Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction In the era of antiretroviral therapy (ART), people living with Human Immunodeficiency Virus (HIV) now have prolonged life spans. An emerging trend of non- acquired immune deficiency syndrome (AIDS) related complications now prevails in the aging HIV infected population. Increased levels of inflammation and chronic immune activation are associated with HIV infection. In the era of ART people living with HIV are at an increased risk of cardiovascular disease (CVD). Platelets play a pivotal role in both inflammation and immune activation and upon activation platelets degranulate and secrete various inflammatory, coagulatory and adhesion molecules. Activated platelets express surface P-selectin (CD62P) and are a key component of the coagulation pathway and serve as a link between inflammation and thrombosis. Activated platelets have been implicated in inflammatory and cardiovascular disease and have been identified as immune cells that play a crucial role in pathogen recognition and modulation of immune cells during infections. Several antiviral and antibacterial properties of platelet alpha granule contents have been established. Platelet aggregometry remains the most widely used technique to evaluate platelet function even though this technique is limited by many pre-analytical variables. Platelet flow cytometry on the other hand offers a rapid measurement of platelet function in their physiological environment with minimal artefactual activation. Few studies have however reported on standardized methods to evaluate platelet function in the context of HIV. Platelet function remains unclear and data on HIV infected treatment naïve individuals remains scarce. The aim of this project was to examine the relationship between platelet function and immune activation in patients with HIV Materials and methods This study consisted of five sub-studies, firstly platelet indices and levels of platelet activation were determined in a cohort of 330 participants (185 HIV infected ARV naïve and 145 uninfected healthy controls) using; flow cytometry and haemotology analyzers. The relationship between these indices and markers of platelet activation, disease progression and immune activation were assessed. Furthermore, levels of platelet activation and aggregation were evaluated in a cohort of 82 participants (41 HIV infected (ARV naïve) individuals and 41 uninfected healthy controls), using a novel whole blood flow cytometry based functional assay. These baseline levels were then correlated with markers of immune activation and disease progression in HIV. In a subsequent study, platelet function in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls was evaluated using flow cytometry. Platelet response was measured post stimulation with adenosine diphosphate (ADP) at concentrations known to induce reversible (0.04mM) and irreversible (0.2mM) platelet aggregation. In order to assess platelet function in HIV, platelet response was evaluated in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls. Platelets were activated using varying concentrations of ADP, arachidonic acid (AA) and collagen and platelet function was measured using flow cytometry. Levels of circulating platelet leukocyte aggregates (PLAs) were also measured using flow cytometry in a cohort consisting of 35 HIV-infected (ARV naïve) individuals and 32 uninfected healthy controls. Associations between PLAs, immune activation and disease progression in HIV infected individuals were determined. The final study evaluated platelet aggregates, platelet derived microparticles (PMPs) and microparticles (MPs) in a cohort consisting of 46 HIV infected (ARV-naïve) and 40 uninfected healthy controls. Associations between MPs, PMPs, platelet aggregates and markers of immune activation and disease progression were evaluated. Results HIV infected individuals showed decreased mean platelet volume levels (HIV mean 7.91 ± 0.85 vs. 8.52 ± 1.12, p<0.0001) that directly correlated with CD4 counts (r=-0.2898, p=0.0075) and viral load (r=0.2680, p=0.0177). Platelet distribution width (PDW) levels directly correlated (r=0.3455, p=0.0362) with active coagulation and inversely correlated (r=-0.3666, p=0.0463) with platelet aggregation. HIV infected individuals showed increased levels of platelet activation (%CD62P median 11.33[5.96-29.36] vs. control group 2.48[1.56-6.04], p=0.0001). In HIV, platelet function is retained and platelets showed increased response to submaximal concentrations of endogenous agonists. HIV infected individuals showed increased levels of circulating platelet monocyte aggregates (25.26[16.16-32.28] vs. control group 14.12[8.36- 18.83], p=0.0001) that directly correlated with markers of immune activation; %CD38/8 (r=0.54624, p=0.0155), viral load (r=0.633, p<0.009). Furthermore we report on increased levels of circulating MPs (median %MPs 1.7[0.95-2.83] vs. Control group 1.12[0.63-1.57], p=0.0160); PMPs (median %PMPs 26.64[11.33-36.62] vs. Control group 20.02[18.08-26.08], p=0.0133); activated PMPs (median CD62P MFI 3.81[3.46-4.54] vs. Control group 3.41[3.16-3.6], p=0.0037) and platelet aggregates (Median %CD62P 14.10[5.49-39.94] vs. Control group 0.17[0.10-10.99], p= 0.0097) in HIV infected asymptomatic individuals. Conclusion This study supports the potential use of the MPV and PDW as readily available markers of platelet activation and immune activation in HIV. We also showed elevated levels of activated platelets in HIV infected individuals that were hyper responsive to endogenous agonists in a concentration dependent manner. Platelet flow cytometry is a rapid and valuable technique in the evaluation of platelet function in HIV. The measurement of platelet function using flow cytometry allows the evaluation of platelet signalling pathways that may be modified in HIV infected individuals. Lastly we describe an optimized whole blood flow cytometry based assay for the evaluation of circulating microparticles (MPs), platelet derived microparticles (PMPs) and levels of activated platelets and aggregates which mimics the in vivo physiological environment of MPs. To the best of our knowledge, this study is the first to report on a novel approach in evaluating platelet function in HIV using a series of optimised whole blood flow cytometry based platelet assays. In addition, minimal work has been performed previously on platelet function in the context of HIV-infection; and particularly in a cohort of asymptomatic, untreated patients as defined for this study. / AFRIKAANSE OPSOMMING: Inleiding In die era van antiretrovirale terapie (ART), het mense wat met die menslike immuniteitsgebreksvirus (MIV) leef, het nou 'n verlengde lewensduur. 'N opkomende neiging van nie-verworwe immuniteitsgebreksindroom (vigs) heers nou in die verouderende MIV-besmette bevolking. Verhoogde vlakke van inflammasie en chroniese immuun aktivering word geassosieer met MIV-infeksie en in die era van ART loop mense wat met MIV leef, 'n verhoogde risiko van kardiovaskulêre siekte (KVS). Plaatjies speel 'n belangrike rol in beide inflammasie en immuun aktivering en met aktivering degranulate en skei plaatjies verskeie inflammatoriese, coagulatory en adhesie molecule af. Geaktiveerde plaatjies druk oppervlak P-selectin (CD62P) is 'n belangrike komponent van die stollings weg en dien as 'n skakel tussen inflammasie en trombose. Geaktiveerde plaatjies is in beide inflammasie en kardiovaskulêre siekte betrokke en is geïdentifiseer as immuun selle wat 'n deurslaggewende rol speel in die patogeen erkenning en modulasie van immuun selle tydens infeksies. Verskeie antivirale en antibakteriese eienskappe van plaatjie alpha korrel inhoud is vasgestel. Plaatjie aggregometry bly die mees gebruikte tegniek om plaatjie funksie te evalueer, alhoewel hierdie tegniek is beperk deur baie pre-analitiese veranderlikes. Plaatjie vloeisitometrie aan die ander kant bied 'n vinnige meting van plaatjie funksie in hul fisiologiese omgewing met 'n minimale artefactual aktivering. Min studies het egter berig op gestandaardiseerde metodes om plaatjie funksie in die konteks van MIV te evalueer. Plaatjie funksie is steeds onduidelik en data oor MIV besmet behandeling naïef individue bly skaars. Die doel van hierdie projek was om die verhouding tussen die plaatjie funksie en immuun aktivering in pasiënte met MIV te ondersoek. Materiaal en metodes Hierdie studie het bestaan uit vyf sub-studies. In die eerste plekis plaatjie indekse en vlakke van plaatjie aktivering bepaal in 'n groep van 330 deelnemers (185 MIV-besmette ARV naïef en 145 onbesmette gesonde kontrole) met behulp van vloeisitometrie en hematologie ontleders. Die verhouding tussen hierdie indekse en merkers van plaatjie aktivering, die siekte se progressive en immuun aktivering is beoordeel. Verder is die vlakke van plaatjie aktivering en samevoeging in 'n groep van 82 deelnemers (41 MIV-besmette (ARV naïef) individue en 41 onbesmette gesonde kontrole) geëvalueer, met behulp van 'n nuwe vol bloed vloeisitometrie gebaseerde funksionele toets. Hierdie basislyn vlakke is dan gekorreleer met merkers van immuun aktivering en die progreessie van die siekte in MIV. In 'n daaropvolgende studie, is plaatjie funksie in 'n groep wat bestaan uit 58 MIV besmet te (ARV naïef) en 38 onbesmette beheer geëvalueer met behulp van vloeisitometrie. Plaatjie reaksie is na stimulasie gemeet met adenosine diphophate (ADP) by konsentrasies bekend omkeer (0.04mM) te oorreed en onomkeerbaar (0.2mm) plaatjie aggregasie. Ten einde plaatjie funksie in MIV te evalueer, is plaatjie reaksie in 'n groep wat bestaan uit 58 MIV-besmette (ARV naïef) en 38 onbesmette kontrole geëvalueer. Die plaatjies is geaktiveer deur gebruik te maak van wisselende konsentrasies van ADP, is aragidoonsuur (AA) en kollageen en plaatjie funksie gemeet met behulp van vloeisitometrie. Vlakke van sirkulerende plaatjie leukosiet gemiddeldes is ook gemeet met behulp van vloeisitometrie in 'n groep wat bestaan uit 35 MIV-positiewe (ARV naïef) individue en 32 onbesmette gesonde kontrole. Assosiasies tussen leukosiet gemiddeldes, immuun aktivering en die progressive van ie siekte in MIV-besmette individue is ook bepaal. Die finale studie het plaatjie-gemiddeldes, plaatjie afgelei mikrodeeltjies en mikrodeeltjies geëvalueer in 'n groep wat bestaan uit 46 MIV besmet (ARV-naïewe) en 40 onbesmette gesonde kontrole. Assosiasies tussen mikrodeeltjies, plaatjie afgelei, plaatjie gemiddeldes en merkers van immuun aktivering en die siekte se progressie is geëvalueer. Resultate MIV-besmette individue het gedaalde gemiddelde plaatjie volume vlakke getoon (HIV gemiddelde 7,91 ± 0,85 8,52 ± 1,12 teen, p <0,0001) wat direk gekorreleer het met CD4-tellings (r = -0,2898, p = 0,0075) en virale (r = 0,2680, p = 0,0177). Plaatjie verspreiding breedte vlakke het direk gekorreleer met (r = 0,3455, p = 0,0362) met 'n aktiewe koagulasie en omgekeerd gekorreleer (r = -0,3666, p = 0,0463) met plaatjie aggregasie. MIV-besmette individue het verhoogde vlakke van plaatjie aktivering getoon (% CD62P mediaan 11,33 [5,96-29,36] teen kontrole groep 2,48 [1,56-6,04], p = 0,0001). In MIV, was plaatjie funksie behou en plaatjies het 'n verhoogde reaksie op submaksimale konsentrasies van endogene agoniste getoon. MIVbesmette individue het verhoogde vlakke van sirkuleer plaatjie monosiet-gemiddeldes gedemonstreer (25.26 [16,16-32,28] teen kontrole groep 14,12 [8,36-18,83], p = 0,0001) wat direk gekorreleer het met merkers van immuun aktivering; % CD38 / 8 (r = 0,54624, p = 0,0155), virale lading (r = 0,633, p <0,009). Verder rapporteer ons op verhoogde vlakke van sirkulerende mikrodeeltjies (mediaan% LP 1.7 [0,95-2,83] teen kontrole groep 1,12 [0,63-1,57], p = 0,0160); PMPs (mediaan% PMPs 26,64 [11,33-36,62] teen kontrole groep 20,02 [18,08-26,08], p = 0,0133); geaktiveer PMPs (mediaan CD62P MFI 3,81 [3,46-4,54] teen kontrole groep 3,41 [3,16- 3,6], p = 0,0037) en plaatjie gemiddeldes (Mediaan% CD62P 14,10 [5,49-39,94] teen 0.17 [0,10- 10,99], p= 0.0097) in MIV besmet asimptomatiese individue. Gevolgtrekking Hierdie studie ondersteun die potensiële gebruik van die MPV en PDW as waardevolle geredelik waardevolle merkers van plaatjie aktivering en immuun aktivering in MIV. Ons het ook getoon verhoogde vlakke van geaktiveer de plaatjies in MIV-besmette individue getoon wat hyper reageer op endogene agoniste was in 'n konsentrasie-afhanklike wyse. Plaatjie vloeisitometrie is 'n vinnige en waardevolle tegniek in die evaluering van plaatjie funksie in MIV. Die meting van plaatjie funksie gebruik vloei cytometry maak die evaluering van plaatjie sein paaie wat in MIVgeïnfekteerde individue verander moontlik. Laastens het ons beskryf 'n hele bloed vloeisitometrie gebaseer de toets vir die evaluering van sirkulerende mikrodeeltjies, plaatjie afgelei mikrodeeltjies en vlakke van geaktiveer plaatjies en gemiddeldes wat lyk soos die in vivo fisiologiese omgewing van MP's. Na die beste van ons kennis, is hierdie studie die eerste om te rapporteer oor 'n nuwe benadering in die evaluering van plaatjie funksie in MIV met behulp van 'n reeks van new hele bloed vloeisitometrie gebaseer de plaatjie toetse. Daarbenewens is minimale werk voorheen uitgevoer op die plaatjie funksie in die konteks van MIV-infeksie; en veral in 'n groep van asimptomatiese, onbehandelde pasiënte soos vir hierdie studie. Hierdie projek het bewyse bygevoeg tot die teorie dat plaatjies, in MIV, kan 'n skakel wees tussen die aktiewe inflammatoriese reaksie en die toename in die aantal trombotische en kardiovaskulêre siekte waargeneem in pasiënte wat met hierdie siekte saamleef.

Background: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. Methods: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. Results: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. Conclusion: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders. / Hintergrund: Die Vitalitätsbestimmung von Blutplättchen ist sowohl für die Analyse angeborener Plättchendefekte als auch für die Qualitätsbestimmung von Plättchenkonzentraten von zentraler Bedeutung. Methoden: In der vorliegenden Arbeit stellen wir eine Methode vor, die mittels einer Kombination von Vitalfarbstoffen und konfokaler «Real time»-Mikroskopie neue Einblicke in die Vitalitätsbestimmung lebender Plättchen ermöglicht. Mittels der Zugabe von FITC-gekoppeltem Weizenkeimlektin (WGA), Tetramethylrhodamin-Methylesterperchlorat (TMRM) und Acetoxymethylester (Rhod-2) wurde bei lebenden Blutplättchen deren Morphologie, mitochondriale Aktivität und Veränderungen im Calcium-Haushalt im Rahmen der Lagerung analysiert. Für die Mikroskopie wurde ein Nipkow-System gewählt, das eine konfokale Mikroskopie lebender Zellen ermöglicht. Ergebnisse: Der Vergleich von 10 humanen Blutplättchenproben zu Beginn bzw. nach 5 und 7 Tagen Lagerung zeigte einen Anstieg der Rhod-2-positiven Plättchen von 3,6 über 47 auf 71%. Die Anzahl der Blutplättchen mit TMRM-positiven Mitochondrien hingegen lag vor der Lagerung bei 95,4% und nach den 7 Tagen Lagerung bei 92,5%. Schlussfolgerung: Die hier vorgestellte Methodik der Bildgebung zur Bestimmung vitaler Parameter von Blutplättchen eignet sich als ergänzende Analysemodalität für eine bessere Bestimmung der Blutplättchenqualität. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

A fosfolipase A2 (PLA2) é uma enzima chave no metabolismo dos fosfolípides de membrana e é um dos principais componentes envolvidos na sinalização celular. Alterações da atividade da PLA2 tem sido descritas no cérebro e no sangue (soro, plasma e plaquetas) de pacientes com diversas doenças neuropsiquiátricas. Neste estudo foi desenvolvido um ensaio radioenzimático para detectar em plaquetas, a atividade dos três principais grupos de PLA2, que são PLA2 secretórias ou PLA2 extracelular dependente de Ca 2+ (sPLA2); PLA2 citósólicas dependentes de Ca 2+ (cPLA2) e as PLA2 intracelulares independentes de Ca 2+ (iPLA2). Para confirmar a presença desses grupos da enzima em plaquetas, algumas variáveis foram testadas, como as diferenças de preferência ao ácido graxo como substrato, o requerimento de cálcio e a inibição seletiva com os inibidores Bromoenol lactone (BEL) e o Methyl Arachidonyl Fluorophosphonate (MAFP). Os resultados obtidos demonstram a presença dos três principais grupos de PLA2 (sPLA2, cPLA2, and iPLA2) em plaquetas. Estes achados sugerem o uso de plaquetas, uma amostra biológica de fácil acesso, como possível modelo periférico de neurônios para o estudo do metabolismo de fosfolípides. / Phospholipase A2 (PLA2) is a key-enzyme in the metabolism of membrane phospholipids and is one of the major components involved in cell signaling. Alterations of PLA2 activity have been reported in brains and blood cells in several neuropsychiatric diseases. In this study we developed a radio-enzymatic assay to detect in platelets the activity of the three main groups of PLA2, which are secretory PLA2 or extracellular calcium dependent PLA2 (sPLA2), cytosolic calcium dependent PLA2 (cPLA2) and intracellular calcium independent PLA2 (iPLA2). To confirm the presence of these PLA2 groups some variables were tested, such as differences in the preferred fatty acid substrate, calcium dependence, and selective inhibition with Bromoenol lactone (BEL) and Methyl Arachidonyl Fluorophosphonate (MAFP). Our findings demonstrate the presence of the three main groups of PLA2 (sPLA2, cPLA2, and iPLA2) in platelets. In addition, this study is in line with others suggesting that platelets, a typical biological sample, can be used as a peripheral model for neurons.

Thesis (Mech.E)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1980. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Bibliography: leaves 355-373. / by Martin J. Forbes. / Mech.E

Fan Boli. / "January 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 219-248). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Orientador: Maria José Hitomi Nagata / Banca: Mário Taba Junior / Banca: Álvaro Francisco Bosco / Banca: Ronaldo Célio Mariano / Banca: Valdir Gouveia Garcia / Resumo: O objetivo do presente trabalho foi avaliar, histologicamente, a influência da associação do plasma rico em plaquetas (PRP) ao enxerto de osso autógeno (OA) no reparo ósseo de defeitos de tamanho crítico (DTC) em calvárias de coelhos. Sessenta coelhos foram divididos em 3 grupos: C (controle), OA (osso autógeno) e OA/PRP (osso autógeno associado ao plasma rico em plaquetas). Um defeito de tamanho crítico, com 15 mm de diâmetro, foi criado na calvária de cada animal. No Grupo C, o defeito foi preenchido somente com coágulo sanguíneo. No Grupo OA, o defeito foi preenchido com osso autógeno particulado. No Grupo OA/PRP, o defeito foi preenchido com osso autógeno particulado associado ao PRP. Todos os grupos foram divididos em subgrupos (n = 10) para eutanásia aos 30 ou 90 dias pós-operatórios. Foram realizadas análises histológica e histométrica. A quantidade de osso neoformado foi calculada como uma porcentagem da área total do defeito original. Os dados em porcentagem foram transformados em raiz quadrada para análise estatística (ANOVA, teste t, p<0,05). Aos 30 dias pós-operatórios, o Grupo OA/PRP apresentou uma quantidade significativamente maior de osso neoformado que o Grupo OA (64,44% e 46,88%, respectivamente). Aos 90 dias, todos os espécimes dos Grupos OA/PRP e OA mostraram fechamento ósseo completo do defeito, com quantidades similares de osso neoformado (77,9% e 75%, respectivamente). Dentro dos limites deste trabalho, pode-se concluir que o PRP acelerou o processo de reparo do enxerto de osso autógeno em defeitos de tamanho crítico em calvária de coelhos. / Abstract: The purpose of this study was to histologically analyze the effect of platelet-rich plasma (PRP) on bone healing in an autogenous bone graft placed in surgically created criticalsize defects (CSD) in rabbit calvaria. 60 rabbits were divided into 3 groups: C (control); AB (autogenous bone graft) and AB/PRP (autogenous bone graft with platelet-rich plasma). A 15 mm diameter CSD was created in the calvarium of each animal. In Group C the defect was filled by blood clot only. In Group AB the defect was filled with particulate autogenous bone. In Group PRP it was filled with particulate autogenous bone in combination with platelet-rich plasma. All groups were divided into subgroups (n=10) and euthanized at either 30 or 90 days post-operative. Histometric, using image analysis software, and histologic analyses were performed. Amount of new bone was calculated as a percentage of the total area of the original defect. Percentage data were transformed into square root for statistical analysis (ANOVA, t test, p<0.05). At 30 days post-op, Group AB/PRP had a statistically greater amount of bone formation than Group AB (64.44% and 46.88%, respectively). At 90 days, complete bone regeneration of the defect was seen in all specimens of Groups AB/PRP and AB, with similar amounts of newly formed bone (77.9% and 75%, respectively). Within the limits of this study, it can be concluded that PRP accelerated the healing of autogenous bone graft in critical-size defects in rabbit calvaria. / Doutor

Abstract not available

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.

Growth factor delivery may be useful to accelerate the rate of tendon healing. We studied Platelet Concentrate, which in effect can be regarded as a cocktail of growth factors relevant for tendon healing. In a rat Achilles tendon transection model, one postoperative injection of Platelet Concentrate resulted in increased strength even 3 weeks later. Mechanical stimulation improves the repair of ruptured tendons. We studied the effects of platelets upon Achilles tendon regenerates in rats 3, 5 and 14 days after transection, either unloaded or mechanically stimulated. At 14 days, physical activity and platelets increased repair independently. Unloading decreased the mechanical properties of the repair tissue to less than half of normal. Moreover, the platelets had no effect without loading. Thrombin, which we used for platelet activation, improved healing of the rat Achilles tendon by itself. Conversely, continuous inhibition of thrombin by low molecular weight heparin (LMWH) inhibited tendon repair. However, intermittent inhibition, similar to clinical thromboprophylaxis, had no effect on tendon healing. Cartilage Derived Morphogenetic Protein-2 (CDMP-2) can improve tendon healing in loaded defect models. We now studied unloaded repair in a rabbit patellar tendon model. Two hours postoperative, the rabbits received CDMP-2 injected into the haematoma. The healing tendon became 65 % stronger than controls. We then studied Achilles tendon healing with CDMP-2 injections in sheep, to get a bigger animal model. There was an unexpectedly high variation of repair in these animals, and the study turned out to be underpowered. Spontaneous ruptures in humans have a more variable geometry than in our sheep model, so humans can also be expected to vary a lot in mechanical characteristics of Achilles tendon repair. This accentuates the importance of individualized rehabilitation programs. In conclusion, both platelet concentrate and CDMP-2 injections might be of interest for clinical use as a complement to surgical or conservative treatment of tendon ruptures. Platelet treatment for tendon ruptures should probably be combined with early physiotherapy.

Arterial thrombus forms from the capture and accumulation of circulating platelets on a stenosis. As the thrombus grows, the lumen becomes further stenotic producing very high shear rates as the blood velocities increase through the narrowed cross-section. This study explores the molecular binding conditions that may occur under these pathologic shear conditions where circulating platelets must adhere quickly and with strong bonds. Platelets binding in an arterial stenosis of >75% are subject to drag forces exceeding 10,000 pN. This force can be balanced by 100 simultaneous GPIb-vWFA1 bonds of 100 pN each. The number and density of GPIb on platelets is sufficiently high; however, platelet capture under high shear would require the density of A1 receptors to be increased to over 416 per square micron. A computational model is used to determine platelet capture as a function of shear rate, surface receptor density, surface contact and kinetic binding rate. A1 density could be increased by a combination of vWF events of: i) plasma vWF attach to the thrombus surface and elongate under shear; ii) the elongated vWF strands create a net with 3-D pockets; and iii) additional vWF is released from mural platelets by activation under shear. With all three events, A1 density matches the existing high GPIbα densities to provide sufficient multivalency for capture at 100,000 s-1 with greater than 170 bonds per platelet. If the on-rate is greater than 108 M-1s-1, then a platelet could be captured within 15 microseconds, the amount of time available to form bonds before the platelet is swept away. This mechanism of platelet capture allows for the rapid platelet accumulation in atherothombosis seen clinically and in high shear experiments.