sulfoxide EXTEM Extrinsic coagulation activator (tissue factor) FDA The Food and Drug Administration FITC Fluorescein isothiocyanate G6PD Glucose-6-phosphate dehydrogenase GPVI glycoprotein VI GSAO (4-(N-(S-glutathionylacetyl) amino) phenylarsonous acid) GSH Reduced glutathione HIV The human immunodeficiency virus HSR Hypertonic shock response xx INTEM Intrinsic coagulation activator (ellagic acid) ISO PAS Isoplate platelet additive solution IVIg Immunoglobulin LTOWB Low titre group O whole blood MCF Maximum clot firmness miRNA MicroRNA MU Mother unit MUFAs Monounsaturated fatty acids NAC N-acetylcysteine NADPH Reduced nicotinamide adenine dinucleotide phosphate NDTCs Non-destructive test containers PAI-1 Plasminogen activator inhibitor 1 PAS Platelet additive solution PC1 Principal component 1 PE Phycoerythrin PF3 Platelet factor 3 PI Pathogen inactivation PROPPR Pragmatic Randomized Optimal Platelet and Plasma Ratios xxi PRP Platelet rich plasma PUFAs Polyunsaturated fatty acids RCCs Red cell concentrates ROS Reactive oxygen species ROS Reactive oxygen species ROTEM Rotational thromboelastometry RPs Room temperature stored platelets SAGM Saline-adenine-glucose-mannitol SAH S-adenosylhomocysteine SCID Severe combined immunodeficiency SO Sodium octanoate STARTEM Coagulation initiator containing calcium chloride TC Temperature cycling TCA The tricarboxylic acid TEG Thromboelastography THOR Trauma, Hemostasis, Oxygenation, and Research supplement tPA Tissue plasminogen activator UBC The University of British Columbia xxii UHPLC-MS Ultra-high pressure liquid chromatography coupled to mass spectrometry UVC Theraflex UV-platelet system VEGF Vascular endothelial growth factor VWF Von Willebrand factor xxiii Acknowledgements I would like to thank my supervisor, Dr. Dana Devine, for her dedication, generosity, and kindness to me. She introduced me to the fascinating world of transfusion medicine and tirelessly gave me guidance and advice throughout my study. I would also like to thank Dr. Katherine Serrano for her supervision and support. She has always been resourceful and dedicated to my success. Many thanks to my supervisory committee members: Dr. Suzanne Vercauteren, Dr. Christian Kastrup, and Dr. Edward Conway for keeping me on track and offering valuable advice and help whenever I am in doubt. My thanks to Dr. Will Lockwood for chairing my committee meetings. I would like to acknowledge the Canadian Blood Services (CBS) for granting me the Graduate Fellowship in support of my study. Furthermore, I also thank the University of British Columbia for awarding me the President’s Academic Excellence initiative PhD Award. Additionally, I would like to expression my gratitude to the UBC Centre for Blood Research for providing opportunities for outreach, annual research symposia and fundings. I would like to acknowledge the folks at the Blood for Research Facility and CBS who have been providing blood product for my research project. I thank Dr. Peter Schubert, the head of the Blood for Research Facility, for his advice on experimental design and platelet storage methodology. Furthermore, I thank Ken McTaggart, the director of Product & Process Development at CBS, for personally making the non-destructive test containers (NDTCs) used in this thesis. Lastly, I would also like to thank Dr. William Sheffield, the director of Centre for Innovation at CBS, for his recommendation on the correlation between cold-stored platelets and PAI-1 level. Most importantly, I extend my most sincere thanks to the blood donors who have generously contributed to my research by giving blood. xxiv I would like to acknowledge our collaborator Dr. Angelo D’Alessandro at the University of Colorado Anschutz Medical Campus in Denver. I thank Dr. D’Alessandro for his advice on platelet storage metabolomics and his generosity in allowing me to visit his laboratory in Denver. Additionally, I thank Davide Stefanoni in Dr. D’Alessandro’s lab for helping me with sample analysis, data graph generation and manuscript writing. I want to give a special acknowledgement to the members of the Devine lab: Dr. Zhongming Chen, Brana Culibrk, Nobu Nakane, Dr. Elena Levin, and Dr. Narges Hadjesfandiari. They have created a fun and welcoming environment for which I enjoy working in very much. To my colleagues and friends in the Devine lab, Kizhakkedathu lab, and Conway lab, you have made this journey far more enjoyable and fulfilling than I could ever imagine. Also, special thanks to my friends who are members of the GR Racing community. I thank you for your company and support during times of difficulties. You are an important part of this journey in my life. Finally, I extend my most sincere thanks to my parents: Donghui Zhao and Dong Wang for pushing me to pursuit my passion in research. I am most grateful for their support and guidance in every step of my life. xxv Dedication To my parents: for your never-ending love, trust and support in me. 1 Chapter 1: Introduction 1.1: Platelets Platelets, or thrombocytes, are small anucleate cells produced by the megakaryocytes in the bone marrows. On average, platelets are 2-4 µM in diameter and have a discoid shape in the blood circulation.1 Typically in normal hemostasis, platelets have a life span of 7-10 days in humans before they are cleared by neutrophils, macrophages and/or hepatocytes.2, 3 Platelets have an integral role in maintaining primary hemostasis by tethering to the site of injury, aggregating with other platelets to form clots and initiate wound healing.4 During this process, platelets also release the contents inside their granules to help with hemostasis. Proteomic analyses have shown that there are over 300 proteins in the platelet α-granules.5 Some of these proteins, such as vascular endothelial growth factor (VEGF), have been associated with wound healing; while others, such as von Willebrand factor (VWF) have been associated with platelet adhesion and aggregation at the site of injury.6, 7 Platelets also have a role in immunity and inflammation.8 Platelets have been recognized as an important part of the innate immune system as they release inflammatory molecules through degranulation. Platelets also express toll-like receptors which can recognize pathogens.9 Additionally, platelets can interact with viruses, such as the human immunodeficiency virus (HIV).10 1.1.1: Platelets in transfusion medicine Platelets have been an indispensable part of transfusion medicine for thrombocytopenic patients since the 1970s.11-14 These patients are prophylactically transfused with platelets to prevent bleeding. Historically, prophylactic transfusions account for the majority of platelet transfusions.15 In the late 1960s, a study by Murphy S and Gardner FH showed room temperature 2 storage of platelets maximized the post transfusion platelet survivability and circulation time.16 Since then, platelets have been stored at 22°C under constant agitation for 5 to 7 days depending on national regulations. Platelets stored in this condition are known as room temperature stored platelets (RPs). 1.1.2: Problems with current platelet storage methodology Although storing platelets at room temperature maximizes post transfusion platelet recoverability and survivability, there are several significant challenges with managing RPs in the blood supply operations. Depending on national regulations, the shelf life of platelet units is 5-7 days. This is an extremely short amount of time considering other blood components, such as packed red blood cells, can be stored for up to 42 days.17 Given the large geographical area, the distribution of platelet units to hospitals outside of metropolitan areas significantly contributes to platelet unit wastages, especially in Canada. Furthermore, evidence from large tertiary hospitals in the United States suggests that platelet wastage due to expiration amount to approximately one million dollars annually.18 In addition to the wastage, RPs also suffer from a significant deterioration of quality exemplified by the increased expression of P-selectin, decreased pH and decreased visible swirl scoring in a process known as the platelet storage lesion.19 There has also been a recent rise in the number of therapeutic platelet transfusions due to increased demand for platelets to treat hemorrhage.20, 21 Hemorrhage is one of the leading preventable causes of death in trauma.22, 23 Actively bleeding patients receiving massive transfusion packages containing a 1:1:1 ratio of red blood cells, plasma and platelets significantly reduced mortality in the Pragmatic Randomized Optimal Platelet and Plasma Ratios (PROPPR) study.24 However, there are concerns about whether RPs are as good for treating trauma patients as they are for prophylactic patients.25 3 1.2: Cold-stored platelets Cold-stored platelets (CPs) are not a new product. Prior to the late 1970s, all platelet units used for transfusions were stored at 4 °C. However, it was recognized in 1969 that CPs were quickly cleared from the circulation, whereas RPs had significantly longer circulation times.16 Despite the rapid in vivo clearance, there has been a renewed interest in bringing back CPs as a product. This recent resurgence can be attributed to three main reasons. First, cold storage significantly reduces the growth of microorganisms, such as bacteria, in the platelet units.26 This allows the platelet units to have a longer shelf life thus reducing the wastage due to shortages. Second, CPs are shown to be hemostatically superior to RPs.27 Lastly, while RPs require constant mechanical agitation during storage, CPs do not seem to require agitation.28 This eliminates the cost of mechanical shakers and improves logistic handling during shipping. Furthermore, recent investigations into the utilization of platelets for transfusion have shown that there is a significant increase in the need for therapeutic platelet transfusions compared to prophylactic transfusions.29, 30 A brief overview of the known characteristics of CPs and the gap in knowledge are summarized in Figure 1-1.With superior hemostatic functions and longer potential shelf life, CPs could be a promising product for actively bleeding patients. 4 Figure 1-1:Summary of known effects of cold storage on platelets and aspects of cold-stored platelets currently under investigations. The box shaded with red and green colors represent varying effects of cold storage on platelet surface receptors. 1.2.1: Cold-stored platelet clearance The rapid clearance of CPs is attributed to one of the significant physiological changes when platelets are exposed to the cold temperature. It was discovered that cold storage induces clustering of platelet surface glycoprotein GPIbα which has been shown to mediate phagocytosis by macrophages in short-term (< 48 hours) CPs.31, 32 Long term CPs (> 48 hours) are cleared by hepatocytes in the liver through interaction of the Ashwell-Morell receptor with the exposed β-GlcNAc moieties on CPs.33 Efforts to prevent the rapid clearance of CPs by galactosylation of the β-GlcNAc moieties have not been successful in humans.34 5 1.2.2: Physiological changes of platelets in the cold It was observed that platelets stored in the cold experience significant morphological changes compared to fresh platelets. In the circulation, non-activated platelets have a thin discoid shape. Upon exposure to 0°C for 10 minutes ex vivo, the majority of the platelets lose their discoid shapes and become spherical.35 Under the electron microscope, platelets exposed to the cold are spherical with many “bumps” and often possess thin pseudopods extending outward. Furthermore, platelets exposed to the cold also lose the circumferential microtubule rings.36 Cold temperature also induces activation of platelets with platelet granule content release and the exposure of phosphatidylserine on the membrane surface.37 One possible mechanism of cold induced platelet activation is the significant accumulation of intracellular calcium during cold storage.38 Furthermore, cold temperature has significant effects on the metabolism of stored platelets. CPs have reduced glucose consumption and lactate production.39 Storage in the cold has also been associated with preserved mitochondrial functions of the platelets.40 1.2.3: Cold-stored platelet in vitro characterization During the late 1960s when platelet storage practice was switched from CPs to RPs, it was known that cold-stored platelets were better for bleeding patients as they resulted in a superior correction in bleeding time.27 The latest in vitro models have revealed new information about the storage characteristics of CPs. Scanning electron microscopy was used to examine the characteristics of clots formed by CPs versus RPs.41 The microscopy images suggested that the clots from CPs had significantly more crosslinking with denser fibers compared to clots formed by RPs. In vitro analysis of CPs stored for 5 days also showed similar clot stiffness to that of fresh platelets. The superior fibre crosslinking in CPs could be mediated by factor XIII through 6 binding on the activated glycoprotein IIb/IIIa on the CPs surfaces.41 In addition, clot histology showed that CPs had higher porosity and more well-defined structure in their clots similar to fresh platelets.42 On the other hand, RPs formed clots with altered structures and decreased porosity. Mitochondria in platelets are essential for platelet energy metabolism and production.43 Mitochondria also play important roles in mediating platelets’ response to stress and activation.43 Reactive Oxygen Species (ROS), which are generated during mitochondrial respiration, were found to be lower in CPs compared to RPs.40 Furthermore, CPs maintained mitochondrial respiration capabilities whereas RPs had decreased mitochondrial respiration after 7 days of storage.40 Finally, cold storage has also been discovered to induce significant changes on the adenosine diphosphate (ADP) receptors of platelets.44 In particular, P2Y1 and P2X1 receptors decreased in CPs compared to RPs. The changes in the ADP receptors of CPs could provide an explanation as to why CPs are less responsive to inhibitory signals, such as nitric oxide and prostaglandin E1, compared to RPs.44 Recent in vitro investigation of CPs also revealed contradictory data with regards to the superiority of the CPs hemostatic functions. For instance, it was shown that the concentration of glycoprotein VI (GPVI), the receptor on the platelet surface for collagen-triggered activation, was lower in CPs versus RPs.45 Subsequently, using a customized microfluidics model designed to measure platelet contractile force under shear stress, CPs were found to have weaker aggregation response to collagen than RPs. In vivo transfusion experiments using human volunteers under dual antiplatelet therapy also showed that post transfusion CPs responded less to collagen compared to RPs, further suggesting the loss of GPVI during storage in vitro.45 However, it is important to note that these experiments were performed with washed platelets, 7 which may affect GPVI levels. There was also a limited number of biological replicates conducted in the human study (N = 8). In addition, there are concerns about whether in vitro assays are reliable in studying CPs. One such example is the use of viscoelastic testing. Viscoelastic tests, such as rotational thromboelastometry (ROTEM) or thromboelastography (TEG), have often been used to show the superior hemostatic effectiveness of CPs over RPs. However, due to the significant changes in the surface receptors, such as glycoprotein IIb/IIIa in CPs, the results of viscoelastic testing on CPs may be misleading. When analyzing CPs on TEG under MAfibrin mode, which was only supposed to reflect the contribution of fibrinogen to clots, the activated glycoprotein IIb/IIIa on CPs contributed to an increase in the maximum amplitude.46 Blocking glycoprotein IIb/IIIa using inhibitors eliminated this effect.46 These data showed the CPs in vitro results should be interpreted with caution and other assays should be used in tandem. 1.2.4: “Omics” and cold-stored platelets Recently, “Omics” technology, such as proteomics and metabolomics, have become more accessible. This is facilitated by the decreased cost of performing these assays and the increased recognition that a large repertoire of information about platelet storage biology can be generated.47 An untargeted metabolomic approach was used to examine the effect of cold temperature on metabolites during platelet storage. Compared to RPs, CPs had significantly decreased oxidative stress compared to RPs. CPs had reduced glutathione consumption during storage as well.48 When examining the trends in metabolite changes using principal component analysis, the metabolomic profile of CPs stored for 21 days clustered closely to day 5 stored RPs.48 Intuitively, the cold temperature should slow down the metabolism of platelets which would lead to less activated metabolomic pathways; however, the metabolomic data suggested 8 otherwise. Some glycolytic pathways in CPs, such as the pentose phosphate pathway and the purine metabolism pathway, had an increase in activity after prolonged storage in the cold.48 Changes in the platelet proteomes during cold storage were also examined. Different storage temperatures of platelets led to differentially expressed proteins important in platelet degranulation, blood coagulation vesicle transport, protein activation cascade and response to stress.49 Interestingly, many of the differentially expressed proteins were located in the membrane bound vesicles of platelets. Some examples of these differentially expressed proteins were GP5, TBXAS1 and FERMT3. These proteins have been found to have a role in regulating platelet activation during storage.50, 51 Storage temperature also had significant impact on the microRNA (miRNA) expression profile of platelets. In a preliminary study, it was found that there were over 100 differentially expressed miRNAs between fresh platelets, CPs and RPs. Furthermore, qPCR confirmed that several miRNAs, such as mir-20a-5p, had significantly increased expression in CPs compared to RPs.52 The significance of the miRNA correlation with platelet quality is still being investigated. 1.2.5: Reduction of CPs storage lesion Results from the recent omics study suggest that oxidative stress plays a significant role in the platelet storage lesion both in RPs and CPs. Hence, to improve CPs storage quality, both synthetic and naturally occurring antioxidants have been used as supplements. N-acetylcysteine (NAC) has been used as a supplement to minimize mitochondrial uncoupling and proton leaks caused by cold-storage.53 NAC supplement also showed a slight effect of reducing reactive ROS in CPs. Both RPs and CPs with NAC supplement had better correction in mice tail bleeding experiment than CPs without NAC.53 However, these experiments had relatively few biological replicates. In addition, the use of platelet additive solution (PAS) could have influenced the 9 results as well. In another study, whole blood derived platelet rich plasma (PRP) supplemented with NAC and stored in the cold seemed to have reduced platelet activation measured by P-selectin expression compared to RPs.54 NAC supplement did not result in differences in the phosphatidylserine expression of CPs or RPs. However, platelets used in this experiment were stored in gas permeable tubes instead of standardized storage containers, making it difficult to draw solid conclusions.54 In a follow up study, the effect of NAC on CPs post transfusion recovery has been tested using the Prkdcscid mouse model.55 There was a slight improvement of platelet recovery 2-hour post transfusion in the CPs with NAC group. However, this improved recovery effect was diminished after 24 hours post transfusion.55 It is important to note that in this mouse study, CPs without NAC supplement were not tested due to aggregation formations in the storage bag. The absence of this control makes data interpretation more challenging as the result could be due to either the storage temperatures or the addition of NAC. Another antioxidant, resveratrol, and a mitochondrial targeting protein, cytochrome c, have been used to reduce oxidative stress in CPs as well.56 Preliminary results from these supplements showed decreased ROS in resveratrol treated CPs.56 However, further investigation of supplemented CPs function and transfusion efficacy are needed. In addition to using antioxidants, synthetic molecules have also been used to reduce the storage lesion defect in CPs. Specialized pro-resolving mediators (SPMs) are biosynthesized molecules derived from essential fatty acids. SPMs have been found to reduce acute inflammation during the host response to invading pathogens.57 Specific SPMs, such as resolving E1, have been found to regulate platelet activation.58 In CPs, the addition of SPMs significantly reduced the glycoprotein 1b expression compared to CPs without SPMs.59 However, in this study there were no RPs with SPMs as a control. It is not clear what other effects SPMs may have in CPs during 10 storage. Similarly, sodium octanoate (SO) was used as a supplement in CPs to improve mitochondrial activity and reduce CPs apoptosis.60 Although CPs with SO seem to trend toward better mitochondrial activity compared to CPs without SO, the results were not statistically significant. Furthermore, although a lower phosphatidylserine expression was observed with CPs with SO on day 5 of storage, further analysis of the caspase-3 level between the treatment groups was not significant. Platelet endocytosis with HepG2 cell models also did not show a difference in SO treated versus non-treated CPs.60 Finally, the integrin inhibitor RGDS, and a calcium ion chelator EGTA were added to improve cold-stored platelet post-transfusion clearance.61 It is important to note that this experiment was performed with mouse platelets and the cold storage period of murine platelets was for a maximum of 24 hours. While the calcium chelator EGTA showed some improvement in cold-stored murine platelet clearance, the effect was not seen when human platelets were used. In addition, EGTA supplementation in human PRP prevented the reduction in platelet count commonly observed in CPs during storage as well as aggregate formation. However, CPs stored in PAS could also achieve similar effects so the exact function of EGTA in CPs is not as straight forward as it seems. 1.2.6: Pathogen inactivation technology and platelet additive solutions in cold-stored platelets Despite stringent donor eligibility criteria, blood products are still exposed to the risk of emerging pathogen contaminations. West Nile virus outbreaks in the past and the recent outbreak of Zika virus in the US are good examples of this.62, 63 Pathogen inactivation (PI) technology has been gaining interest in further securing the safety of blood components.64 This technology targets and damages the nucleic acids of pathogens using ultraviolet light, thus preventing pathogen replication in blood products. PI RPs have been licensed in many countries including 11 the US, Spain, and Switzerland, according to a report by AABB.65 CPs, due to the lower storage temperature, have been shown to slow the growth of bacteria.26 However, psychrophilic bacteria, such as Yersinia enterocolitica, can survive at 4 °C and proliferate to a clinically significant level.66 Yersinia enterocolitica have been associated with transfusion reaction in red blood cell transfusions.67 Thus, there are incentives to investigate the effect of PI on CPs. Recent data show that CPs in PAS treated with Intercept PI (Cerus Corporation, Concord, CA) had less activation level and aggregation in response to agonists than non-PI treated CPs.68 However, when tested in microfluidic devices, PI treated CPs had better fibrin formation rate.68 One caveat for this study is that the platelets were only stored for up to 5 days in the cold. When Intercept PI treated CPs were stored for an extended period up to 21 days, there was no major difference in the metabolism of the platelets in terms of count, pH, glucose or lactate compared to non-PI treated CPs.69 Interestingly, PI treatment resulted in significant changes to CPs function. There was a significant reduction in clot retraction capabilities in PI treated CPs. There was also an elevated phalloidin staining in PI treated CPs. Furthermore, there was also elevated lysis index at 30 minutes measured by ROTEM in PI treated CPs.69 These data suggest that PI may adversely affect CPs during storage. A different PI system, Theraflex UV-platelet System (UVC), was also used on CPs.70 Similar to the effects of the Intercept system, this technology also resulted in damage to the CPs as shown by the decreased hypertonic shock response (HSR). In addition, PI treated CPs also had higher activation shown by annexin V staining and higher release of microparticle compared to untreated CPs70. Together, these results show that PI does have significant effects on CPs. More data on post transfusion effectiveness of PI treated CPs are needed to fully understand the feasibility of these platelets in the clinic. 12 PAS is a chemical solution that can replace plasma in platelet storage. Different types of PAS used in studying CPs have been summarized in Table 1. PAS provides several benefits. First, PAS can be used to replace some of the plasma in platelet storage. The replaced plasma can then be used for fractionation into intravenous immunoglobulin (IVIg) to help with the ongoing shortages.71, 72 In addition, reducing the amount of plasma in a platelet unit lowers the risk of allergic transfusion reactions.73 This adds an additional layer of safety for the transfusion recipients. In CPs, there have been suggestions that PAS could be beneficial.74 It was first established that much like PAS in RPs, a residual of 35% plasma is optimal for CPs in stored PAS.75 Based on this result, the effects of different PAS on CPs storage quality have been investigated. For example, two FDA approved collections and PAS storage systems of platelets (Trima apheresis collection system in Isoplate PAS versus Amicus collection system in Intersol PAS) stored in the cold were compared.76 The results showed that there were only minor differences in the platelet count over 15 days of storage between the two systems. CPs produced from the two systems also had similar aggregation response profiles76. It is important to note that the minor differences observed could be contributed by either the different apheresis collection instruments or by the different PAS. To further differentiate the storage quality of CPs in PAS versus in plasma, ISO PAS platelets were stored for 19 days and compared to apheresis platelets in 100% plasma.77 CPs stored in PAS had significantly reduced glucose consumptions versus CPs in plasma. This is likely due to the substitution of acetate as energy source instead of glucose, while also reducing lactate production. Day 19 CPs PAS also had acceptable pH which would meet FDA pH criteria.77 One criticism for this work is that the CPs in 100% plasma were only stored for 3 days making parallel comparison to day 19 CPs in PAS difficult. CPs stored in PAS-E in also maintained pH above 6.4 for 21 days.78 The aggregation response to agonists is 13 the same between CPs in PAS versus CPs in plasma. However, it is important to note that the addition of PAS to CPs reduced fibrinogen concentration and other coagulation factors, which could be detrimental to the recipient upon transfusion. In addition, PAS could provide additional benefits specific to CPs. The lowered fibrinogen concentration in PAS leads to a significant reduction of aggregate formation while preserving in vitro function in CPs.39 Additionally, PAS may help in maintaining mitochondrial integrity by reducing the depolarization of the inner membrane potential in CPs.79 Finally, the type of PAS used in CPs could also affect the post-transfusion recovery. The recoveries of CPs stored in either 100% plasma, Intersol or Isoplate PAS were compared in an in vivo transfusion of autologous radiolabeled platelets into healthy donors. Interestingly, CPs stored in Intersol had better recoveries than CPs in Isoplate. However, CPs stored in 100% plasma still had better than CPs stored in either PAS.80 Table 1-1: Different platelet additive solutions (PAS) used in recent investigations of cold-stored platelets (CPs) PAS Author Ref Main Usage T-PAS Nair et al 42 Investigation of the clot structure of CPs T-PAS Reddoch-Cardenas et al 77 Compared CPs stored in PAS versus RPs and CPs in plasma. T-PAS and Intersol Getz et al 39 Reduce aggregate formation in CPs using PAS. SSP+ Six et al 68 Pathogen inactivation (Intercept). SSP+ Johnson et al 70 Pathogen inactivation (Theraflex UV-platelet System). SSP+ Marini et al 75 Plasma replacement and test for the percentage of residual plasma required. SSP+ Wood et al 81 Delayed cold storage. PAS-E Hegde et al 53 NAC supplementation in CPs PAS-E Johnson et al 78 Prolonged platelet cold storage. InterSol Agey et al 69 Pathogen inactivation (Intercept). Isoplate Reddoch-Cardenas et al 79 Preserving mitochondrial function in CPs. Isoplate and Intersol Reddoch-Cardenas et al 76 Compare the CPs storage characteristics in two FDA approved PAS. 14 Isoplate and Intersol Stolla et al 80 PAS in CPs in vivo recoveries using autologous radiolabelled platelet transfusion. PAS-IIIM Braathen et al 82 Delayed cold storage. 1.2.7: Changes in cold storage methodologies: delayed cold storage and temperature cycling One benefit of CPs is that we can alleviate the platelet wastage due to the CPs’ longer shelf life. However, having two platelet inventories to manage adds complexity to the blood operators and hospitals. There are already many challenges associated with managing the current platelet inventories83, 84. One way to mitigate these problems would be to store platelets at room temperature until close to the end of their shelf life and then transferring them to the cold with the hope to extend their viability and reduce platelet wastage. This idea is known as the delayed cold storage of platelets. The possibility of delayed storage and the quality of delayed cold-stored platelet product have been assessed. In a recent study, platelets in PAS were split into two identical units. One unit was immediately stored at 4 ° C after production, while the other unit was stored at room temperature for 4 days prior to being transferred to 4 ° C. Both units were tested for in vitro parameters for up to 21 days81. It was shown that delayed CPs had similar count and mean platelet volume compared to the CPs. However, PAS was likely the main contributor here. Intuitively, due to first being stored at room-temperature, the delayed CPs had worse blood gas parameters consisting of higher glucose consumption, lactate production and lower pH compared to platelets that were stored in the cold immediately after production. Interestingly, delayed CPs had better hypotonic shock response recovery compared to CPs, suggesting a potential benefit of delayed cold storage81. Similarly, compared to CPs, apheresis platelets in PAS that were transferred to the cold after 7 days at room temperature showed worse in vitro parameters82. Furthermore, platelet functional analysis by TEG and aggregometry showed that delayed CPs had worse clot dynamics and aggregation response to agonists, 15 respectively82. Although delayed cold storage looks to be the best of both worlds in terms of reducing platelet wastage while not adding the extra complexity of a dual inventory, the quality and usability of delayed CPs need to be carefully examined. In addition to delayed cold storage, another change in CPs storage method that may improve quality is temperature cycling (TC). This idea is based on the findings that continuous cold storage of platelets is deleterious to platelet storage quality85. By alternating between cold and room temperatures during storage, the platelets would have the opportunity to recover at warmer temperatures and partially reverse the changes caused by cold storage86. When platelets were cycled between 4 °C storage for 12 hours followed by 37 °C storage for 30 minutes, the platelets showed superior recovery in a SCID mouse model compared to non-TC RPs or CPs87. To reduce logistic efforts of manual temperature cycling, an automated temperature cycling machine which cycles between 11 hours of 5 °C storage followed by 1 hour of recovery at 22 °C was used to perform further studies. In the in vitro analyses, TC platelets showed better storage characteristics compared to CPs but worse than RPs. When the TC platelets were tested using SCID mouse model of transfusion, they showed comparable recovery to RPs. CPs showed the worst post-transfusion recovery88. The in vivo recovery and survival TC platelets were then compared to RPs and CPs in healthy human volunteers. In humans, the results were not as optimal as they were in the mouse model. TC platelets transfused to humans showed recovery better than CPs, but worse than that of RPs89. A P38 mitogen activated protein kinase inhibitor (VX-702) has been used to try to improve the TC platelets. This inhibitor seems to improve the in vitro storage characteristics of TC platelets but not CPs. When used in a SCID mouse transfusion model, the addition of the inhibitor to TC platelets initially had better recovery than TC without the inhibitor. However, the effect was temporary and quickly diminished90. While 16 the idea of temperature cycling is attractive, it requires additional equipment implementation which could be costly. Further investigation of post platelet transfusion effectiveness, possible increased risk of bacterial growth, and hemostatic efficacy of TC platelets are also needed to fully understand the utility of TC platelets. 1.2.8: CPs in the clinics: regulatory approval, application and where in vitro analyses fit in The inconsistent correlation of in vitro data to in vivo results has always been one of the most difficult obstacles in studying platelet storage91. The relevance of many in vitro analyses of stored platelets to in vivo effects has often been questioned92. One such example recently came from the investigation of CPs in restoring vascular endothelial functions. When added to an in vitro leaky endothelial cell culture model, CPs had superior reductions of endothelial cell permeability compared to RPs. When tested in a mouse model, however, the superior effect of CPs diminished compared to RPs upon transfusion into the mice. CPs still provided significant leak reduction compared to non-transfused mice, but the effect was not comparable to RPs transfused mice93. GSAO (4-(N-(S-glutathionylacetyl) amino) phenylarsonous acid) is a tumor metabolism inhibitor which has been used to label procoagulant platelets in conjunction with P-selectin. When investigating the procoagulant subpopulation of platelets, defined as CD62P+ and GSAO+ by Hua et al94 , it was initially discovered that while cold storage increases the proportion of procoagulant platelets, their response in vitro to agonists stimulation seems to be blunted compared to fresh in vitro. However, when these platelets were transfused, their response to agonist stimulation were restored95. This does not mean that investigations of the in vitro storage characteristics of CPs are meaningless, however. Perhaps focusing on in vivo results using radiolabeled platelets transfusion into healthy donors are not the best way to assess the accuracy of in vitro results96. 17 Since CPs have the greatest potential to be a superior product for actively bleeding patients, in vivo results from therapeutic transfusion using CPs should be prioritized. However, it has been challenging to implement CPs in the clinics due to the process of regulatory approval. In the Mayo Clinic Rochester, CPs have been utilized for bleeding patients since 2015 following the FDA outline of a maximum storage duration of 3 days. Due to this regulation, there was a large discard rate and only 19.1% of the collected CPs were transfused97. This centre has been trying to obtain regulatory approval from both the AABB and FDA since 2013 to store apheresis platelets in the cold for 5 days without agitation. However, this goal was not achieved, and FDA only approved 3-day cold storage without the need for bacterial testing in the end97. Recently, during the early periods of the COVID-19 pandemic, there were serious concerns about blood product shortages98, 99. Given the short storage life of RPs, this has pushed the FDA to grant several emergency approvals for extending product shelf life using CPs. In February 2020, FDA has granted the Texas Blood & Tissue Center license to make and distribute platelets stored in the cold for 14 days. In addition, in Mayo Clinic Rochester, pathogen inactivated RPs after 5 days of storage were allowed to be transferred to cold storage for an additional 9 days100. A total of 61 units of these delayed cold-stored platelets were transfused to 40 patients. The majority of these patients were receiving cardiac surgeries. The results from these transfusions showed that the transfusion recipients had adequate surgical hemostasis comparable to patients receiving RPs. There were also no documented transfusion reactions related to cold-stored platelet transfusion100. More evidence from a pilot clinical trial of CPs further demonstrates the feasibility of cold-stored platelets in treating bleeding during cardiothoracic surgeries. In adults receiving complex cardiothoracic surgery, the median chest tube drain output of patients who received to day 14 CPs was similar to median chest tube drain 18 output of patients receiving day 7 RPs. Secondary outcomes such as platelet function, total blood usage, immediate and long-term adverse events, length of stay in intensive care and mortality were comparable between patients receiving the two different methods of stored platelets101. 1.3: Research question, hypothesis, and specific aims Given the potential benefits including superior hemostatic function, reduction in product wastage, increased transfusion recipient safety of CPs and poor quality of previous experiments, we conducted a systematic investigation of CPs. 1.3.1: Research question What are the differences between CPs and RPs in their storage characteristics and functions? 1.3.2: Hypothesis Cold-stored platelets are a promising product in addition to the currently standard room temperature stored platelets. We hypothesize that cold-stored platelets may be a superior product for bleeding patients. 1.3.3: Specific aims To investigate the in vitro characteristics and functions of CPs compared to RPs, we generated the following aims. 1.3.3.1: Aim 1: Instrument and test container validations In this aim, we assess the internal variability of the instruments we used to test the in vitro characteristics and functions of CPs and RPs. Identical platelet samples were analyzed on the instrument multiple times to identify the measurement variability (technical repeat). The same process was repeated for different platelet units on different days on the same instruments (biological repeats). In addition, we validated the platelet storage characteristics in specifically 19 designed miniature storage bags against platelets stored in standard bags. These small bags help reduce the number of platelets required and the concentration of inhibitors used in the later aim of this study. 1.3.3.2: Aim 2: In vitro characterization and metabolomic analysis of CPs In this aim, we conducted a thorough investigation of the in vitro characteristics of CPs, such as platelet count, lactate concentration, and P selectin expression using modern assay techniques. Additionally, the functional parameters of CPs were also assessed and compared to RPs. To understand the biological mechanisms that underline the storage characteristics and functional differences between CPs and RPs, we conducted an untargeted metabolomic analysis of CPs and RPs during storage of up to 14 days. These experiments were conducted in collaboration with Dr. Angelo D’Alessandro and Dr. Davide Stefanoni at the University of Colorado Anshutz School of Medicine in Denver. 1.3.3.3: Aim 3: Effectiveness of CPs in trauma CPs have been shown to have superior hemostatic functions over the standard RPs in vitro. There is a need to investigate whether the superior hemostatic functions of CPs translate to trauma patients. We used an in vitro trauma transfusion model first designed by Dr. Cedric Carter, UBC Department of Pathology & Laboratory Medicine, to compare the effect of CPs in restoring the hemostatic parameters of trauma patient’s blood. 1.3.3.4: Aim 4: The role of pentose phosphate pathway in CPs Our metabolomic data showed that both CPs and RPs experience oxidative stress during storage. The pentose phosphate pathway is responsible for generating reduced glutathione which can neutralize oxidative stress. In this aim, we inhibited the key enzyme, glucose-6-phosphate 20 dehydrogenase, of the pentose phosphate pathway using commercially available inhibitors. We then performed platelet in vitro characterization to observe the effect of pentose phosphate pathway in CPs and RPs. 21 Chapter 2: Instrument and non-destructive test container validation 2.1: Introduction In order to accurately detect the differences between CPs and RPs, a preliminary analysis of the internal variability of instruments used to measure platelet in vitro characteristics is necessary. Currently, standard platelet assays include platelet count measured by hematology analyzer, platelet unit biochemical properties measured by blood gas analyzer, platelet surface marker expression measured by flow cytometry as well as platelet function measured by ROTEM. Although largely improved over their predecessors, many of these assays contain internal variability which would hinder our exploration of the research question. For example, flow cytometry analysis is a complex technique where the operator can introduce a lot of variability in addition to variabilities from reagents and the flow cytometer itself.102 Additionally, viscoelastic testing machines, such as ROTEM, have been shown to have variability between devices.103 In addition to the instruments’ internal variabilities, platelet units can also vary due to donor differences. In this study we used buffy coat platelet concentrates derived from whole blood. Even though each platelet concentrate produced in this method is derived from four donor’s buffy coats, it is still necessary to investigate the variability from one unit to another. In addition to validating the instruments used to study CPs, we also needed a model to maximize the usage of platelet concentrates when studying CPs and RPs. A Canadian Blood Services internal research project led by Dr. Peter Schubert produced a series of small platelet storage bags that mimic the platelet storage quality of the standard bags used in the clinics. These small bags are known as the non-destructive test containers (NDTCs). An example of NDTCs is shown in Figure 2-1. The average volume required in these small bags is 12 mL versus 320-350 mL in the standard platelet storage bags. Additionally, in the NDTCs it is necessary to have 2mL of air 22 bubble to allow for the gentle agitation of platelets stored at room temperature. The validation of RPs stored in NDTC versus standard bags has been performed by Dr. Schubert’s team. We need to assess the storage characteristics of CPs in NDTCs versus standard bags. Additionally, we also need to verify whether the air bubble is required NDTCs stored at cold temperature. Platelet units are the most expensive blood component to produce compared to the other standard blood products, and it is essential that we can take full advantage of a platelet unit while testing multiple conditions at the same time.104 Figure 2-1: Examples of non-destructive test containers (NDTCs) 2.2: Materials and methods 2.2.1: Instrument validation 2.2.1.1: Platelet handling, storage, and sampling Standard buffy coat derived platelet concentrates were obtained from the Canadian Blood Services Blood for Research Facility in Vancouver, BC. The platelet concentrates were placed at 4°C in a walk-in fridge within 4 hours of production. A new platelet concentrate was obtained 23 for each biological replicate performed. A total of 7 biological replicates were performed (N = 7). Platelet concentrates were sampled aseptically in a biological safety cabinet on day 1, 2, 8 and 14. Day 1 sampling was done on freshly produced platelets prior to placing the platelet concentrate at 4°C. 2.2.1.2: Instrument validation: In vitro assay variability assessment For count, platelets were sampled in EDTA vacutainers (3mL; BD Biosciences, Franklin Lakes, NJ). Platelets in vacutainers were counted on a Sysmex XN-550 hematology analyzer in manual mode (Sysmex Corporation, Kobe, Hyogo, Japan). The same platelet tube was analyzed 3 times. For biochemical properties, platelets were sampled into 3 mL sterile syringes and analyzed on the blood gas analyzer (GEM Premier 4000, Instrumentation Laboratory, Bedford, MA). The same syringe containing platelets was analyzed 3 times on the blood gas analyzer. Parameters tested included pH, the glucose concentration, and the lactate concentration. For platelet surface marker expression analysis using flow cytometry, platelets were sampled into Eppendorf tubes and then diluted to 200 × 109/L with phosphate buffered saline. Then the 45 µL of diluted platelets were stained with 5 µL of staining reagents and incubated for 30 minutes at room temperature in the dark. The staining reagents used were phycoerythrin (PE) labelled CD62P (Beckman Coulter, Indianapolis, IN), and fluorescein isothiocyanate (FITC) labelled Annexin V (BD Biosciences, Franklin Lakes, NJ). The increase in CD62P expression of platelets in response to physiological agonist ADP was also assessed by flow cytometry. In these tubes 48 µL of diluted platelets were mixed with 6 µL of 100 µM of ADP before being stained with 6 µL of CD62P antibody. All flow cytometry samples were independently prepared in triplicates prior to analysis on a FacsCanto II flow cytometer (BD BioSciences). 24 For platelet clotting dynamics measured by ROTEM (Instrumentation Laboratory), platelets were sampled into Eppendorf tubes and diluted to 100 × 109/L with thawed fresh frozen AB+ plasma. The diluted platelets were analyzed on ROTEM using manual mode by mixing 300 µL of diluted platelets with 20 µL of coagulation activator and 20 µL of 0.2 M calcium chloride. The coagulation activator used was INTEM (ellagic acid). Common ROTEM parameters and their definitions are shown in Table 2-1. A typical graph of a ROTEM run is also shown in Figure 2-2 Table 2-1: Major ROTEM parameters and their definition ROTEM Parameter Definition CT: Clotting time The time it takes for the clot to reach 2mm of amplitude CFT: Clot formation time The time it takes for the clot to reach 20mm of amplitude from 2mm. MCF: Maximum clot firmness The maximum amplitude the clot reaches during the test. ML: Maximum Lysis The decrease in clot amplitude as a percentage of MCF. Figure 2-2: A typical ROTEM result graph 25 2.2.2: Non-destructive test container validation of CPs 2.2.2.1: Platelet handling, storage, sampling, and experimental setup Per biological replicate, two buffy coat platelet units were obtained from the Canadian Blood Services Blood for Research Facility in Vancouver BC. The two platelet units were pooled and split into two identical units. One platelet unit was stored at 4°C in a walk-in fridge. This unit was labelled as the Mother Unit (MU). The other platelet unit was used to fill NDTCs. There were two types of NDTCs. Type 1 NDTCs had a maximum fill volume of 17 mL, optimal fill volume of 12 mL with 2 mL of air bubble inside. Type 1 NDTCs were labelled as P17-12-2. Type 2 NDTCs had a maximum fill volume of 17 mL, optimal fill volume of 12 mL with 0 mL of air bubble inside. Type 2 NDTCs were labelled as P17-12-0. Per experiment, 3 NDTCs of each type were filled on day one as NDTCs were one-time use only and the sampling dates were day 1, 2, 8 and 14. Day 1 sampling was done prior to filling the NDTCs and cold storage. The NDTCs were stored at 4°C in a walk-in fridge after filling. A total of 5 biological replicates were performed (N = 5). A graphical depiction of the experimental setup is shown in Figure 2-3. 2.2.2.2: In vitro characterization of NDTCs versus MU The MU was sampled aseptically in a biological safety cabinet. Type 1 and type 2 NDTCs were sampled by cutting the tubing on the top of the bag and transferring the platelets into a sterile 15 mL falcon tube (Corning Inc. Corning, New York). This procedure was performed in a biological safety cabinet. Sampled platelets were counted on a hematology analyzer (PocH-100i, Sysmex Corporation). The platelet biochemical properties were analyzed by a blood gas analyzer (GEM400, Instrumentation Laboratory). The platelet P-selectin expression, increase in P-selectin expression in response to ADP and phosphatidylserine expression were measured by flow cytometry. The staining reagents and procedures used were identical to the method described in 26 section 2.2.1.2. ROTEM was used to analyze the dynamic clot formation of sampled platelets using INTEM as the coagulation activator. Figure 2-3: Non-destructive test container experimental setup overview *Day 1 platelets are sampled prior to filling NDTCs and cold storage. 2.2.3: Statistical analysis For instrument validation, the results were graphed as the mean ± 1 standard deviation of 7 independent biological replicates. For NDTCs validation of CPs, the results were graphed as the mean ± 1 standard deviation of 5 independent biological replicates. Two-way ANOVA for repeated measures was used to 27 compare the significant differences between platelets from MU, type 1 NDTCs (P17-12-2) and type 2 NDTCs (P17-2-0) on each sampling day. Statistical significance is shown by the number of asterisks in the graphs. * = P < 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, and **** = P ≤ 0.0001. All graphs and statistical analyses were performed on GraphPad Prism 6 (GraphPad Software, Inc, La Jolla, CA). 2.3: Results 2.3.1: Instrument validation Figure 2-4: Variability of platelet counts measured by the hematology analyzer Platelets were sampled into vacutainers containing EDTA and ran on manual mode of the Sysmex hematology analyzer. Each platelet sample was counted three times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 28 Figure 2-5: Variability of pH measured by the blood gas analyzer Platelets were sampled into sterile syringes and analyzed by the GEM4000 blood gas analyzer. Each platelet sample was analyzed three times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 29 Figure 2-6: Variability of glucose concentration measured by the blood gas analyzer Platelets were sampled into sterile syringes and analyzed by the GEM4000 blood gas analyzer. Each platelet sample was analyzed three times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 30 Figure 2-7: Variability of the lactate concentration measured by the blood gas analyzer Platelets were sampled into sterile syringes and analyzed by the GEM4000 blood gas analyzer. Each platelet sample was analyzed three times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 31 Figure 2-8: Variability of P-selectin expression measured by flow cytometry Platelets were sampled into Eppendorf tubes and diluted to 200 × 109/L with phosphate buffered saline. The diluted platelets were stained with CD62P antibody and incubated at room temperature in the dark for 30 minutes prior to being analyzed on the flow cytometer. Each platelet sample was analyzed three times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 32 Figure 2-9: Variability of the increase in P-selectin expression of platelets in response to ADP stimulation Platelets were sampled into Eppendorf tubes and diluted to 200 × 109/L with phosphate buffered saline. A final concentration of 10 µM of adenosine diphosphate (ADP) was added to diluted platelets which were then stained with CD62P antibody. The tubes were incubated at room temperature in the dark for 30 minutes prior to being analyzed on the flow cytometer. Each platelet sample was analyzed three times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 33 Figure 2-10: Variability of phosphatidylserine expression of platelets measured by flow cytometry Platelets were aliquoted into Eppendorf tubes and diluted to 200 × 109/L with phosphate buffered saline. The diluted platelets were stained with annexin V antibody and incubated in the dark for 30 minutes prior to being analyzed on the flow cytometer. Each platelet sample was analyzed three times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 34 Figure 2-11: Variability of platelet clotting time measured by ROTEM Platelets were aliquoted into Eppendorf tubes and diluted to 100 × 109/L with thawed fresh frozen AB+ plasma. The diluted platelets are then analyzed by ROTEM under manual pipette mode using INTEM coagulation initiator (ellagic acid). Each sample was analyzed 4 times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 35 Figure 2-12: Variability of platelet clot formation time measured by ROTEM Platelets were sampled into Eppendorf tubes and diluted to 100 × 109/L with thawed fresh frozen AB+ plasma. The diluted platelets are then analyzed by the rotational thromboelastometry under manual pipette mode using INTEM coagulation initiator (ellagic acid). Each sample was analyzed 4 times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 36 Figure 2-13: Variability of maximum clot firmness measured by ROTEM MCF = maximum clot firmness. Platelets were sampled into Eppendorf tubes and diluted to 100 × 109/L with thawed fresh frozen AB+ plasma. The diluted platelets are then analyzed by the rotational thromboelastometry under manual pipette mode using INTEM coagulation initiator (ellagic acid). Each sample was analyzed 4 times. Data were shown as the mean. The platelet concentrate used in the third independent repeat (B3) was discarded after day 8 due to the discovery that the unit was contaminated with HPV virus. 37 Figure 2-4 shows the result of variability of platelet count measured by the hematology analyzer. Overall, the hematology analyzer had small internal variability as shown by the closely clustered results on for each platelet unit. However, there was considerable variability in the platelet count of different platelet concentrates. For example, even prior to storage on day 1, the first platelet concentrate (B1) had an average platelet count of about 700 × 109/L, whereas B7 had a count of 1000 × 109/L. This trend of variability in platelet count between the different platelet concentrates continued throughout the storage. Figures 2-5, 2-6 and 2-7 show the results of variabilities in the platelet biochemical parameters measured by the blood gas analyzer. Overall, there is low variability in the measurement using blood gas analyzer. For pH (Figure 2-4) there is also low variability between different platelet concentrates throughout 14 days of storage. The glucose concentration (Figure 2-5), on the other hand, had more variability from one platelet concentrate to another. This variation seems to increase with increased storage duration. For lactate concentration (Figure 2-6) there seems to be little variability between platelet concentrates on day 1 and day 2 of storage. However, the variability increased as storage duration increased to day 8 and 14. Figures 2-8, 2-9 and 2-10 show the results of variabilities in the platelet surface marker expression measured by the flow cytometer. For P-selectin expression measured by CD62P antibody (Figure 2-8), the flow cytometer had little variation as shown by the closely clustered results of the three technical repeat measurements for any given platelet concentrate. There was also a small variation between the platelet concentrates in their P selectin expression. The increase in P-selectin expression of platelets in response to ADP (Figure 2-9) followed similar trend as Figure 2-8 where there was both little variation in the flow cytometer measurement as well as between platelet concentrates. The only exception is on sampling day 14 where B6 38 platelet concentrate had larger than normal internal variation. For phosphatidylserine expression measured by the annexin V protein (Figure 2-10), there was relatively small variation between measurements and platelet concentrates for day 1 and 2 of storage. However, the variability largely increased as storage continued. Day 8 and 14 showed large variability of annexin V binding within each platelet concentrate tested. Figures 2-11, 2-12, and 2-13 show the results of variabilities in the platelet clotting dynamics measured by ROTEM. The clotting time reflects the time it takes from the beginning of the experiment to when 2 mm of clot amplitude is formed. As seen in Figure 2-10, on day 1 there were large internal variabilities in several platelet units (e.g. B1, B4 and B7). However, this was not observed in day 2, 8 or 14 except for B4 on day 8. The clotting time between each platelet concentrate was relatively consistent. The clot formation time shows the time it takes for the clot to reach 10 mm of amplitude from 2 mm. In Figure 2-11, large internal variabilities were observed on day 1 for platelet unit B1, B6 and B7. Additionally, on day 8 B4 platelet unit had large variability. Overall, each platelet unit had similar clot formation time. Finally, the maximum clot firmness (MCF) represents the highest clot amplitude reached by the platelets during the experiment. Figure 2-11 showed that in all sampled days, there were small internal variabilities of maximum clot firmness. Different units of platelets also had similar MCF on different storage days. 39 2.3.2: NDTCs validation of CPs Figure 2-14: Count and biochemical properties of platelet stored in NDTCs versus MU Platelets stored in the mother unit (MU) and non-destructive test containers (NDTCs) were sampled aseptically in a biological safety cabinet. Platelet count was performed on a hematology analyzer. Platelet pH, glucose concentration and lactate concentration were analyzed by a blood gas analyzer. Statistical significance is denoted by the number of asterisks (*). ** = P ≤ 0.01, and **** = P ≤ 0.0001. The results were shown as the mean ± 1 Standard deviation. A total of 5 independent biological replicates were performed (N = 5). 40 Figure 2-15: Activation level and response to ADP of platelet stored in NDTCs versus MU measured by flow cytometry Platelets stored in the mother unit (MU) and non-destructive test containers (NDTCs) were sampled aseptically in a biological safety cabinet. Platelets were diluted with PBS to 200 × 109/ L prior to being stained with antibodies. The P-selectin expression of stored platelets were measured using CD62P antibodies. Additionally, the platelet’s ability to upregulate P-selectin expression in response to ADP (10 µM) was also measured and labelled as ADP response. Phosphatidylserine expression is measured by Annexin V antibody adherence. The results were shown as the mean ± 1 Standard deviation. A total of 5 independent biological replicates were performed (N = 5). 41 Figure 2-16: ROTEM parameters of platelets stored in NDTCs versus MU Platelets stored in the mother unit (MU) and non-destructive test containers (NDTCs) were sampled aseptically in a biological safety cabinet. Platelets were diluted to 100 × 109/L with AB+ plasma. Twenty µL of 0.2 M calcium chloride and INTEM reagents were mixed with 300 µL of diluted platelets and analyzed on ROTEM. The results were shown as the mean ± 1 Standard deviation. A total of 5 independent biological replicates were performed (N = 5). 42 Figure 2-14 shows the platelet count measured by the hematology analyzer as well as the pH, lactate and glucose of stored platelets measured by blood gas analyzer. Over 14 days of storage, there were no significant differences between CPs stored in MU versus CPs stored in NDTCs. Significant differences were observed in the pH. On sampling day 2, CPs stored in NDTCs had significantly higher pH (7.2) compared to CPs stored in MU (pH 7.1) (P < 0.01). Similar differences were observed on day 8 and 14 as well. Figure 2-15 shows the platelet degranulation level, response to ADP and phosphatidylserine expression measured by flow cytometry. Overall, CPs stored in either MU or NDTCs had an increase in P-selectin expression (measured by CD62P) and a reduction in the response to ADP agonist. Phosphatidylserine expression also gradually increased throughout storage. No significant difference between CPs stored in NDTCs versus CPs stored in MU were observed. Figure 2-16 shows the CPs clotting dynamics measured by ROTEM using the INTEM coagulation activator. There was no significant difference in the clotting time, clot formation time and maximum clot firmness between CPs stored in NDTCs versus CPs stored in MU. 2.4: Discussion In order to properly assess the in vitro storage characteristics of CPs and how they compare to RPs, we conducted a thorough validation of our current platelet characterization assays. Our data showed that although the internal variabilities of our assays/instruments are mostly minimal, there is significant platelet unit variability. As an example, we observed large variations in the platelet count in different platelet units. Platelet units have been shown to have high variability that could affect transfusion outcomes.105, 106 One way to minimize the variability in platelet units is to employ the pool and split model. In this model, two or more platelet concentrates are 43 obtained and mixed together before splitting into identical platelet units. This model should further help with minimizing the unit-to-unit variability shown by our results. Additionally, we observed high internal variability in the platelet phosphatidylserine expression assay which was measured by using annexin V binding and analyzed by flow cytometry. The internal variability was particularly large on storage day 8 and 14. There are several potential causes to this observation. First, annexin V assay requires calcium ions (Ca2+) as a co-activator. Calcium has a well-known role in platelet activation.107 We suspect that the additional calcium in the annexin V assay is activating the CPs leading to aggregation. This may be correlated to the observation that the flow rate on the flow cytometer of CPs stained with annexin V and calcium was abnormally low. We suspect that the activated CPs were blocking the flow channel leading to this event. One potential solution to this problem is to find another protein that has high affinity for phosphatidylserine without needing calcium as a co-activator. Lactadherin is a milk protein with such properties. Lactadherin has also been shown to be more sensitive than annexin V in an in vitro comparative study.108 In our future investigation of CPs, we will be switching to lactadherin to test for phosphatidylserine expressions. There were also internal variabilities measured in the ROTEM assay of CPs. In particular, the parameters related to the time of clot formation seem to have the biggest internal variabilities. This could in part be explained by the method of ROTEM assay setup. In this experiment, we initiated ROTEM reactions with the manual mode. This means that we added the platelets and coagulation initiators manually before starting the ROTEM reaction. ROTEM has an automatic program where the machine automatically aspirates the amount of platelets and coagulation initiators needed, mixes the sample and then starts the reaction. This automatic program not only streamlines the reaction setup to reduce operator errors, it also may improve the variability in the 44 time measurement. In future experiments, we will be using only the automatic program to run ROTEM samples. Our results from the NDTCs versus MU experiment showed that CPs stored in both types of NDTCs had similar in vitro storage characteristics as the CPs stored in MU. The only difference observed during storage was in the pH of the platelets. CPs in NDTCs or in MU both maintained acceptable pH throughout storage, however, CPs stored in NDTCs had a significantly more basic pH in later days of storage. This difference in pH may be due to the change in surface to volume ratio between NDTCs and MU. It is also important to note that in this experiment we only measured selected platelet storage characteristics. There are other parameters, such as platelet hypertonic shock response, which were not measured. Overall, our results showed that NDTCs remain a reasonable model to use in order to maximize the usage of platelet units and reduce the amount of inhibitors/drugs needed to test multiple treatments of CPs. 45 Chapter 3: In vitro characterization and metabolomic analysis of cold-stored platelets 3.1: Introduction Platelet transfusions are an important component of modern transfusion therapy. In Canada, most platelets for transfusion are produced by the buffy coat method and resuspended in plasma with storage at 22°C with constant agitation for up to 7 days. RPs have a short storage life compared to other blood components. RPs demonstrate significant changes in quality during storage known as the platelet storage lesion.19, 109 With current shelf life, platelet wastage due to expiration is a significant problem in many blood operations and creates an inventory management challenge for transfusion services.110-112 With storage at room temperature and without using pathogen inactivation technology, contaminated platelet units may have relatively unrestricted bacterial growth posing risk to transfusion recipients.26, 113 CPs have recently been reconsidered as an alternative storage method.114 Cold storage can slow bacterial growth and has the potential to extend shelf life. CPs are not a new product. Prior to the late 1960s, all platelet units were stored at 4°C. However, an in vivo transfusion survival and recovery study showed that CPs are quickly cleared from the recipient’s circulation after transfusion.16 Several attempts were made to improve the in vivo survival and recovery. When platelets are exposed to cold temperature, they lose their discoid resting shape and become spherical. It was initially thought that by preserving the platelet discoid morphology using drugs, such as cytochalasin B, refrigerated platelet survival would improve. However, Hoffmeister et al. demonstrated that platelet in vivo survival and recovery are independent of platelet shape.115 The platelet surface glycoprotein GP1b clusters in response to cold storage. This clustering is also accompanied by desialylation of the GP1b receptor exposing β-N-acetylglucosamine.115 Efforts 46 to re-glycosylate cold-stored platelets showed relatively good improvement in post-transfusion recovery and survival of short-term (<2 hours) refrigerated murine platelets.116 However, when human platelets were refrigerated for 48 hours, galactosylation did not prevent their clearance.34 RPs remain in the circulation for much longer than CPs. For this reason, room temperature became the storage standard for platelet concentrates. This switch in practice was done in consideration of patients receiving prophylactic platelet transfusions in order to maintain platelet counts and decrease the frequency of transfusion. However, RPs are not necessarily the best product for actively bleeding patients. Even in the 1970s, CPs were shown to have superior hemostatic function compared to RPs.117 In recognition of the different needs of actively bleeding patients, there is renewed interest in CPs. Thus, there is a need to investigate the storage characteristics of CPs compared to RPs using current assay techniques. There is also a need to understand the changes in biological processes associated with different storage temperatures. In this study, using a panel of analytical assays we report the in vitro characteristics of CPs and RPs derived from whole blood donations using buffy coat methodology. In addition, we explored the effect of agitation in cold storage. Taking advantage of the recent advancement of ‘omics’ technologies which have significantly contributed to platelet storage research,118, 119 we employed an untargeted metabolomic approach to examine platelet biochemical changes with storage at 4°C. 3.2: Materials and methods 3.2.1: Sample processing and experimental setups Whole blood was collected by Canadian Blood Services from donors providing signed informed consent. Buffy coat platelet concentrates suspended in plasma were produced by the Canadian Blood Services Blood4Research Facility in Vancouver, Canada. For each biological replicate, 47 three buffy coat platelet units were pooled and split into three identical platelet units to minimize the effect of donor variability. One unit (RP) was stored at 22°C under constant agitation (Helmer Scientific, PC1200-Pro Platelet Incubator, Noblesville, IN). One unit (CPA) was stored in a walk-in fridge (4°C) in a platelet agitator (Forma platelet agitator, Thermo Electron Corporation. Waltham, MA). The last unit (CP) was stored in walk-in fridge (4°C) without agitation. Platelet units were sampled through a coupling port (Fresenius Kabi, Bad Homburg, Germany) using a sterile syringe in a biosafety cabinet. Platelet units were sampled on days 1, 2, 4, 7, 9, 11, and 14 of storage. This study was approved by both the University of British Columbia Ethics Committee (H17-02708) and the Canadian Blood Services Ethics Board (2018-003). 3.2.2: Platelet in vitro assays Flow cytometry: Platelets were sampled into K2EDTA vacutainers (3 mL; BD Biosciences, Franklin Lakes, NJ). Platelets were counted using a Sysmex XN-550 hematology analyzer in automatic sampler mode (Sysmex Corporation, Kobe, Hyogo, Japan). Platelet biochemical analysis was performed using a blood gas analyzer GEM Premier 4000 (Instrumentation Laboratory, Bedford, MA). Reagents used for flow cytometry staining were: PE labelled CD62P (Beckman Coulter, Indianapolis, IN), PE labelled CD42b (Beckman Coulter), and FITC labelled lactadherin (Haematologics Technologies, Essex Junction, VT). Platelet samples were diluted with phosphate-buffered saline to 100 × 109/L and 45 µL of diluted platelets were incubated with 5 µL of staining reagent for 30 min at room temperature in the dark. One milliliter of phosphate-buffered saline was added to each sample prior to analysis on a FacsCanto II flow cytometer (BD BioSciences). 48 ROTEM (Instrumentation Laboratory): Platelets were diluted to 100 × 109/L with fresh frozen AB+ plasma. Three hundred µL of diluted platelets were added to the ROTEM cup and 20 µL each of STARTEM and INTEM reagents were added automatically by the ROTEM machine. Reactions ran for 1 hour. Light transmission aggregometry: Platelet samples were diluted with fresh frozen AB+ plasma to 250 × 109/L and added to glass vials containing a magnetic stir bar (BioData Corporation, Horsham, PA). All platelet samples were incubated at 37°C for 1 min in the aggregometer (Platelet Aggregation Profiler®, Model PAP-8E, BioData Corporation) before physiological agonists were added directly into the glass vials. The agonists used were ADP (10 µM final concentration) or a combination of epinephrine (2 µM final concentration) with collagen (2.5 µg/mL final concentration) (Chrono-log Corporation, Havertown, PA). 3.2.3: Metabolomic analysis of stored platelet concentrates Fifty microliters of previously frozen platelets were extracted 1:10 in ice cold extraction solution (methanol:acetonitrile:water 5:3:2 v/v) or pure methanol, in the presence of stable isotope-labeled internal standards, as described.120 Samples were vortexed and insoluble material sedimented prior to further metabolomic and lipidomic analysis via ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS – Vanquish-QExactive, Thermo Fisher), as extensively described in prior methodological publications.121 Briefly, we used a 5 min C18 gradient method in positive and negative modes exactly as described previously in method papers and applied to the study of metabolic changes in platelets as a function of human aging or storage in the blood bank.48, 120, 121 Peak integration and metabolite assignment were performed using Maven (Princeton University) against the KEGG database, confirmed against high-resolution chemical formula determination from isotopic patterns and accurate mass, and 49 validated against experimental retention times for >650 standard compounds (Sigma Aldrich; MLSMS, IROATech, Bolton, MA, USA). Instrument stability was assessed using replicate injections of a quality control mix injected every 10 runs, while samples were run in randomized order. Detailed metabolomic methods is provided in appendix A. 3.2.4: Statistical analysis In vitro data were compared for statistical significance using a two-way ANOVA for repeated measures. Multiple comparisons were made between RPs, CPAs and CPs on each sampling day. Multiple comparisons were corrected for using Bonferroni’s correction. In vitro data statistical analyses and graphs were generated using GraphPad Prism 6. Metabolomic graphs and statistical analyses (either unpaired t-test or repeated measures ANOVA) were prepared with GraphPad Prism 8.0 (GraphPad Software, Inc, La Jolla, CA) or MetaboAnalyst 4.0 (https://www.metaboanalyst.ca/). Spearman’s correlation analyses were performed to identify metabolic correlates to storage temperature, agitation, duration and functional assays. 50 3.3: Results 3.3.1: Platelet in vitro storage characteristics Figure 3-1: Changes in count and in vitro biochemical parameters of stored buffy coat platelet concentrates. RPs= room-temperature stored platelets, CPAs=cold-stored platelets with agitation, CPs=cold-stored platelets without agitation. * = statistical significance between RPs and CPAs. † = statistical significance between RPs and CPs. Significance level is indicated by the number of symbols. i.e: * = P<0.05; ** = P<0.01; *** = P<0.001; **** = P<0.0001. Data were compiled from 8 independent biological replicates (n=8). a: Platelet count of RPs, CPAs and CPs by Sysmex hematology analyzer. b: pH of stored platelets measured by blood gas analyzer. c: Platelet glucose concentration measured by blood gas analyzer. d: Platelet lactate concentration measured by blood gas analyzer. No significant difference between CPAs and CPs was observed in all the above parameters. 51 Table 3-1: Flow cytometry analysis of buffy-coat derived platelets stored in different conditions. Storage Condition Sample Timepoint P-selectin (%) P-selectin (%) + ADP(10µM) GP1bα (%) Lactadherin Binding (%) RPs Day 1 18±3.1 49±6.5 96±4.9 2±0.8 Day 2 19±4.1**** 39±5.6** 95±2.2 3±1.8 Day 4 28±4.6**** 29±4.6** 94±2.0 3±0.6 Day 7 41±4.6**** 18±4.8 92±2.1 6±3.6 Day 9 47±4.6** 14±4.0 91±1.9 7±1.6*** Day 11 56±4.7 9±3.3 90±2.8 9±2.2*** Day 14 65±6.5** 6±3.1 87±3.3* 14±3.8*** CPAs Day 1 18±3.1 49±6.5 96±4.9 2±0.8 Day 2 49±6.9 31±6.5 95±2.6 4±2.4 Day 4 59±6.3 21±6.6 94±2.5 5±2.3 Day 7 59±4.9 15±5.1 89±4.0 10±4.2 Day 9 55±4.0 12±5.0 87±2.9 14±4.3 Day 11 58±4.3 11±2.3 86±3.1‡‡ 17±4.0 Day 14 56±5.1 9±3.7 79±11.8 22±5.5 CPs Day 1 18±3.1 49±6.5 96±4.9 2±0.8 Day 2 49±6.8†††† 31±6.0†† 96±2.6 3±2.4 Day 4 60±5.6†††† 20±6.1†† 94±2.8 5±3.1 Day 7 60±3.8†††† 14±6.2 91±2.9 11±7.4† Day 9 54±7.1† 15±7.1 88±4.2 12±4.5† Day 11 53±13.6 9±4.2 77±18.4†††† 15±5.2†† Day 14 56±2.8†† 5±4.0 81±5.5 20±5.2†† RPs = room temperature with agitation; CPAs = 4°C with agitation; CPs = 4°C without agitation. On day 1, platelet units are tested immediately after production. P-selectin (%) = percentage of platelets expressing P-selectin measured by CD62P antibody. P-selectin (%) + ADP (10µM) = additional platelet P-selectin expression after stimulating with 10µM adenosine diphosphate (ADP). GP1bα (%) = percentage of GP1bα positive platelets measured by CD42b antibody. Lactadherin binding (%) = percentage of lactadherin positive platelets. Results are expressed as the mean of the percentage of positively labelled cells ± 1 standard deviation from a total of 8 biological repeats (n=8). Statistical analysis was conducted using 2-way ANOVA with Bonferroni correction on Graphpad Prism 6. * = statistical significance between RPs and CPAs. † = statistical significance between RPs and CPs. ‡ = 52 statistical significance between CPAs and CPs. Significance level is indicated by the number of each symbol. i.e: * = P<0.05; ** = P<0.01; *** = P<0.001; **** = P<0.0001. Figure 3-2: ROTEM analysis of stored buffy coat platelet concentrates. RPs= room-temperature stored platelets, CPAs=cold-stored platelets with agitation, CPs=cold-stored platelets without agitation. * = statistical significance between RPs and CPAs. † = statistical significance between RPs and CPs. ‡ = statistical significance between CPAs and CPs. Significance level is indicated by the number of each symbol. i.e: * = P<0.05; ** = P<0.01; *** = P<0.001; **** = P<0.0001. Data were compiled from 7 independent biological replicates (n = 7). ROTEM reactions were initiated with STARTEM and INTEM reagents. a: MCF = maximum clot firmness. b: A20= clot amplitude at 20 min. c: Alpha = alpha angle. 53 Figure 3-3: Aggregation responses of stored platelets stimulated with physiological agonists. RPs= room-temperature stored platelets, CPAs=cold-stored platelets with agitation, CPs=cold-stored platelets without agitation. Data were compiled from 7 independent biological replicates for a and b (n = 7); 4 independent biological replicates for c (n=4). * = statistical significance between RPs and CPAs. † = statistical significance between RPs and CPs platelets. Significance level is indicated by the number of each symbol. i.e: * = P<0.05; ** = P<0.01; *** = P<0.001; **** = P<0.0001. a: Area under the aggregation curve (AUC) of platelets stimulated with 10µM of adenosine diphosphate (ADP). b: Maximal aggregation response of platelet induced by 10µM of ADP c: Platelet aggregation responses when stimulated with 2µM epinephrine and 2.5µg/mL collagen. 54 Platelet count and biochemical parameters during storage are illustrated in Figure 3-1. There were no significant differences in platelet count among RPs, CPAs, or CPs for the first 7 days of storage. However, by day 9, CPAs and CPs had a significant decrease in platelet count compared to RPs (Figure 3-1a). There were no significant differences in count between CPAs and CPs. RPs had an increase in pH for the first 4 days of storage. After that, RPs pH steadily decreased until the end of storage. CPAs and CPs had slightly increased pH throughout 14 days of storage (Figure 3-1b). Figures 3-1c and 3-1d show the glucose and lactate concentrations of platelet units during storage, respectively. RPs had significantly faster glucose consumption and lactate production compared to CPs. No significant differences were observed between CPAs and CPs. Flow cytometry results are summarized in Table 1. Compared to RPs, both CPAs and CPs rapidly increased P-selectin expression at the beginning of storage reaching a peak on day 4 and staying relatively unchanged until day 14. RPs had a gradual increase of P-selectin expression. ADP (10 µM) was used to induce further P-selectin expression. RPs had a significantly better response to ADP compared to CPAs and CPs on days 2 and 4. However, this effect diminished as storage continued. The platelet surface receptor for von Willebrand factor GP1bα was also measured. On day 14 of storage, RPs had significantly higher GP1bα expression than CPAs. On day 11, CPAs had significantly higher GP1bα expression than CPs. Finally, lactadherin binding was assessed for stored platelets. Lactadherin has a high affinity for phosphatidylserine122. From day 9 to 14, RPs lactadherin binding was significantly less than CPAs and CPs. Platelet coagulation dynamics were assessed by ROTEM and the results are shown in Figure 3-2. The maximum clot firmness (MCF; Figure 3-2a) of CPAs and CPs was significantly lower than that of RPs from day 4 until the end of storage. There was no significant difference between MCF of CPAs and CPs. Similarly, the amplitude at 20 min (A20; Figure 3-2b) of RPs was higher 55 than CPAs and CPs after day 4. On day 2, RP A20 was also significantly higher than CPAs and CPs. Finally, no significant difference was observed among the alpha angles of stored platelets. The platelets’ ability to aggregate in response to physiological agonists was tested by light transmission aggregometry (Figure 3-3). In Figure 3-3a, platelets were activated by 10 µM of ADP. RPs stored for 7 days had an average aggregation area under the curve of 255 ± 196 which was significantly less than both CPAs (715 ± 522; P <0.05) and CPs (831 ± 424; P <0.01). No significant difference was observed between CPAs and CPs on any tested days. The maximal aggregation of stored platelets stimulated with 10 µM of ADP was assessed (Figure 3-3b). CPAs had significantly higher maximal aggregation than RPs on days 4, 9 and 11. On day 14, CPAs (71% ± 43) and CPs (70% ± 41%) both had significantly higher maximal aggregation than RPs (16% ± 12%) (P <0.01). Platelet response to a combination collagen (2.5 µM/mL final concentration) and epinephrine (2 µM final concentration) was also tested (Figure 3-3c). Prior to storage, platelets had 38 ± 6 sec of lag phase before aggregation initiation. Throughout storage, RPs retained a relatively constant lag phase. In contrast, past day 2, CPAs and CPs had minimal lag phase following agonist addition. 56 3.3.2: Metabolomic analysis of stored platelets Figure 3-4: The impact of storage condition on metabolomic phenotypes of platelets. RPs= room-temperature stored platelets, CPAs=cold-stored platelets with agitation, CPs=cold-stored platelets without agitation. Platelets were sampled for metabolomic analysis on days 1,2,7 and 14. A total 57 of 8 biological repeats were analyzed (n=8). Principal component analysis (a) of stored platelet metabolites over 14 days. b: heat map based on ANOVA’s best metabolites. These figures were generated using MetaboAnalyst 4.0 under “Time-series/Two-factor Analysis” mode with Interactive PCA Visualization (a) and Heatmap Visualization (b). 58 59 Figure 3-5: Changes of metabolites in platelets stored at different conditions. Platelets were sampled for metabolomic analysis on days 1,2,7 and 14. A total of 8 biological repeats were analyzed (n=8). Changes of metabolites involved in energy related metabolic pathways (glycolysis a and the citric acid cycle b). c: changes in metabolites of purine metabolism. Changes in metabolites 60 involved in amino acid methionine (d), arginine (e) and tryptophan (f) metabolism. Graphs were created with the help of Davide Stefanoni. Figure 3-6: Changes in amino acid level during platelet storage. 61 Platelets were sampled for metabolomic analysis on days 1,2,7 and 14. A total of 8 biological repeats were analyzed (n=8). a: amino acid levels of platelets stored at different conditions. b: proteolytic markers of platelets stored at different conditions. c: schematic of amino acid participation in metabolism. Graphs were created with the help of Davide Stefanoni. 62 Figure 3-7: Lipidomic analysis of stored platelets. MUFAs= monounsaturated fatty acids. PUFAs = polyunsaturated fatty acids. Platelets were sampled for metabolomic analysis on days 1,2,7 and 14. A total of 8 biological repeats were analyzed (n=8). a: the levels of acylcarnitines. b: the levels of saturated fatty acids. c: the levels of MUFAs and PUFAs. Graphs were created with the help of Davide Stefanoni. 63 Figure 3-8: Correlation between metabolites and in vitro platelet parameters. Spearman’s correlation analyses were performed to identify metabolic correlates. For platelet count, pH and P-selectin%, in vitro data from 8 biological replicates (n = 8) were used to perform correlation with metabolomic results. For maximal aggregation and maximum cloth firmness, in vitro data compiled from 7 biological replicates (n = 7) were used to perform correlation. Platelet in vitro functions (Maximal aggregation a) and maximum clot firmness b) were correlated with metabolite levels. Platelet in vitro characteristics (c-e. P-selectin expression percentage, pH and count, respectively) were correlated with metabolite levels. Graphs were created with the help of Davide Stefanoni. 64 3.3.2.1: Overview of the effect of storage temperature and time on the platelet metabolomic profile Multivariate analyses were performed to examine the pattern of metabolomic changes in buffy-coat derived platelets stored at different conditions. The result of principal component analysis is shown in Figure 3-4a. Principal Component 1 (PC1) explained 29.9% of total metabolomic sample variance. Looking at PC1, there was clustering of elements in the metabolomic profile based on platelet storage time as well as temperature. RPs stored for 14 days had a distinct metabolomic profile compared to the rest of the data. Furthermore, with respect to PC1, day 14 CPAs and CPs clustered closely with day 7 RPs. The PCA loadings plot for Figure 3-4a was provided in appendix B. Hierarchical clustering analysis showing the top significant metabolites by ANOVA is shown in Figure 3-4b. 3.3.2.2: Changes in metabolites of platelets stored under different conditions The relative levels of metabolites involved in glycolysis of stored platelets (Figure 3-5a) indicate that glycolysis metabolites were relatively stable for the first 7 days for all platelet groups. However, further storage significantly altered the metabolite profile, especially in RPs. RPs had a significantly lower level of glucose compared to CPAs and CPs on day 14. The level of glucose-6-phosphate decreased between day 7 and day 14 of storage in all three storage conditions. RPs had a lower level of 1,3-bisphosphoglycerate and higher 2/3-P-glycerate (isobaric isomers) than CPAs and CPs on day 14. RPs had a significant decrease in pentose phosphate during storage compared to CPAs and CPs. The change in metabolites involved in the tricarboxylic acid cycle (TCA) of stored platelets (Figure 3-5b) included higher levels of α-ketoglutarate and succinate in RPs compared to CPAs and CPs at each tested day. In addition, the glutamine level in RPs 65 dropped dramatically during storage. By day 14, glutamine was almost undetectable in RPs. In comparison, CPAs and CPs had much slower rates of glutamine consumption. The levels of malate did not significantly differ except for day 14 where the level in RPs increased. Purine metabolism of stored platelets is shown in Figure 3-5c. During storage, high energy phosphate compounds decreased similarly between storage conditions for the first 7 days. With extended storage to 14 days, RPs had lower levels of adenosine triphosphate (ATP), ADP and adenosine monophosphate (AMP) than CPAs and CPs. The level of hypoxanthine also steadily increased during storage for all three storage conditions. Interestingly, the increase in xanthine was significantly higher in RPs compared to CPAs and CPs during storage. The methionine pathway metabolites (Figure 3-5d) showed the level of methionine steadily increased in RPs while the level in CPAs and CPs remained relatively unchanged. There was also an increase in the level of methionine sulfoxide (Meth SOxide) in RPs. There was a decreased level of S‐adenosylhomocysteine (SAH) in RPs compared to CPAs and CPs on day 14. Metabolites involved in arginine metabolism and the urea cycle (Figure 3-5e) showed that RPs had higher levels of arginine and ornithine after day 7. The levels of arginine and ornithine in CPAs and CPs remained similar to day 2 stored platelets throughout the storage period. On day 14, RPs had a significant drop in citrulline level which was not observed in CPAs or CPs. In addition, the polyamine spermidine steadily increased throughout storage in RPs but not in CPAs or CPs. Differences in tryptophan metabolite levels (Figure 3-5f) were reflected in an increased tryptophan level in RPs as early as day 7 of storage. The tryptophan levels of CPAs and CPs remained relatively constant. Furthermore, there was also an increased level of kynurenine in RPs on day 14. 66 Storage temperature had a significant impact on platelet amino acid metabolism (Figure 3-6a). Overall, RPs had mostly increased amino acid levels during storage. CPAs and CPs amino acid level remained relatively stable. In addition, acetyl-methionine was only elevated in RPs but not in CPAs or CPs (Figure 3-6b). The schematic of amino acid participation in metabolism is illustrated in Figure 3-6c. The differences in fatty acid profile of platelets stored in different conditions are shown in Figure 3-7. The levels of fatty acids in CPAs and CPs remained relatively constant. In RPs, the level of free carnitine was reduced significantly over 14 days of storage (Figure 3-7a). Fatty acids are activated with carnitine for transportation into the mitochondria for β-oxidation. In RPs, there were reduced levels of acylcarnitines, such as C18:2, C14:0, and C10, suggesting a higher need for energy via β-oxidation. Furthermore, there were higher levels of both saturated and unsaturated fatty acids in RPs (Figure 3-7c). One notable exception was C4:0 which is small enough to diffuse into mitochondria without carnitine transport (Figure 3-7b). 3.3.2.3: Correlation of metabolites with platelet in vitro characteristics and function Regression analysis of platelet metabolites with platelet in vitro characteristics and functions was conducted. The top metabolites that positively correlated with platelet maximal aggregation measured by aggregometry are adenine, L-cystine, L-carnitine, ascorbate and alpha-D-ribose-1-phosphate. On the other hand, L-proline, L-arginine, L-tyrosine, cis-p-coumarate and guanine correlated negatively with maximum aggregation (Figure 3-8a). MCF measured by ROTEM highly correlated with glucose-6-phosphate, high energy phosphate metabolites (ATP, ADP and AMP) and the TCA intermediate succinate. ATP, ADP and AMP were all present at higher levels in CPAs and CPs than RPs in the metabolomic analysis. RPs had higher levels of succinate. Aspartate, bilirubin, hypoxanthine, butanoic acid, N-acetylneuraminate and 1-3-67 bisphosphoglycerate correlated negatively with MCF (Figure 3-8b). Platelet P-selectin expression correlated positively with purine pathway metabolites (xanthine and hypoxanthine), glycolysis metabolites (lactate and 2/3-phospho-D-glycerate), polyunsaturated fatty acids (icosatrienoic acid and eicosatetraenoic acid) and nicotinate ribonucleotide. Metabolites related to physiological energy and antioxidants correlated with lower P-selectin expression (Figure 3-8c). The top negative correlates are ATP and glucose. Platelet pH correlation with metabolites is shown in Figure 3-8d. Metabolites related to antioxidant pathways (alpha-D-ribose 1-phosphate and S-adenosyl-L-homocysteine), sugar molecules (D-glucose and D-ribose), 2-methyleneglutarate, 1-4-beta-D-xylan and butanoic acid correlated positively with platelet storage pH. Metabolites involved in energy metabolism (glycolysis, TCA cycle, and fatty acid β-oxidation) are correlated negatively with pH. The top negative correlates were choline and eicosatetraenoic acid. Platelet count (Figure 3-8e) correlated negatively with metabolites of the purine metabolism pathway (hypoxanthine and L-aspartate), glycolysis (D-glyceraldehyde 3-phosphate and 1-3-bisphosphoglycerate). High energy phosphate metabolites (AMP, guanosine diphosphate, ADP and ATP) and metabolites involved in oxidative balance (ascorbate and hexose 6-phosphate) correlated positively with platelet count. Correlation coefficients and P values for each metabolite are provided in appendix C. 68 3.4: Discussion In this study, we explored the in vitro characteristics and metabolomic phenotypes of agitated and non-agitated cold-stored platelets and how they differ from standard room-temperature stored platelets. We then correlated the metabolic changes with in vitro results. Whereas our study was conducted with buffy coat platelets in plasma, most studies of cold-stored platelets in the literature used apheresis platelets that were stored in plasma or in PAS. Our in vitro characterization results showed that CPAs and CPs have decreased count, reduced glucose consumption, lactate production and more stable pH than RPs. CPAs and CPs also had rapid increase in P-selectin expression and more lactadherin binding than RPs. These findings are similarly observed in both buffy coat and apheresis cold-stored platelets in PAS.76, 82, 123 The ROTEM findings (INTEM initiated) of clot dynamics were unanticipated. Our results showed RPs had better clot firmness than CPAs and CPs. This finding is not supported by the microscopic data of Nair et al..41 In addition, in Getz et al.’s ROTEM study, CPs have consistently higher or equal MCFs compared to RPs, although the reaction was initiated by EXTEM instead of INTEM.39 INTEM contains ellagic acid, which is an intrinsic coagulation pathway initiator. Further analysis into INTEM coagulation process may be required to understand this observation. Our aggregometry data suggest both CPAs and CPs had a better aggregation response than RPs. This result has been reported by others.37 In addition to the magnitude of aggregation, we saw a reduced lag phase when CPAs and CPs were treated with a combination of physiological agonists. This more rapid response could be beneficial for patients who need immediate coagulation to stop bleeding. Our metabolomic data show that compared to RPs, CPAs and CPs metabolite levels were relatively stable during storage especially in glycolysis and the TCA cycle. This is likely due to 69 the slower kinetics of enzymes as a function of lower storage temperature. For example, RPs had much lower 1,3-bisphosphoglycerate and higher 2/3-P-glycerate suggesting that RPs have higher energy demand than CPAs and CPs. Furthermore, RPs largely reduced pyruvate to lactate on days 7 and 14. This is probably required to replenish NAD+ to feed back into glycolysis. In RPs, there were higher levels of α-ketoglutarate and fumarate suggesting higher mitochondrial activity. High mitochondrial activity leads to ROS production which could be detrimental to platelet function and aggregation.40 RPs storage is associated with increased mitochondrial dysfunction124 and mitochondrial metabolism is key to platelet activation and hemostatic function.120 Having preserved mitochondrial metabolism due to lower storage temperature, CPAs and CPs may be more beneficial for treating active bleeding. The oxidative balance in platelets is complex and has been an area of hot debate. On the one hand, oxidative stress is a factor that contributes to the loss of platelet viability and function during storage.125 High oxidative stress has also been associated with platelet surface marker expression changes resulting in platelet dysfunction.126 ROS produced by platelets also inhibit platelet aggregation.127 On the other hand, superoxides can augment platelet aggregation.128 Nitric oxide is also an important player in inhibiting platelet aggregation and is derived from L-arginine metabolism.129 Our metabolomic data suggest that RPs actively manage oxidative stress by upregulating redox pathways such as the pentose phosphate pathway, the methionine metabolism pathway and the SAH homocysteine pathway. ROS can react with methionine to form methionine sulfoxide effectively scavenging methionine.130 SAH is the precursor for homocysteine, which can be further metabolized to generate reduced glutathione (GSH) for oxidative balance.131 In comparison, CPAs and CPs maintained relatively stable pools of GSH and pentose phosphate and had similar levels of arginine at the start of storage. Having a stable 70 pentose phosphate pool may also be advantageous for transfusion as it was recently shown that G6P and ribose phosphate are the top positive correlates to post-transfusion recovery and survival upon platelet transfusion.132 Relatively stable GSH levels in apheresis derived cold-stored platelets have also been shown in previous work.48 Different storage conditions also led to significant changes in stored platelet amino acid metabolism. Specifically, glutamine was extensively used as an energy source in RPs. This process generates ammonia as a byproduct and ammonia accumulates in stored RPs.133 This effect was significantly reduced in CPAs and CPs. In addition, there was also evidence of elevated protein lysis in RPs as demonstrated by a higher level of acetyl-methionine. A high arginine level in RPs also had an effect of polyamine production. Specifically, spermidine has an anti-platelet aggregation effect in a rabbit model.134 This could in part explain the storage lesion and loss of coagulability of RPs. Furthermore, there were higher levels of tryptophan and kynurenine in RPs. Kynurenine can pass through the blood-brain barrier and can be further degraded to kynurenic acid or quinolinic acid. These metabolites have neuroactive properties and can change brain activity.135 A high level of kynurenine in transfused RPs could have implications on the recipient’s neuroactivity and mood.136 While other groups have demonstrated characterization of CPs metabolism compared to RPs,48 in this study we provide new insight into the effect of agitation in cold storage as well as metabolites correlation to platelet functional parameters. We showed that agitation in the cold had minimal impact on both the in vitro platelet functions as well as metabolite levels compared to non-agitated cold storage. L-carnitine, which correlated positively with platelet maximal aggregation in our studies, has been shown to increase platelet aggregation in uremic patients,137 suggesting an important role for fatty acid mobilization and transport in the platelet aggregation 71 response. Other top positive correlates such as ascorbate and alpha-D-ribose-1-phosphate can interfere with oxidative species in platelets.138 Negative correlates such as proline and arginine are end products of glutamine catabolism suggesting an inverse relationship of purine metabolism with platelet aggregation.139 MCF, on the other hand, correlated positively with platelet metabolites involved in the pentose phosphate pathway (hexose-6-phosphate) and the TCA cycle and high energy nucleoside compounds. This suggests the importance of physiological energy for clot strength. Metabolites that are the result of energy breakdowns and purine catabolism correlated negatively with clot strength (hypoxanthine, N-acetylnueraminate). The platelet P-selectin expression associated with platelet degranulation140 correlated positively with metabolites of glycolysis, pyruvate fermentation and purine catabolism. This suggests energy stress or increased energy requirements for platelet degranulation. Metabolites important in oxidative stress balance correlated positively with platelet storage pH. pH is an important in vitro marker of platelet quality.141 Decreasing pH during storage is usually an indication of platelet storage lesion. The accelerated decrease in RPs pH could be related to oxidative stress and consumption of sugar molecules such as glucose. Finally, correlation of platelet count during storage with metabolites suggests that high energy phosphate compounds and antioxidant metabolites may be important for maintaining platelet count during storage, whereas energy consumption through glycolysis and purine catabolism are associated with decreased platelet count. One limitation of this study is not distinguishing whether the metabolites came from the platelet cytosol or mitochondrial compartment. Future targeted studies should be conducted to specifically investigate the metabolites in different compartments of stored platelets. Another limitation of this study originates from the fact that in vitro functional characterization of stored 72 platelets does not perfectly correlate with post-transfusion effectiveness. In future studies it will be desirable to measure the in vivo functions of transfused cold-stored platelets and correlate the results with metabolomic findings. 73 Chapter 4: The functions of cold-stored platelets assessed by the in vitro transfusion model 4.1: Introduction Platelets have been an indispensable part of transfusion medicine for thrombocytopenic patients since the 1970s.11-14 These patients are prophylactically transfused with platelets to prevent bleeding. Historically, prophylactic transfusions account for the majority of platelet transfusions.15 In the late 1960s, a study by Murphy S and Gardner FH showed room temperature storage of platelets maximized the post transfusion platelet survivability and circulation time.16 Since then, platelets have been stored at 22°C under constant agitation for 5 to 7 days depending on national regulations. Recently, there has been a significant rise in the number of therapeutic platelet transfusions due to increased demand for platelets to treat hemorrhage.20, 21 Hemorrhage is one of the leading preventable causes of death in trauma.22, 23 Bleeding patients receiving massive transfusion packages containing a 1:1:1 ratio of red blood cells, plasma and platelets had significantly reduced mortality in the Pragmatic Randomized Optimal Platelet and Plasma Ratios (PROPPR) study.24 However, there are concerns about whether RPs are as good for treating trauma patients as they are for prophylactic patients.25 CPs, which were the gold standard of platelet storage prior to the late 1970s, have been gaining renewed interest in treating hemorrhage due to several advantages over RPs. First, CPs have superior hemostatic function compared to RPs.27, 37, 41, 117, 142 It has been shown in mice that CPs are also quickly cleared from circulation by the hepatic Ashwell-Morrell receptors after transfusion.143, 144 Given that hemorrhaging patients likely do not have underlying coagulopathy, the superior hemostatic function and quick clearance of CPs may be beneficial. Second, the cold storage temperature significantly reduces most bacterial growth in 74 the platelet units.26 This potentially allows cold-stored platelets to have an extended shelf life, reducing the current platelet wastage due to outdate. Lastly, evidence suggests that CPs do not require mechanical agitations.28 This allows for improved logistics in platelet unit shipping and distribution and eliminates the cost of platelet mechanical agitators. These properties of cold-stored platelets have made them an attractive option to treat actively bleeding patients. While previous studies have investigated the in vitro characteristics and functions of CPs compared to RPs, few have compared their differences in treating actively bleeding patients in trauma. To understand the difference between using RPs versus CPs to treat actively bleeding patients, we designed an in vitro transfusion model mimicking the massive transfusion protocol. 4.2: Materials and methods 4.2.1: Blood collection, storage, and research ethics Blood products were provided by the Blood for Research Facility of the Canadian Blood Services in Vancouver, BC, Canada. Platelet concentrates used in this study were generated by the buffy coat method following the manufacturing protocol of the Canadian Blood Services. Briefly, whole blood was collected from healthy donors and left to rest overnight. The next day, platelets were produced from the whole blood and mixed with 100% plasma. There was no exclusion criterion on the blood groups of the platelet concentrates used in this study. Red cells concentrates were type O and stored in SAGM. The plasma was type AB+ and frozen at -30°C. Whole blood was drawn via venipuncture from donors into vacutainers (Becton, Dickinson and Company; Franklin Lakes, NJ) containing citrate anticoagulant. To our best knowledge, the blood products used in this study came from different donors in the Canadian Blood Service system. 75 Per biological replicate, two units of platelet concentrates were pooled and split into two identical units. One unit was stored at 22°C under constant agitation (RPs). The other unit was stored at 4°C in a walk-in fridge without agitation (CPs). Platelet units were placed into cold storage in less than 4 hours after production. Red cell concentrates were stored stationary at 4°C in a walk-in fridge. AB+ plasma was stored in a -30°C freezer. This study was approved by the UBC Research Ethics Board (H17-02708) and the Canadian Blood Services Research Ethics Board (2018.003b4r). 4.2.2: In vitro transfusion model setup On storage days 1, 7 and 14, RPs, CPs and red cell concentrates were sampled aseptically in a biological safety cabinet. AB+ plasma was thawed in a 37°C water bath. All blood components were allowed to reach room-temperature before mixing. On each sampling day, platelets were counted on a hematology analyzer (Sysmex Corporation, Kobe, Hyogo, Japan). To simulate transfusion packages used in Massive Transfusion Protocol, RPs and CPs mixed with equal volumes of thawed fresh frozen plasma and stored red blood cells to make the transfusion package (1:1:1 volume ratio). Transfusion packages made with RPs were labelled warm packages. Transfusion packages made with CPs were labelled cold packages. Whole blood from donors were modified to simulate hemodiluted or hyperfibrinolytic coagulation states. For hemodilution, whole blood was first counted on the hematology analyzer (Sysmex Corporation) to obtain the hematocrit. Then donor whole blood was diluted with 0.9% saline (Baxter, Mississauga, Ontario, Canada) to 20% hematocrit. For hyperfibrinolysis, a final concentration of 8.8 µg/mL of recombinant tissue plasminogen activator (Hyphen BioMed, Neuville-sur-Oise, France) was added. Then in vitro transfusion was performed by combining 70% of warm or cold transfusion package with 30% (by volume) of modified whole blood. A graphical depiction of 76 the in vitro transfusion model setup is shown in Figure 4-1. Figure 4-1a depicts the setup of the warm transfusion package and Figure 4-1b depicts the setup of the cold transfusion package. Figure 4-1: Schematics of in vitro transfusion model. a) Warm transfusion package was made by combining equal volumes of red cell concentrates (RCCs), plasma and room-temperature stored platelets (RPs). RPs and RCCs were sampled on days 1, 7 or 14 and combined plasma to make warm transfusion packages. Donor whole blood was either hemodiluted to 20% hematocrit (hemodilution) or mixed with recombinant tissue plasminogen activator to a final concentration of 8.8 µg/mL (hyperfibrinolysis). By volume, 70% of warm package was mixed with 30% of modified whole blood. The mixture was then analyzed on rotational thromboelastometry (ROTEM) with either INTEM or EXTEM coagulation initiators. b) Cold transfusion package set up was identical to the procedures described in a) other than the transfusion package was made from mixing cold-stored platelets (CPs) with RCCs and plasma on days 1, 7 or 14 instead of RPs. 77 4.2.3: Rotational thromboelastometry (ROTEM) ROTEM (Instrumentation Laboratory, Bedford, MA) was used to analyze the dynamic clot formation of the mixed transfusion products. Samples were re-calcified with STARTEM reagent, and coagulation was initiated by either INTEM (ellagic acid) or EXTEM (tissue factor) reagents (Instrumentation Laboratory). The reagents and samples were added via the automatic pipetting program on ROTEM while following on-screen instructions. ROTEM samples were run for 1 hour at 37°C. 4.2.4: Plasminogen activator inhibitor-1 (PAI-1) assay The supernatant of warm and cold packages prior to in vitro transfusions was collected and analyzed for PAI-1 activity. Briefly, the transfusion packages were made according to steps specified under the in vitro transfusion model setup section of the methods. The transfusion packages in 1.7mL Eppendorf tubes (Eppendorf, Hamburg, Germany) were centrifuged at 300 x g for 15 minutes at room temperature. The supernatant was collected and frozen at -70°C for PAI-1 analysis. To measure PAI-1 activity in the supernatant, the PAI-1 Activity Assay Kit (Abcam, Cambridge, UK) was used. The PAI-1 activity was assessed following the manufacturer’s protocol. 4.2.5: Statistical analysis Two-way ANOVA was used to compare the difference between trauma whole blood transfused with either warm or cold transfusion package. Results are shown as mean ± 1 standard deviation for all figures. The Bonferroni correction was used to correct for multiple comparisons. Statistical significance is denoted by the number of asterisks. * = P<0.05; ** = P<0.01; *** = P<0.001 and **** = P<0.0001. A total of 6 independent biological replicates were performed for the in vitro transfusion experiment with hemodiluted donor whole blood. A total of 8 biological 78 replicates were performed for the in vitro transfusion experiment with hyperfibrinolytic whole blood. A total of 7 independent biological replicates were performed for the transfusion package PAI-1 activity assay. A total of 18 independent biological replicates were performed for ROTEM analysis of hemodiluted whole blood without transfusion packages. A total of 6 independent biological replicates were performed for ROTEM analysis of hyperfibrinolytic whole blood without transfusion packages. Statistical analyses and graphs were generated on GraphPad Prism 9 (GraphPad Software, Inc, La Jolla, CA). The mean and standard deviation of platelet counts as well as whole blood without any in vitro transfusion packages were analyzed on Microsoft Excel (Microsoft Corporation, Redmond, WA). 4.3: Results Figure 4-2: ROTEM analysis activated with INTEM of hemodiluted donor whole blood (diluted with saline to 20% hematocrit) transfused with warm or cold package. 79 Warm package ( ); cold ( ) package. Compiled ROTEM parameters of the 6 biological replicates (N = 6) were shown in a) coagulation time b) clot formation time c) maximum clot firmness and d) alpha angle. Statistically significant differences between warm and cold package transfusions were denoted by asterisk (*). *** = P ≤ 0.001; **** = P ≤ 0.0001. Results were shown as mean ± 1 standard deviation. The solid horizontal line denoted the mean of hemodiluted whole blood without the addition of any transfusion packages. The two dotted lines showed the ±1 standard deviation from the solid horizontal line. A total of 18 vials of non-transfused hemodiluted whole blood were analyzed (N = 18). Figure 4-3: ROTEM analysis activated with EXTEM of hemodiluted donor whole blood (diluted with saline to 20% hematocrit) transfused with warm or cold package. Warm package ( ); cold ( ) package. Compiled ROTEM parameters of the 6 biological replicates (N = 6) were shown in a) coagulation time b) clot formation time c) maximum clot firmness and d) alpha angle. Statistically significant differences between warm and cold package transfusions were denoted by asterisk (*). * = P < 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001. Results were shown as mean ± 1 standard deviation. The solid horizontal line denoted the mean of hemodiluted whole blood without the addition of any transfusion packages. The two dotted lines showed the ±1 standard deviation from the solid horizontal line. A total of 18 vials of non-transfused hemodiluted whole blood were analyzed (N = 18). 80 Figure 4-4: ROTEM analysis activated with INTEM of hyperfibrinolytic donor whole blood (mixed with recombinant tissue plasminogen activator to a final concentration of 8.8 µg/mL) transfused with warm or cold package. Warm package ( ); cold ( ) package. Compiled ROTEM parameters of the 8 biological replicates (N = 8) were shown in a) coagulation time b) clot formation time c) maximum clot firmness and d) maximum lysis. Statistically significant differences between warm and cold package transfusions were denoted by asterisk (*). * = P < 0.05; ** = P ≤ 0.01; **** = P ≤ 0.0001. Results were shown as mean ± 1 standard deviation. The solid horizontal line denoted the mean of hyperfibrinolytic whole blood without the addition of any transfusion packages. The two dotted lines showed the ±1 standard deviation from the solid horizontal line. A total of 6 vials of hemodiluted whole blood were analyzed (N = 6). 81 Figure 4-5: ROTEM analysis activated with EXTEM of hyperfibrinolytic donor whole blood (mixed with recombinant tissue plasminogen activator to a final concentration of 8.8 µg/mL) transfused with warm or cold package. Warm package ( ); cold ( ) package. Compiled ROTEM parameters of the 8 biological replicates (N = 8) were shown in a) coagulation time b) clot formation time c) maximum clot firmness and d) maximum lysis. Statistically significant differences between warm and cold package transfusions were denoted by asterisk (*). * = P < 0.05; **** = P ≤ 0.0001. Results were shown as mean ± 1 standard deviation. The solid horizontal line denoted the mean of hyperfibrinolytic whole blood without the addition of any transfusion packages. The two dotted lines showed the ±1 standard deviation from the solid horizontal line. A total of 6 vials of hemodiluted whole blood were analyzed (N = 6). 82 Figure 4-6: The activity of plasminogen activator inhibitor 1 (PAI-1) in in vitro transfusion packages. PAI-1 activity per mL of warm ( ) and cold ( ) transfusion packages were measured by commercially available colorimetric assay kit. A total of 7 biological replicates (N = 7) were performed. Statistically significant differences between warm and cold package PAI-1 activity were denoted by asterisk (*). ** = P ≤ 0.01; *** = P ≤ 0.001. Results were shown as mean ± 1 standard deviation. The results of transfused hemodiluted whole blood initiated with INTEM are shown in Figure 4-2. There were no significant differences in the coagulation times of samples after either warm or cold package transfusions (Figure 4-2a). Transfusing cold package made from day 7 or day 14 CPs to hemodiluted whole blood resulted in significantly longer clot formation time compared to warm packages made from day 7 or day 14 RPs (Figure 4-2b). There were significant decreases of maximum clot firmness for hemodiluted whole blood transfused with day 7 and day 14 cold packages (Figure 4-2c). Finally, the alpha angle of hemodiluted whole blood after cold package 83 transfusion was significantly less than warm package transfused samples on day 7 and 14 (Figure 4-2d). The results of transfused hemodiluted whole blood initiated with EXTEM are shown in Figure 4-3. Similar to INTEM results, there were no significant differences between in the coagulation time of whole blood transfused with either package (Figure 4-3a). Hemodiluted whole-blood transfused with cold packages, however, resulted in significantly longer clot formation time compared to whole blood transfused with warm packages on both days 7 and 14 (Figure 4-3b). Unlike results initiated with INTEM, there were no significant differences in the maximum clot firmness of hemodiluted whole blood when transfused with either package (Figure 4-3c). Similar to INTEM, there were significant decreases in the alpha angels of hemodiluted whole blood transfused with cold packages compared to warm packages on both days 7 and 14 (Figure 4-3d). For Figures 2 and 3, the solid horizontal line in each graph denotes the mean and the dotted lines denotes ±1 standard deviation of hemodiluted whole blood without any transfusion packages tested with either INTEM or EXTEM. The results of transfused hyperfibrinolytic whole blood initiated using INTEM are shown in Figure 4-4. There were no significant differences in coagulation time and maximum clot firmness between samples transfused with warm or cold packages (Figure 4-4a and 4-4c). Whole blood transfused with cold packages resulted in significantly longer clot formation time on day 7 and day 14 samples compared to warm package transfused whole blood (Figure 4-4b). Finally, there was a significant reduction in the maximum lysis percentage in whole blood transfused with cold packages compared to whole blood transfused with warm packages on day 7 and day 14 (Figure 4-4d). EXTEM initiated results are shown in Figure 4-5. Similar to results in Figure 4, there were no significant differences in coagulation time and maximum clot firmness between warm and cold package transfused hyperfibrinolytic whole blood (Figure 4-5a and 4-5c). Cold 84 package transfused whole blood resulted in longer clot formation time on both day 7 and 14 (Figure 4-5b). The reduction of maximum lysis percentage was observed in hyperfibrinolytic whole blood transfused with day 14 cold package but not with warm package (Figure 4-5d). For Figures 4-4 and 4-5, the solid horizontal line in each graph denotes the mean and the dotted lines denotes ±1 standard deviation of hyperfibrinolytic whole blood without any transfusion packages tested with either INTEM or EXTEM. Additionally, the platelet concentrations of RPs and CPs during storage have been summarized in Table 1. After 14 days of storage, CPs had significantly lower platelet concentration than RPs. Table 4-1: Changes in platelet count throughout storage in room and cold temperature. RPs (×109 platelets / L) CPs (×109 platelets / L) Day 1 1012 ± 64 1012 ± 64 Day 7 953 ± 71 993 ± 68 Day 14 923 ± 99* 811 ± 69 Platelets were sampled aseptically in biosafety cabinet and counted on a hematology analyzer. RPs: room-temperature stored platelets; CPs: cold-stored platelets. A total of 8 biological replicates were performed. Results are expressed as the mean ± 1 standard deviation. * denotes significant differences (P < 0.05) between RPs and CPs count on day 14 of storage. Finally, to understand why transfusion packages made from cold-stored platelets significantly reduced the maximum lysis of whole blood, the PAI-1 activity of the transfusion packages was analyzed (Figure 4-6). In Figure 4-6, the PAI-1 activity in cold packages were significantly higher than that of warm package on both platelet storage day 7 and day 14. 85 4.4: Discussion In this study, we explored the effect of using CPs in an in vitro transfusion model which mimics the massive transfusion protocol for hemorrhaging patients. During a massive bleeding scenario, patients experience both physiological and iatrogenic hemodilution.145 In order to control bleeding and restore tissue perfusion, hemorrhaging patients are often aggressively resuscitated using crystalloid or colloid146. This fluid transfusion has significant impact on the hemostasis of patients as blood plasma proteins, enzymes and coagulation factors are effectively diluted.147, 148 In our model, we simulated this condition through hemodilution of normal donor whole blood with saline. When we transfused hemodiluted whole blood with transfusion packages, we noticed that cold package transfusion led to longer clot formation time and decreased alpha angle in both INTEM and EXTEM initiated blood clotting. This difference is somewhat surprising and contradictory to in vitro data in the literature where CPs were shown to have superior clot and aggregation compared to RPs.27, 41, 149 One possible explanation for this difference is that there were fewer active platelets in the cold packages compared to warm packages. CPs have been shown in this study (Table 1) as well as previous studies to have significantly decreased platelet count over the storage period.28, 37 The reduction of platelet count in CPs could be attributed to the higher chances of aggregation formation in cold-stored platelets prepared in 100% plasma. Our transfusion packages were made with 1:1:1 volume ratio of platelets, plasma, and red blood cells so there would be less platelets in the cold packages compared to warm packages. Another possible explanation is that platelets used in this study were produced by the buffy coat method and stored in 100% plasma, whereas other studies often used apheresis platelets stored in platelet additive solutions. The contributions of different manufacturing techniques and storage solutions to the quality of CPs should be considered in future studies. Despite these differences, we found 86 that in our study both warm and cold packages transfusion led to significant improvements of the ROTEM parameters compared to hemodiluted whole blood without any transfusion packages. In particular, this result was observed with transfusion packages made from platelets that have been stored for 7 and 14 days. In practice, RPs beyond 7 days of storage cannot be used for transfusion due to the risk of bacterial growth. CPs, on the other hand, have the potential to be stored for up to 20 days.150 Our results here demonstrated that day 14 stored CPs will likely be effective in massive transfusion protocol to improve patient blood hemostasis. Recent results from a small clinical trial in cardiothoracic surgery by Strandenes, G et al. further demonstrated the feasibility of extended storage CPs in trauma when patients administered with CPs stored for 14 days have similar median chest drain output as patients transfused with RPs stored for 7 days.101 Furthermore, during the COVID-19 pandemic, several blood centres have transitioned to cold storage of platelets to preserve platelet resources and reduce waste. At the Mayo Clinic in Rochester, Minnesota, CPs have been transfused to bleeding patients and adequately maintained their hemostasis.100 Our results from the hemodilution experiments provide further evidence that CPs could be considered for use in severely bleeding patients. Hyperfibrinolysis is a common coagulopathic condition that follows a massive bleeding episode.151, 152 With vascular injuries, the serine protease, tissue plasminogen activator (tPA) stored in endothelial cells is released and activates plasminogen into plasmin which then breaks down blood clots as part of the fibrinolysis process.153-155 Particularly in trauma patients, an increased level of tPA has been associated with poor clinical outcomes.156 In our in vitro transfusion model, we simulated the hyperfibrinolytic state by adding recombinant tPA to the donor whole blood. After transfusing with either day 7 or day 14 transfusion packages (both warm and cold), there was a significant improvement in the maximum clot firmness compared to 87 hyperfibrinolytic whole blood without any transfusion packages. This result further demonstrates that platelets stored in the cold for an extended period of 14 days could still be functional in the massive transfusion protocol. Similar to results observed with hemodiluted whole blood, cold package transfused hyperfibrinolytic whole blood had significantly longer clot formation time compared to whole blood transfused with warm packages. This could be attributed to the lower concentration of platelets between the warm and cold package as described previously. Interestingly in our model, cold package transfusions of hyperfibrinolytic whole blood resulted in decreased maximum lysis shown on ROTEM compared to warm package transfusion. This result was observed with both INTEM and EXTEM clot initiators. A previous study has shown that CPs have superior percent correction in bleeding time compared to RPs.27 Our findings here suggest that one possible mechanism is that CPs reduce the fibrinolysis induced by elevated level of tPA. Furthermore, plasminogen activator inhibitor 1 (PAI-1) is a potent inhibitor of tPA.157 Under normal hemostasis, PAI-1 tightly regulates tPA activities. Given platelets are one of the main storage pools of PAI-1, we hypothesized that the reduction of maximum lysis observed with cold package transfusion may be related to the PAI-1 level in the transfusion package. Indeed, our results showed that PAI-1 activity of cold package supernatant is significantly higher than that of warm packages on both day 7 and day 14 of storage. This suggests that cold storage of platelets may lead to more PAI-1 release. Increased PAI-1 levels in cold packages could be beneficial to actively bleeding patients as more PAI-1 is available to inhibit the increased tPA level in severely injured trauma patients.158 However, more research into the effect of increased PAI-1 in trauma patients is needed as several groups have reported that PAI-1 is associated with increased injury severity in traumatic brain injury patients.159, 160 88 As with any other in vitro models, our transfusion model has several limitations. First, the model does not take into account the intricate interactions between cells and vasculature of the endothelial system in humans. Second, we simulated the blood of trauma patients by modifying whole blood from healthy donors (through hemodilution or tPA addition). We acknowledge that there are differences between our modified whole blood compared to the blood from real trauma patients. In the future, our model can be improved if we could obtain blood from actual trauma patients. Lastly, although ROTEM analysis has been used to guide clinical transfusion decisions,161 it has its detection limits and it also does not take into account the sheer stress experienced by the blood cells during blood flow in the human vasculature. Microfluidic devices that accurately recapitulate the flow rate and shear stress of blood flow have been successfully used to study platelet function.162 In the future, we hope that these microfluidic devices will be used to complement our in vitro transfusion model and enable us to dig deeper into the application of cold-stored platelets in massively bleeding patients. In addition, there are other emerging blood products that could be beneficial to trauma patients. One such example is the low titer group O whole blood (LTOWB) which has been used to treat trauma patients in austere military settings where the availability of component therapy is lacking.163 In future studies, it will be interesting to compare the effectiveness of LTOWB to the 1:1:1 ratio of red cells, plasma and platelets (RPs or CPs) in our in vitro transfusion model. 89 Chapter 5: The role of the pentose phosphate pathway in platelet storage 5.1: Introduction During storage, platelets are subjected to significant oxidative stress.125 Oxidative stress has been linked to platelet quality deterioration during storage.164 One of the major pathways that platelets have to combat oxidative stress is the pentose phosphate pathway. A brief diagram of the pentose phosphate pathway is shown in Figure 5-1. The pentose phosphate pathway metabolizes glucose-6-phosphate to produce reduced nicotinamide adenine dinucleotide phosphate (NADPH) as well as ribose 5-phoshate.165 The NADPH is then used to generate reduced form of glutathione, which neutralizes hydrogen peroxides and reduces oxidative stress. It has been shown that RPs are more susceptible to oxidative stress during storage than CPs due to the breakdown of reduced glutathione.166 Furthermore, our own metabolomic analysis showed that reduced glutathione were consumed in both RPs and CPs.28 One of the key enzymes of the pentose phosphate pathway is glucose-6-phosphate dehydrogenase (G6PD), which converts glucose-6-phoshate into 6-phosphoglucono-δ-lactone while generating NADPH in the process. G6PD is also the rate-limiting enzyme of the pentose phosphate pathway.167 Clinically, G6PD activity has been positively correlated with tumor growth and development.168-170 While previous research found correlation between the pentose phosphate pathway metabolites and platelet storage quality, the exact role of the pentose phosphate pathway in platelet storage is not clear. In this study, we used G6PDi-1 and 6-Aminonicotinamide (6-AN), which are specific inhibitors of G6PD, in both RPs and CPs to investigate the effect of pentose phosphate pathway inhibition in platelet storage quality. 90 Figure 5-1: Overview of the pentose phosphate pathway The diagram was made with the help of Davide Stefanoni. 5.2: Materials and methods 5.2.1: Inhibitor preparation, platelet handling, and experimental setup The G6PD inhibitors G6PDi-1 (SKU: SML2980) and 6-AN (SKU: A68203) were purchased from Sigma-Aldrich (St.Louis, Missouri, USA). G6PDi-1 was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 57 mg/mL (200.46 mM). 6-AN was dissolved in DMSO at the concentration of 27 mg/mL (196.88 mM). Both inhibitors were aliquoted and frozen at -70°C. Per biological replicate, one buffy coat derived platelet concentrate was obtained from the Blood4Research facility in Vancouver, BC. To test for multiple storage and treatment conditions, NDTCs were used. NDTCs were filled with 12 mL of platelets plus 2 mL of air bubbles. Half of the NDTCs were mixed with final concentrations of 200 µM G6PD inhibitors (G6PDi-1 or 6-AN). The other half of NDTCs were mixed with DMSO in the same volume as the inhibitors. These NDTCs were labelled as carrier controls. NDTCs with inhibitors and carrier controls were 91 stored at both room temperature and cold temperature for up to 14 days. Platelets in the NDTCs were sampled on day 7 and 14 for in vitro platelet quality testing. Day 1 platelets were sampled from the platelet concentrate and analyzed prior to storage. An overview of the experimental setup is illustrated in Figure 5-2. Figure 5-2: Inhibition of G6PD in platelet storage using NDTCs experimental setup Non-destructive test containers (NDTCs) were filled with platelets and either G6PD inhibitors (G6PDi-1 or 6-aminonicotinamide) or same volume of dimethyl sulfoxide (Carrier control). For day 1 sampling, platelets were taken from the buffy coat platelet concentrate before filling the NDTCs and storage. 92 5.2.2: Platelet in vitro testing On storage day 7 and 14, platelets in the NDTCs were sampled in a biological safety cabinet into 15 mL conical tubes (Corning Inc., New York, USA). The platelets were counted using a hematology analyzer (XN-550, Sysmex Corporation) in the manual mode. Platelets were diluted to 100 × 109/L with AB+ plasma for ROTEM analysis using the INTEM initiator. Platelets were diluted to 300 × 109/L with AB+ plasma for EXTEM analysis. All samples were recalcified with STARTEM reagent. ROTEM samples were ran for 1 hour. For flow cytometry analysis of platelet surface activation marker expression, platelets were diluted to 200 × 109/L with PBS and stained with P) labelled CD62P (Beckman Coulter) or FITC labelled lactadherin (Haematologics Technologies, Essex Junction, VT). To test for the ability of platelets to further degranulate in response to physiological agonist, 10 µM final concentration of ADP was added to the diluted platelets prior to staining with PE labelled CD62P. One milliliter of phosphate-buffered saline was added to each sample prior to analysis on a FacsCanto II flow cytometer (BD BioSciences). Platelet aggregation profile was analyzed using light transmission aggregometer (Platelet Aggregation Profiler®, Model PAP-8E, BioData Corporation). Platelets were diluted to 250 × 109/L with AB+ plasma. Diluted platelets were added to glass vials containing a magnetic stir bar and incubated for 2 minutes at 37°C while stirring. The aggregation was induced by adding 20 µM of ADP. 5.2.3: Statistical analysis Results were shown as the mean of three independent biological replicates. Graphs were produced on Graphpad Prism 8 (San Diego, California, USA). 93 5.3: Results 5.3.1: G6PDi-1 in platelet storage Figure 5-3: Platelet count and blood gas analysis of stored platelets with G6PDi-1 inhibitor. Non-destructive test containers (NDTCs) were filled with platelets on day 1 and mixed with either G6PDi-1 or equivalent volumes of dimethyl sulfoxide. A final concentration of 200 µM of G6PDi-1 was used in RT-G6PDi-1 and 4C-G6PDi-1 NDTCs. Platelets were sampled for NDTCs on day 7 and 14. Day 1 data is collected before NDTCs filling (pre-storage). Results were shown as the mean of 3 independent biological replicates. 94 Figure 5-4: Surface marker expression and ROTEM function of stored platelets with G6PDi-1. Non-destructive test containers (NDTCs) were filled with platelets on day 1 and mixed with either G6PDi-1 or equivalent volumes of dimethyl sulfoxide. A final concentration of 200 µM of G6PDi-1 was used in RT-G6PDi-1 and 4C-G6PDi-1 NDTCs. Platelets were sampled for NDTCs on day 7 and 14. Day 1 data is collected before NDTCs filling (pre-storage). Flow cytometry was used to analyze the P-selectin and PS (phosphatidylserine) expression of stored platelets. Rotational thromboelastometry (ROTEM) was used to assess the maximum clot firmness (MCF) of stored platelets. Both INTEM (ellagic acid) and EXTEM (tissue factor) initiators were used in ROTEM. Results were shown as the mean of 3 independent biological replicates. 95 As shown in Figure 5-3, the addition of G6PDi-1 to stored platelets did not result in significant differences in the platelet count and pH. There seemed to be a storage temperature dependent difference on day 14 in the platelet count and pH but the results were not statistically significant. While there were significant differences shown in the glucose and lactate concentrations of the different platelet samples, there were no specific significant differences between stored platelets with the G6PDi-1 compared to the carrier control under the same storage temperature. The results of platelet surface marker expression assessed by flow cytometry and platelet clot dynamics measured by ROTEM are shown in Figure 5-4. Overall, the addition of G6PDi-1 did not result in significant differences in the P-selectin or the phosphatidylserine expressions of platelets stored at room temperature or cold temperature. The maximum clot firmness measured by ROTEM showed that there were significant differences between RPs and CPs on day 14 (INTEM initiated) and day 7 and 14 (EXTEM initiated). No significant differences were measured between platelets with G6PDi-1 versus carrier control within the same storage temperature. 96 5.3.2: 6-aminonicotinamide in platelet storage Figure 5-5:Platelet count and surface marker expression of stored platelets with 6-AN inhibitor. Non-destructive test containers (NDTCs) were filled with platelets on day 1 and mixed with either 6-aminonicotinamide (6-AN) or equivalent volumes of dimethyl sulfoxide. A final concentration of 200 µM of 6-AN was used in RT6AN and 4C6AN NDTCs. Platelets were sampled for NDTCs on day 7 and 14. Day 1 data is collected before NDTCs filling (pre-storage). ADP = adenosine diphosphate. Platelets count were measured by a hematology analyzer. P-selectin and phosphatidylserine expression of stored platelets were analyzed by flow cytometry. Results were shown as the mean of 3 independent biological replicates. 97 Figure 5-6: Aggregation profile of stored platelets with 6-AN. Non-destructive test containers (NDTCs) were filled with platelets on day 1 and mixed with either 6-aminonicotinamide (6-AN) or equivalent volumes of dimethyl sulfoxide. A final concentration of 200 µM of 6-AN was used in RT6AN and 4C6AN NDTCs. Platelets were sampled for NDTCs on day 7 and 14. Day 1 data is collected before NDTCs filling (pre-storage). Aggregation was initiated with a final concentration of 20 µM of adenosine diphosphate (ADP). Results were shown as the mean of 3 independent biological replicates. 98 Figure 5-7: Maximum clot firmness of stored platelets with 6-AN. Non-destructive test containers (NDTCs) were filled with platelets on day 1 and mixed with either 6-aminonicotinamide (6-AN) or equivalent volumes of dimethyl sulfoxide. A final concentration of 200 µM of 6-AN was used in RT6AN and 4C6AN NDTCs. Platelets were sampled for NDTCs on day 7 and 14. Day 1 data is collected before NDTCs filling (pre-storage). Platelets were re-calcified and then mixed with either Intem (ellagic acid) or Extem (tissue factor) to initiate coagulation. Results were shown as the mean of 3 independent biological replicates. 99 Figure 5-5 shows the count and surface marker expression levels of stored platelets with 6-AN. The addition of 6-AN did not result in significant differences in the platelet count of RPs or CPs. RPs with 6-AN seemed to have the highest P-selectin expression on day 14, however, there was no statistical significance when compared to the RPs mixed with DMSO. When 10 µM of ADP was added to platelets, RPs with 6-AN seemed to have the lowest increase in P-selectin expression in response to the ADP. This result, however, was not statistically significant compared to RPs with DMSO. Finally, the addition of 6-AN did not result in different phosphatidylserine expression level in RPs and CPs. The phosphatidylserine expression differences between RPs and CPs are likely due to storage temperature, instead of the effect of the inhibitor. Figure 5-6 shows the aggregation profile of stored platelets with 6-AN. Overall, the addition of 6-AN to RPs or CPs did not result in significant differences in the Area under the curve, maximum aggregation or final aggregation when activated with 20 µM of ADP. Figure 5-7 shows the MCF of stored platelets with 6-AN. In Intem activated samples, RPs had lower MCF compared to CPs on day 14. However, the addition of 6-AN did not result in significant differences between RPs or CPs with 6-AN compared to their respective controls. In Extem activated samples, RPs had lower MCF compared to CPs on both day 7 and day 14. However, similar to results shown in Intem, the addition of 6-AN to either RPs or CPs did not result in significant differences in MCF compared to the controls. 5.4: Discussion G6PDi-1 is a small molecule non-competitive inhibitor of G6PD. It has been shown to specifically inhibit the G6PD activity in a variety of cell lines including CD4+ and CD8+ T cells, as well as red blood cells.171 100 In this study, we explored the effect of G6PDi-1 in platelet storage. Overall, we did not observe any significant changes to platelet in vitro characteristics and functions with the addition of G6PDi-1 in either RPs or CPs. There could be several explanations on why G6PDi-1 did not have any effect. First, the G6PDi-1 concentration of 200 µM may not be enough in significantly inhibiting the G6PD activity in platelets. G6PDi-1 is a relatively new inhibitor and although its inhibitory effects of G6PD has been shown at a relatively low dosage of 10-50 µM in lymphocytes and cancer cell lines,171 no data on its inhibitory effect on platelets are currently available. Additionally, it is also possible that our panel of in vitro platelet assays does not accurately captures the changes in stored platelets with G6PDi-1. Under normal circumstances, the pentose phosphate pathway generates NADPH which is important in creating reduced glutathione to combat oxidative stress. However, NADPH also has an important function in fatty acid synthesis.172 Platelets contain a variety of lipids, including phospholipids, which correlate with platelet functions such as activation and aggregation.173 Platelets have been shown to synthesize lipids during storage and prime NADPH oxidase to produce superoxides as well.174 Therefore, the effect of G6PDi-1 in platelet storage may be best measured when examining the changes to the lipidomic of stored platelets. Similar to results of stored platelet with G6PDi-1, we did not see any significant differences in stored platelets with 6-AN. However, although not statistically significant, RPs with 6-AN appeared to have higher P-selectin expression and lower response to ADP on day 14. This could be the result of accumulating oxidative stress due to suppression of the pentose phosphate pathway. 101 This study has a few limitations which should be addressed in the future. First, both G6PDi-1 and 6-AN experiments had limited number of biological replicates (N = 3). Specifically in the 6-AN experiment, more biological replicates may be able to reveal the statistically significant differences between platelets stored with the inhibitor versus the control. Furthermore, the biological effect of DMSO, which is the solvent for both inhibitors, could be masking the effects of the inhibitors in our assay. DMSO has been shown to negatively affect platelet aggregation profile and function in vitro.175 The maximum percentage of DMSO used in our studies is 0.1%. Future studies should aim at further decreasing the concentration of DMSO used in the system. 102 Chapter 6: Conclusion For the last 50 years, RPs have been an integral part of transfusion medicine. The essential role of platelet inventory established by blood operators cannot be understated as countless patients rely on platelet transfusions for their health. However, as we learn more about platelets and their behaviours ex vivo, it is now understood that the current platelet methodology needs to be improved upon. As stated before, the current method of storing platelets at room temperature leads to significant product wastage, microorganism growth and deterioration of quality. None of these is optimal to platelet transfusion recipients. Furthermore, although RPs maximizes the post-transfusion platelet recoverability, their actual effectiveness in the transfusion recipients are often overlooked. To improve on the current platelet methodology, CPs have been proposed as an alternative product to RPs. The current paradigm in the field of platelet storage methodology is shifting from the single product (RPs) platelet inventory towards a dual inventory (RPs and CPs). It is currently thought that CPs will not be able to completely replace RPs as the optimal platelet survivability and reconvertibilities are still preferred in hematological patients with thrombocytopenia. However, for actively bleeding patients requiring immediate hemostatic effectiveness, CPs should be the better product for these patients. Based on our current understanding of platelet storage, this thesis set out to critically compare CPs with RPs. 6.1: Significance of this thesis in the field of platelet storage The results presented in Chapter 2 first illustrated that the current methods used to assess platelet in vitro characteristics are reliable and repeatable (instrument validation). More importantly, we found that buffy coat derived platelets, which are made from buffy coats of 4 different donors, can still have significant variabilities in their in vitro characteristics, such as platelet counts and surface marker expressions. To further produce reliable results, our data from Chapter 2 showed 103 that a pool and split model should be used to reduce the donor variabilities in studying platelet storage. These results serve as a good reminder that platelet products are not identical, and we must do our best to minimize product variability in research. This is especially important as most platelet research conducted in the United States use apheresis platelets, which have single donor origins. Furthermore, Chapter 2 also contains results on the validation of NDTCs (non-destructive test containers) in cold storage of platelets. We showed that platelets in NDTCs stored in the cold largely mimic the storage characteristics of CPs stored in standard platelet bags. NDTCs are a cost-effective system to test inhibitors, additives, or different storage conditions in studying platelet storage methodology. Here we add to our current understanding by providing a research tool that can be used by other investigators to study different platelet storage conditions and maximize their materials while minimizing the effect that storage containers have on the results. The in vitro characterization data shown in Chapter 3 largely complement the current understanding of RPs and CPs. These results further demonstrate that our assay methodologies are reliable. We significantly added to the understanding of platelet storage biology by conducting an untargeted metabolomic approach to stored platelets. In this method, we are moving beyond platelet in vitro quality assessment into understanding the biological mechanisms of RPs and CPs by studying metabolite changes. Several other groups have also studied the metabolomics of CPs.48, 118 However, our results combined the in vitro measurements of stored platelets with metabolomic results using correlations to pinpoint the relationship between the two. We significantly added to the understanding that metabolites associated with physiological energy and oxidative stress have a strong correlation with platelet functions, such as aggregation and clot firmness. Furthermore, by conducting the untargeted metabolomic analysis, our data 104 also revealed metabolic differences in a number of pathways in RPs and CPs. This experiment generated many potential hypotheses for future studies. In Chapter 4, we assessed the potential of using CPs compared to RPs in a Massive Transfusion Protocol. This was performed to investigate whether CPs provide additional benefits to actively bleeding patients and whether the in vitro superior hemostatic functions of CPs can translate to better functions in bleeding patients. Our model closely simulates the blood characteristics of an active bleeding patient (hemodiluted and hyperfibrinolytic). This study allows us to move beyond the basic in vitro characterization into CPs application. A recent pilot clinical trial showed that cardiac thoracic surgery patients who received day 14 stored CPs had similar clinical outcomes as those receiving day 7 RPs.101 Our data from Chapter 4 complements this result by demonstrating that day 14 CPs are just as good as day 7 RPs at restoring the hemostatic potential of hemodiluted and hyperfibrinolytic whole blood. Specifically, in the hyperfibrinolytic whole blood, CPs were shown to have superior resistance to clot lysis compared to RPs. This confirms the hypothesis that CPs may be a superior transfusion product to bleeding patients. Finally, in Chapter 5 we explored the role of the pentose phosphate pathway in platelet storage by targeting the enzyme G6PD. This is because our metabolomic results showed that there was a large consumption of reduced glutathione in RPs compared to CPs, demonstrating a high degree of oxidative stress in the RPs. In theory, inhibiting the recycling of reduced glutathione by inhibiting the pentose phosphate pathway will result in more oxidative stress in the stored platelets. However, we did not see any difference in the in vitro quality measurements of stored platelets with G6PD inhibitors. This result shows that there needs to be more optimization when studying the role of oxidative stress and metabolic pathways in platelet storage. Our current assays of platelets largely assess the platelet’s ability to utilize energy for functions such as 105 degranulation, aggregation and clot formation. However, in the case of pentose phosphate pathway, we need to change our focus onto platelet lipid synthesis and how the bioactive lipids in platelets contribute to overall platelet function. A great example in the literature can be seen when platelet factor 3 (PF3) a phospholipid, was seen to be lowered in patients with G6PD deficiency.176 The exact effect of PF3 deficiency is yet to be explored. 6.2: Future directions of cold-stored platelets 6.2.1: Cold-stored platelet production and storage methodologies As CPs gain increased research interest, it is important to keep in mind that as a collaborative research community, we should work together to eliminate as many confounding factors as possible in the preparation and storage of CPs. This will ensure the reliable and replicable results for the use of CPs. This issue is particularly challenging when considering different countries have different approved methods of platelet production. For example, in Canada, the only approved platelet products for transfusion are whole blood derived buffy coat platelets in 100% plasma. In the United States, on the other hand, platelets are made by the apheresis method and often stored in 65-70% of platelet additive solution. These two different platelet products have already been shown to have different in vitro characteristics and post transfusion efficacies in the room temperature storage condition. Different platelet products stored in the cold should be compared to better understand the effects of platelet production methods have on CPs. Furthermore, guidelines on the actual storage of platelets in the cold should also be investigated. Currently in Canada, there are no guidelines on the time between platelet production and the beginning of cold storage. Compared to RPs, there are also no guidelines that specify whether CPs should be agitated in the cold. Our in vitro evidence presented in this thesis suggested that 106 agitation did not result in significant differences in CPs. 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British Journal of Haematology. 1965;11(3):258-68. 120 Appendices Appendix A: Detailed method on sample processing, metabolite extraction, data processing, and metabolite assignment. Sample processing and experimental setups Whole blood was collected by Canadian Blood Services from donors providing signed informed consent. Buffy coat platelet concentrates suspended in plasma were produced by the Canadian Blood Services Blood4Research Facility in Vancouver, Canada. For each biological replicate, three buffy coat platelet units were pooled and split into three identical platelet units to minimize the effect of donor variability. One unit (RP) was stored at 22°C under constant agitation (Helmer Scientific, PC1200-Pro Platelet Incubator, Noblesville, IN). One unit (CPA) was stored in a walk-in fridge (4°C) in a platelet agitator (Forma platelet agitator, Thermo Electron Corporation. Waltham, MA). The last unit (CP) was stored in walk-in fridge (4°C) without agitation. Platelet units were sampled through a coupling port (Fresenius Kabi, Bad Homburg, Germany) using a sterile syringe in a biosafety cabinet. Platelet units were sampled on days 1, 2, 4, 7, 9, 11, and 14 of storage. This study was approved by both the University of British Columbia Ethics Committee (H17-02708) and the Canadian Blood Services Ethics Board (2018-003). Sample processing and metabolite extraction: A volume of 50μl of platelet concentrates were extracted at a 1:10 dilution in methanol:acetonitrile:water (5:3:2, v/v/v). After vortexing at 4°C for 30 min, extracts were separated from the protein pellet by centrifugation for 10 min at 10,000g at 4°C and stored at −80°C until analysis. For polar metabolites, half of the extracts were dried down via SpeedVac prior to resuspension in ddH2O + 0.1% formic acid, to facilitate ionization and chromatographic resolution of certain polar metabolites (e.g., betaine, kyurenine, etc.).(1, 2) 121 UHPLC-MS UHPLC-MS metabolomics analyses were performed as described,(1, 3) using a Vanquish UHPLC system coupled online to a high-resolution Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7 µm; Phenomenex, Torrance, CA, USA) at 45°C. A volume of 10 μl of sample extracts was injected into the UHPLC-MS. Each sample was injected and run with two different chromatographic and MS conditions, as previously described (1) and detailed as follows: Positive ion mode 1) using a 5 minute gradient at 450 µL/minute from 5-95% B (A: water/0.1% formic acid; B:acetonitrile/0.1% formic acid) and the MS was operated in positive mode and Negative ion mode 2) using a 5 minute gradient at 450 µL/minute from 5-95% B (A: 5% acetonitrile, 95%water/1 mM ammonium acetate; B:95%acetonitrile/5% water, 1 mM ammonium acetate) and the MS was operated in negative ion mode. The UHPLC system was coupled online with a Q Exactive (Thermo, San Jose, CA, USA) scanning in Full MS mode at 70,000 resolution in the 60-900 m/z range, 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in negative or positive ion mode (separate runs). Quality control and data processing: Calibration was performed prior to analysis using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Acquired data was then converted from .raw to .mzXML file format using Mass Matrix (Cleveland, OH, USA). Samples were analyzed in randomized order with a technical mixture (generated by mixing 5 μl of all samples tested in this study) injected every 10 runs to qualify instrument performance. This technical mixture was also injected three times per polarity mode and analyzed with the 122 parameters above, except CID fragmentation was included for unknown compound identification (10 ppm error for both positive and negative ion mode searches for intact mass, 50 ppm error tolerance for fragments in MS2 analyses – further details about the database searched below). Metabolite assignment and relative quantitation: Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ, USA), (4) against an in house library of deuterated lipid standards (SPLASH® LIPIDOMIX® Mass Spec Standard, Avanti Lipids) and in house libraries of 3,000 unlabeled (MSMLS, IROATech, Bolton, MA, USA; IroaTech ; product A2574 by ApexBio; standard compounds for central carbon and nitrogen pathways from SIGMA Aldrich, St Louis, MO, USA). References 1. Nemkov, T.; Reisz, J. A.; Gehrke, S.; Hansen, K. C.; D'Alessandro, A., High-Throughput Metabolomics: Isocratic and Gradient Mass Spectrometry-Based Methods. Methods Mol Biol 2019, 1978, 13-26. 2. Reisz, J. A.; Zheng, C.; D'Alessandro, A.; Nemkov, T., Untargeted and Semi-targeted Lipid Analysis of Biological Samples Using Mass Spectrometry-Based Metabolomics. Methods Mol Biol 2019, 1978, 121-135. 3. Thomas, T.; Stefanoni, D.; Reisz, J. A.; Nemkov, T.; Bertolone, L.; Francis, R. O.; Hudson, K. E.; Zimring, J. C.; Hansen, K. C.; Hod, E. A.; Spitalnik, S. L.; D'Alessandro, A., COVID-19 infection alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status. JCI Insight 2020, 5, (14). 4. Clasquin, M. F.; Melamud, E.; Rabinowitz, J. D., LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine. Curr Protoc Bioinformatics 2012, Chapter 14, Unit14 11. 123 Appendix B: PCA loadings plot with selective annotations for Figure 3-4a. 124 Appendix C: Correlation coefficient (r) and P value (P) of the Correlation analysis between in vitro parameters and metabolites. * = P<0.05; ** = P<0.01; *** = P<0.001; **** = P<0.0001 In vitro Parameters Positive Correlates Negative Correlates Metabolite r P Metabolite r P Maximal Aggregation Ascorbate 0.53 **** L-proline -0.64 **** L-Carnitine 0.50 **** L-arginine -0.59 **** L-cystine 0.43 **** L-tyrosine -0.57 **** Alpha-D-Ribose 1- phosphate 0.42 *** Cis-p-Coumarate -0.57 **** Adenine 0.41 *** Guanine -0.52 **** Maximum Clot Firmness Hexose 6-phosphate 0.60 **** Aspartate -0.68 **** Succinate 0.50 **** 1-3- Bisphosphoglycerate -0.60 **** ADP 0.49 **** N-Acetylneuraminate -0.58 **** Maltose 0.47 **** Bilirubin -0.53 **** ATP 0.46 **** Butanoic acid -0.53 **** Hypoxanthine -0.52 **** P-Selectin % Hypoxanthine 0.68 **** ADP -0.64 **** Icosatrienoic acid 0.62 **** ATP -0.62 **** 2/3-Phospho-D-glycerate 0.59 **** Gamma-L-Glutamyl-D- alanine -0.58 **** Lactate 0.59 **** GDP -0.56 **** Eicosatetraenoic acid 0.57 **** (5-L-Glutamyl)-L- glutamine -0.55 **** Nicotinate ribonucleotide 0.55 **** S-Adenosyl-L- homocysteine -0.55 **** Xanthine 0.53 **** D-Glucose -0.54 **** Hypotaurine -0.52 **** D-Ribose -0.52 **** Ascorbate -0.50 **** 125 In vitro Parameters Positive Correlates Negative Correlates Metabolite r P Metabolite r P pH Adenosine 0.79 **** Choline -0.69 **** Alpha-D-Ribose 1- phosphate 0.71 **** N-Succinyl-L-glutamate 5-semialdehyde -0.64 **** 2-Methyleneglutarate 0.61 **** Eicosatetraenoic acid -0.61 **** D-Glucose 0.58 **** UDP-N-acetyl-D- glucosamine -0.59 **** 1-4-beta-D-Xylan 0.56 **** D-Arabitol -0.58 **** D-Ribose 0.55 **** Malate -0.58 **** Butanoic acid 0.53 **** Lactate -0.57 **** S-Adenosyl-L-homocysteine 0.48 **** 2/3-Phospho-D- glycerate -0.57 **** Cytidine -0.57 **** L-lysine -0.56 **** Xanthine -0.56 **** Platelet Count Hexose 6-phosphate 0.81 **** Hypoxanthine -0.64 **** ATP 0.68 **** L-aspartate -0.62 **** ADP 0.66 **** Biliverdin -0.54 **** GDP 0.65 **** 1-3- Bisphosphoglycerate -0.54 **** AMP 0.64 **** D-Glyceraldehyde 3- phosphate -0.54 **** Ascorbate 0.60 ****
Thesis/Dissertation
eng
Pathology and Laboratory Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Devine, Dana V.
The evaluation of cold-stored platelets
Text