L’identification des gènes impliqués dans les thrombopénies apporte des éléments importants pour la compréhension des voies de régulation de la production et des fonctions des plaquettes voire de l’hématopoïèse. Notre laboratoire a développé une stratégie d’identification de gènes à l’origine de thrombopénies sans hypothèse a priori par séquençage d’exomes. Cette stratégie a permis de mettre en évidence des mutations touchant les gènes ETV6, ITGA2B et ITGB3.Les thrombopénies à volume plaquettaire normal sont particulièrement importantes à détecter en raison d’un risque d’évolution vers une pathologie onco-hématologique. L’origine génétique de cette catégorie de thrombopénie s’est pendant longtemps limitée aux mutations du gène runx1. Le travail que j’ai effectué au cours de ma thèse a contribué à impliquer le gène etv6 dans ce groupe de thrombopénies.Concernant le gène etv6 6 familles ont présenté des mutations pathogènes. Toutes ces mutations sont à l’origine d’une perte de l’activité répressive du gène et un nombre élevé de cellules CD34+ circulant dans le sang révélant le rôle d’ETV6 dans la prédisposition onco-hématologique. De plus la mégacaryopoïèse présente deux principales anomalies. Elles associent une augmentation du nombre de colonies progéniteurs mégacaryocytaires à une formation de proplaquettes réduites. Concernant les gènes itga2b et itgb3 3 familles ont été étudiées.Des défauts quantitatifs ou qualitatifs du récepteur αIIbβ3 conduisant à sa perte de fonction se retrouvent dans la thrombasthénie de Glanzmann (GT) caractérisée par une thrombopathie mais un nombre de plaquettes et une morphologie normale. / The identification of the genes involved in thrombocytopenia provides important elements for understanding the pathways of regulation of the production and functions of platelets or even hematopoiesis. Our laboratory has developed a strategy for identifying genes causing thrombocytopenia without a priori hypothesis by sequencing exomes. This strategy has been applied to families with autosomal dominant thrombocytopenia and has demonstrated mutations in the genes etv6, ​​itga2b and itgb3. Normal platelet thrombocytopenia are particularly important to detect because of the risk of developing onco-hematological pathology. The genetic origin of this category of thrombocytopenia has long been limited to mutations in the runx1 gene. More recently, mutations on the ankrd26 promoter have been reported. The work I did during my thesis helped to involve the etv6 gene in this group of thrombocytopenia. Concerning this gene six families have pathogenic mutations. All these mutations are the cause of a loss of the repressive activity of the gene and a high number of CD34+ cells circulating in the blood revealing the role of ETV6 in the onco-hematological predisposition. In addition, megakaryopoiesis has two main anomalies. They associate an increase in the number of megakaryocytic progenitor colonies with the formation of reduced proplatelets.Concerning the itga2b and itgb3 genes, 3 families were studied. These genes encode the αIIbβ3 integrin. Integrin αIIbβ3 is a platelet receptor for fibrinogen and Von Willebrand factor, and plays a crucial role in thrombosis and hemostasis.

Investigation of platelet adhesion, platelet activation, and platelet morphology was performed on Hexafluoroethane coated glass coverslips, Fluorinated Ethylene Propylene (FEP), and Polymethyl Methacrylate (PMMA) thin film polymers. Hexafluoroethane was coated on glass coverslips using Radio Frequency Glow Discharge (RFGD) polymer deposition and was supplied by researchers at the University of Washington in Seattle. For several years now, analogously prepared Fluoropolymer surfaces have been reported to exhibit unique protein adsorption properties and reduced adherent platelet concentrations compared to standard fluoropolymers and non-fluorinated polymers (Kiaei, 1995). Platelet interactions were assessed using a novel, dynamic, and physiologically relevant experimental system. In-vitro tests were performed on polymer surfaces mounted in a light transparent flow cell by pumping whole blood through the apparatus to deposit platelets over the polymer surface. Red blood cells were rinsed from the flow cell with plasma immediately after platelet deposition to permit observation of adherent cells. Dynamic, real time single cell morphology observation was made under low flowing plasma conditions using light microscopy and recorded using a computer image acquisition system. Physiologic conditions were maintained using a low plasma flow rate which ensured available nutrients for platelets as tests were performed for up to 90 minutes. Compared to PMMA and FEP, Hexafluoroethane surfaces exhibited the lowest platelet surface concentrations with overall mean adherent platelet concentrations of 8165, 6895, and 4387 platelets per mm², respectively. PMMA is a more activating surface compared to the fluoropolymers tested, however, the rate of progress of platelet activation and morphological trends are similar between the Hexafluoroethane and FEP polymer surfaces. Activation parameters, a quantification of the state of platelet activation, support experimental observations made concerning morphological change information on Hexafluoroetbane, FEP, and PMMA. A Scanning Electron Microscopy study involving all three test surfaces, fixed after 60 or 90 minute plasma flow maintained experiments, support the hypothesis that the pancake platelet evolves from a spreading platelet. Routinely observed, pancake platelets are circular, 3-5 μm in diameter, and have round raised protuberances at the periphery of the cell membrane. Images showing several stages of spread cell retraction on Fluoropolymers tested without added thrombin stimulant give greater detail of peripheral fragmentation and further support a mechanism for polymer surface induced platelet microparticle formation. / Thesis / Master of Engineering (ME)

Platelet-rich plasma (PRP) is a popular treatment for equine tendon and ligament injuries; however, commercial PRP systems are expensive. Development of a safe, inexpensive alternative would make PRP therapy more widely available to horse owners. The purpose of this study was to evaluate the quality and bacteriologic safety of PRP produced by three simple, inexpensive tube centrifugation methods and compare the results to a commercial system. Citrated blood collected from 26 normal horses was processed by four methods: blood collection tubes centrifuged at 1200 and 2000 x g, a 50ml conical tube, and a commercial system. Platelet and cell counts and mean platelet volume (MPV) in whole blood and PRP were determined using an automated hematology analyzer. Results were analyzed using mixed model ANOVA with post-hoc comparisons (MPV and fold change for RBC, WBC, and platelets) and binary logistic generalized estimating equations with horse as a blocking factor (absolute numbers of WBC, and platelets). Aerobic and anaerobic cultures were performed. Significance was set at p<0.05. Mean platelet concentrations ranged from 1.55 to 2.58 fold. The conical tube method produced the highest number of PRP samples with platelet concentrations of greater than 2.5-fold and within the clinically acceptable range of >250,000 platelets/?l. WBC counts were lowest using the commercial system and unacceptably high using the red top methods. The incidence of bacterial contamination was low (2.1%). Based on these results, the conical tube method may be a suitable alternative to commercial PRP systems in cases with budgetary constraints. / Master of Science

<p>Haematology instruments using optical and fluorescence techniques have improved the platelet count in domestic animals. There are still some difficulties present, especially when counting cat thrombocytes due to their ability to aggregate and the occurrence of large platelets.</p><p>The objective of this study was to evaluate and compare the platelet count, mean platelet volume and platelet crit in dogs, horses and cats on Advia 2120 and Sysmex XT-2000iV.</p><p>Fresh blood samples from 64 dogs, 40 horses and 39 cats with various medical conditions were analysed on both instruments. Manual blood smears of all feline samples were scrutiniously analysed to evaluate the aggregation warning flag from Advia.</p><p>There was good agreement between the instruments for the optical platelet count in dogs and cats. Slightly higher values were reported from Advia. Samples from horses presented poor correlations for all studied parameters. Platelet clumps appeared in 70% of the 37 scrutinized feline blood smears, while 46% of the samples generated aggregation warning flags from the Advia instrument.</p><p>Advia and Sysmex showed good agreement for platelet counts in blood from dogs and cats. Mean platelet volume and platelet crit need further evaluation before conclusions can be made concerning their clinical relevance. The sensitivity of the platelet aggregation warning flag from the Advia instument needs further elevation.</p>

Haematology instruments using optical and fluorescence techniques have improved the platelet count in domestic animals. There are still some difficulties present, especially when counting cat thrombocytes due to their ability to aggregate and the occurrence of large platelets. The objective of this study was to evaluate and compare the platelet count, mean platelet volume and platelet crit in dogs, horses and cats on Advia 2120 and Sysmex XT-2000iV. Fresh blood samples from 64 dogs, 40 horses and 39 cats with various medical conditions were analysed on both instruments. Manual blood smears of all feline samples were scrutiniously analysed to evaluate the aggregation warning flag from Advia. There was good agreement between the instruments for the optical platelet count in dogs and cats. Slightly higher values were reported from Advia. Samples from horses presented poor correlations for all studied parameters. Platelet clumps appeared in 70% of the 37 scrutinized feline blood smears, while 46% of the samples generated aggregation warning flags from the Advia instrument. Advia and Sysmex showed good agreement for platelet counts in blood from dogs and cats. Mean platelet volume and platelet crit need further evaluation before conclusions can be made concerning their clinical relevance. The sensitivity of the platelet aggregation warning flag from the Advia instument needs further elevation.

Platelet rich plasma (PRP) is a popular autologous biological therapy used for the treatment of various equine ailments, including tendon and ligament injuries, osteoarthritis, and cutaneous wounds. A number of commercial products are available for producing PRP, each generating a slightly different product. Variations in platelet numbers and white blood cell (WBC) counts are believed to be the most critical variables, as they are directly related to concentrations of growth factors and inflammatory cytokines. Accurate documentation of platelet numbers is essential for prospective evaluation of clinical outcomes, but can be problematic in platelet concentrates depending on the counting method employed. The objectives of this study were to compare the performance of four platelet counting technologies in equine platelet concentrates and to evaluate the ability of the Magellan PRP system to concentrate equine platelets. We hypothesized that there would be no differences in platelet counts among the four counting technologies and that the Magellan system would generate platelet concentrations greater than 500,000/μL. Citrated whole blood was collected from 32 horses and platelet, WBC, and red blood cell concentrations were measured using a commercial hematology analyzer (Advia 2120) prior to preparation of PRP using the Magellan system. Platelets were quantified in individual identical aliquots of equine PRP produced by the Magellan system (n=32) using three different technologies: optical scatter (Advia 2120), impedance (CellDyn 3700), and hand count using direct microscopy (Thrombo-TIC). An immunofluorescent counting method was performed on a subset of 15 of the 32 samples using a mouse monoclonal anti-sheep antibody against integrin alpha αIIbβ₃ (anti-CD41/CD61) and a fluorescent secondary antibody. Measured platelet concentrations were compared using Passing and Bablok regression analyses and mixed model ANOVA. The Magellan PRP system yielded mean (± SD) platelet and WBC counts of 893,090 ± 226,610/μL and 35,806 ± 9,971/μL, respectively. Platelet counts generated by optical scatter were consistently higher than those generated by impedance. Systematic and proportional biases were observed between these two automated methods. No bias (systematic or proportional) was observed among any of the other counting methods. Despite the bias detected between the two automated systems, there were no significant differences on average among the four counting methods evaluated, based on the ANOVA. All four platelet counting methods tested are therefore suitable for quantifying platelets in equine PRP for clinical applications. The Magellan PRP system consistently generated desirably high platelet concentrations as well as higher than expected WBC concentrations. The high platelet concentrations served as a good test medium for the study; however, the concurrent high WBC counts may be undesirable for selected orthopedic applications. / Master of Science

by Chu, Pui-ya. / Thesis (M.Phil.)--Chinese University of Hong Kong. / Bibliography: leaves 115-126. / ACKNOWLEDGEMENT / LIST OF ABBREVIATIONS / ABSTRACT --- p.1 / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- PLATELET-ACTIVATING FACTOR (PAF) --- p.4 / Chapter 1.1.1 --- Metabolism of PAF --- p.4 / Chapter 1.1.1.1 --- Biosynthesis of PAF --- p.4 / Chapter 1.1.1.2 --- Degradation of PAF --- p.9 / Chapter 1.1.2 --- Systemic effects of PAF --- p.12 / Chapter 1.1.3 --- Structure - activity relationship of PAF --- p.16 / Chapter 1.2 --- PAF RECEPTORS --- p.19 / Chapter 1.2.1 --- PAF receptor on platelets --- p.19 / Chapter 1.2.2 --- PAF receptor on leukocytes --- p.20 / Chapter 1.2.3 --- PAF receptor on macrophages --- p.20 / Chapter 1.2.4 --- Antagonists of PAF --- p.20 / Chapter 1.2.4.1 --- Nonspecific inhibitors of PAF --- p.21 / Chapter 1.2.4.2 --- Specific antagonists of PAF --- p.21 / Chapter 1.3 --- SECOND MESSENGER SYSTEMS OF PAF --- p.22 / Chapter 1.3.1 --- Adenylate cyclase - cyclic adenosine mono- phosphate system --- p.26 / Chapter 1.3.2 --- Phospholipase C - phosphatidylinositol system --- p.28 / Chapter 1.3.3 --- Intracellular calcium --- p.29 / Chapter 1.3.4 --- The role of prostaglandins and leukotrienes --- p.30 / Chapter 1.3.5 --- Protein kinase C --- p.30 / Chapter 1.3.5.1 --- Dual action of PKC --- p.31 / Chapter 1.3.5.2 --- Down regulation of PKC --- p.33 / Chapter 1.4 --- THE FUNCTION OF MACROPHAGES --- p.33 / Chapter 1.4.1 --- Phagocytosis --- p.34 / Chapter 1.4.2 --- Antigen presentation --- p.34 / Chapter 1.4.3 --- Release of cytokines --- p.34 / Chapter 1.4.4 --- Respiratory burst --- p.35 / Chapter CHAPTER 2 --- METHODS / Chapter 2.1 --- PREPARATION OF PERITONEAL MACROPHAGES --- p.37 / Chapter 2.1.1 --- Preparation of reagents --- p.37 / Chapter 2.1.2 --- Preparation of peritoneal macrophages --- p.37 / Chapter 2.2 --- RADIOLIGAND BINDING ASSAY --- p.38 / Chapter 2.2.1 --- Reagents --- p.38 / Chapter 2.2.2 --- [3H]-PAF binding to TG-PEC --- p.39 / Chapter 2.2.2.1 --- Preparation of thioglycollate-elicited peritoneal macrophages --- p.39 / Chapter 2.2.2.2 --- Preparation of working solutions --- p.39 / Chapter 2.2.2.3 --- Assay of [3H]-PAF binding --- p.40 / Chapter 2.2.3 --- [3H]-PAF binding to resident PEC --- p.40 / Chapter 2.2.3.1 --- Preparation of resident peritoneal mcrophages --- p.41 / Chapter 2.2.3.2 --- Preparation of working solutions --- p.41 / Chapter 2.2.3.3 --- Assay of [3H]-PAF binding --- p.41 / Chapter 2.2.4 --- Preparation of drugs --- p.41 / Chapter 2.2.5 --- Data analysis --- p.42 / Chapter 2.3 --- MEASUREMENT OF INOSITOL PHOSPHATES --- p.42 / Chapter 2.3.1 --- Preparation of reagents --- p.42 / Chapter 2.3.2 --- Dowex column preparation --- p.43 / Chapter 2.3.3 --- Cell plating in 24 - well plastic trays --- p.44 / Chapter 2.3.4 --- Determination of total inositol phosphates --- p.44 / Chapter 2.3.5 --- Column separation --- p.45 / Chapter 2.3.6 --- Determination of inositol trisphosphate (IP3) --- p.46 / Chapter 2.4 --- MEASUREMENT OF INOSITOL PHOSPHATES ACCUMULATION AFTER PROLONGED PMA PRETREATMENT --- p.46 / Chapter 2.4.1 --- Preparation of phorbol-12-myristate-13-acetate (PMA) solutions --- p.47 / Chapter 2.4.2 --- Preparation of cells with PMA --- p.47 / Chapter 2.5 --- DETERMINATION OF SUPEROXIDE ANION PRODUCTION --- p.47 / Chapter 2.5.1 --- Preparation of drugs --- p.47 / Chapter 2.5.2 --- Assay of superoxide anion production --- p.48 / Chapter 2.5.3 --- Data analysis --- p.48 / Chapter 2.6 --- STATISTICAL METHOD --- p.48 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- [3H]-PAF BINDING TO TG-PEC --- p.50 / Chapter 3.1.1 --- General properties --- p.50 / Chapter 3.1.2 --- Stereospecific of PAF receptor --- p.57 / Chapter 3.1.3 --- Comparison of [3H]-PAF binding to TG-PEC and TG-PMΦ from Balb/c mice --- p.57 / Chapter 3.1.4 --- Inhibition of specific binding of [3H]-PAF to murine and guinea pig TG-PEC by PAF --- p.57 / Chapter 3.1.5 --- Effects of PAF antagonists on the binding of [3H]- PAF to TG-PEC from Balb/c mice and guinea pigs --- p.60 / Chapter 3.1.6 --- Scatchard analysis of [3H]-PAF binding to murine and guinea pig TG-PMΦ --- p.65 / Chapter 3.2 --- SUPEROXIDE ANION PRODUCTION IN PERITONEAL MACROPHAGES --- p.69 / Chapter 3.2.1 --- Superoxide anion production induced by PAFin murine and guinea pig TG-PMΦ --- p.69 / Chapter 3.2.2 --- Effect of PMA on superoxide anion production in murine and guinea pig TG-PMΦ --- p.69 / Chapter 3.2.3 --- Effect of calcium ionophore on superoxide anion production in murine and guinea pig TG-PMΦ --- p.75 / Chapter 3.2.4 --- Effect of PAF antagonists on PAF - induced superoxide anion production in guinea pig TG-PMΦ --- p.75 / Chapter 3.3 --- PHOSPHOLIPASE C - PHOSPHATIDYLINOSITOL SYSTEM IN PERITONEAL MACROPHAGES --- p.79 / Chapter 3.3.1 --- Effect of PAF on the accumulation of inositol phosphates in the absence of LiCl --- p.79 / Chapter 3.3.2 --- Effect of PAF on the accumulation of inositol phosphates in the presence of LiCl --- p.79 / Chapter 3.3.2.1 --- Time course of [3H]-inositol phosphates accumulation induced by PAF in TG-PMΦ --- p.86 / Chapter 3.3.2.2 --- Effect of PAF on the accumulation of [3H]-IPs in TG- PMΦ from Balb/c mice and guinea pigs --- p.86 / Chapter 3.3.2.3 --- Effect of PAF antagonists on PAF-induced [3H]-IPs accumulation in TG-PMΦ from Balb/c mice and guinea pigs --- p.86 / Chapter 3.3.2.4 --- Effect of PMA on PAF-induced [3H]-IPs formation in TG-PMΦ from Balb/c mice and guinea pigs --- p.90 / Chapter 3.3.2.5 --- Effect of prolonged PMA pretreatment on PAF and PMA-induced [3H]-IPs accumulation in murine and guinea pig TG-PMΦ --- p.90 / Chapter 3.4 --- STUDIES OF RESIDENT PEC FROM Balb/c MICE --- p.96 / Chapter 3.4.1 --- Binding of 2nM [3H]-PAF to Balb/c mice resident PEC --- p.96 / Chapter 3.4.2 --- PAF-induced [3H]-IPs accumulation in murine resident PMΦ --- p.100 / Chapter 3.4.3 --- Binding of 0.2 nM [3H]-PAF to Balb/c mice resident PEC --- p.100 / Chapter 3.4.4 --- Superoxide anion production induced by PAF and PMA in murine resident PMΦ --- p.104 / Chapter CHAPTER 4 --- DISCUSSIONS / Chapter 4.1 --- PAF RECEPTOR ON PERITONEAL MACROPHAGES --- p.125 / Chapter 4.1.1 --- [3H]-PAF binding to peritoneal macrophages --- p.105 / Chapter 4.1.2 --- Expression of PAF receptor on Balb/c mice peritoneal macrophages --- p.107 / Chapter 4.2 --- SUPEROXIDE ANION PRODUCTION IN PERITONEAL MACROPHAGES --- p.108 / Chapter 4.3 --- PAF RECEPTOR AND POLYPHOSPHATIDYLINOSITOL SYSTEM IN PERITONEAL MACROPHAGES --- p.109 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.112 / REFERENCES --- p.115

Air pollution is increasingly recognised as an important and modifiable risk factor for cardiovascular disease. Exposure is associated with a range of adverse cardiovascular events including hospital admissions with angina and myocardial infarction, and with cardiovascular death. The main arbiter of these adverse health effects appears to be combustion-derived nanoparticles that incorporate reactive organic and transition metal components. Through the induction of cellular oxidative stress and pro-inflammatory pathways, these nanoparticles exert detrimental effects on platelets, vasculature and myocardium, and can augment the development and progression of atherosclerosis. Over the last 10 years there has been remarkable progress in the development of targeted engineered nanoparticles as contrast agents to enhance cellular and molecular imaging. Ultra-small paramagnetic iron oxide (USPIO) nanoparticles (<100 nm) produce disruptions in the magnetic field of magnetic resonance imaging (MRI) scanners, and a decrease in image intensity in areas where the particles accumulate. USPIO particles are phagocytosed by cells of the monocyte-macrophage system throughout the body including within atheromatous plaques. USPIOs have regulatory approval in the United Kingdom for imaging lymph nodes in breast and prostate cancer as well as FDA approval for parenteral iron-replacement therapy in chronic kidney disease. There is great interest in developing USPIO and other nanoparticle contrast agents for imaging atherosclerosis. The delivery of engineered nanoparticles (ENPs) directly into the bloodstream to provide enhanced imaging of the unstable atheromatous plaque may assist in the diagnosis of plaque rupture and may ultimately permit targeted delivery of therapies directly to the site of vascular injury. However, these particles once blood-borne may alter monocyte-macrophage function and activate circulating platelets with adverse effects on clinical outcomes. Previously it has been shown that inhalation of combustion-derived nanoparticles results in increases in platelet-monocyte aggregation and thrombus formation in healthy volunteers. These combustion derived nanoparticles share structural similarities with engineered nanoparticles designed for intravascular infusion. This raises an obvious paradoxical question: can engineered nanoparticles designed for medical use mediate similar effects to combustion derived nanoparticles in susceptible populations? My thesis addresses this question and describes a series of complimentary experimental and clinical studies to investigate the effects of engineered nanoparticles on platelet function and thrombogenesis using commercial and clinically available nanoparticles. I found that cationic nanoparticles caused platelet activation and aggregation in vitro, whereas, anionic nanoparticles caused inflammation and up-regulated adhesion molecule ICAM-1 in monocyte derived macrophages indicating that nanoparticles have different toxicological properties in different biological conditions. Using an ex vivo model of thrombus formation, the Badimon chamber, I observed that USPIO nanoparticles added to flowing native whole blood in an extra-corporeal circuit increased platelet rich thrombus formation under high shear conditions compared to saline control in healthy volunteers. These studies were repeated in patients with abdominal aortic aneurysms who received intra-venous systemic infusions of USPIO to enhance MRI imaging. I demonstrated up-regulation in markers of platelet activation and more platelet rich thrombus formation in the Badimon chamber one hour following systemic delivery of USPIO. In summary I have demonstrated that medical nanoparticles influence platelet activation in patients with cardiovascular disease and have pro-thrombotic effects in an ex-vivo model of in both healthy persons and susceptible patients. In light of this data and to ensure the safe future development of engineered nanoparticles for medical use platelet activation assays and follow-up monitoring of patients should be considered routine in both the developmental and clinical stages of engineered nanoparticle use.

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