Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder which results from fetal platelet destruction by maternal antibodies against platelet antigens, including GPIIbIIIa (αIIbβ3 integrin) and GPIbα. β3 integrin is also expressed by angiogenic endothelial cells (ECs) and is required for angiogenesis. Therefore, we investigated whether anti-β3 antibodies in FNIT cross-react with blood vessels of the fetus/neonate and contribute to pathogenesis. Antibodies to GPIbα were used as controls. To mimic human FNIT, β3 integrin- or GPIbα-deficient female mice were immunized with wild-type platelets and bred with wild-type male mice. Pups in both groups had thrombocytopenia but intracranial hemorrhage was only observed in anti-β3-mediated FNIT. Anti-β3-mediated FNIT pups had increased apoptosis in the brain and impaired vascularization of the brain and retina. In addition, anti-β3 sera inhibited proliferation and vascular-like tube formation by ECs in vitro. Therefore, anti-β3 antibodies in FNIT likely impair angiogenesis in the developing fetus/neonate.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder which results from fetal platelet destruction by maternal antibodies against platelet antigens, including GPIIbIIIa (αIIbβ3 integrin) and GPIbα. β3 integrin is also expressed by angiogenic endothelial cells (ECs) and is required for angiogenesis. Therefore, we investigated whether anti-β3 antibodies in FNIT cross-react with blood vessels of the fetus/neonate and contribute to pathogenesis. Antibodies to GPIbα were used as controls. To mimic human FNIT, β3 integrin- or GPIbα-deficient female mice were immunized with wild-type platelets and bred with wild-type male mice. Pups in both groups had thrombocytopenia but intracranial hemorrhage was only observed in anti-β3-mediated FNIT. Anti-β3-mediated FNIT pups had increased apoptosis in the brain and impaired vascularization of the brain and retina. In addition, anti-β3 sera inhibited proliferation and vascular-like tube formation by ECs in vitro. Therefore, anti-β3 antibodies in FNIT likely impair angiogenesis in the developing fetus/neonate.

Die Adipositasprävalenz hat in den letzten Jahren in den Industrieländern signifikant zugenommen. Fettleibigkeit ist ein unabhängiger Risikofaktor für das Auftreten verschiedener Erkrankungen wie Schlaganfall, Lungenembolie, Myokardinfarkt und verschiedene Tumoren. Da kardiovaskuläre Erkrankungen in Industrieländern die häufigste Todesursache darstellen, stellt die Adipositas ein wachsendes Gesundheitsproblem dar und sollte Schwerpunkt intensiver Forschungsarbeit sein. Da die Adipositas in der Mehrzahl der Fälle von einer Hyperleptinämie begleitet ist, war das Ziel der vorliegenden Arbeit, die mögliche Rolle von Leptin als Thrombozytenagonist auf verschiedenen Ebenen zu analysieren. Die durchgeführten Experimente konnten eine Leptin-induzierte Aktivierung des Integrins αIIbβ3, dem thrombozytären Fibrinogenrezeptor, und eine Steigerung der Adhäsion von Thrombozyten an immobilisiertem Fibrinogen zeigen. Leptin ist weiterhin in der Lage, die ADP-induzierte Thrombozytenaggregation signifikant zu steigern. Das Adipokin führt ebenfalls zu einer Steigerung der Thromboxan-B2-Freisetzung aus Thrombozyten. Neben der Untersuchung des Einflusses von Leptin auf die Thrombozytenfunktion sollte in der vorliegenden Studie ebenfalls die thrombozytäre Signalkaskade unter Wirkung des Proteohormons beleuchtet werden. Zentrales Molekül in diesem Signalweg ist JAK2. Für diese Tyrosinkinase konnte eine biologische Relevanz mit Hilfe von in vitro Versuchen zu Adhäsion und Aggregation von Thrombozyten unter Einfluss von Leptin demonstriert werden. Auch STAT3 wird zeit- und dosisabhängig durch Leptinstimulation in Thrombozyten phosphoryliert, wobei seine Bedeutung für die Thrombozytenaktivierung noch ungeklärt ist. Weitere an der Signaltransduktion in Thrombozyten unter Wirkung von Leptin beteiligte Kinasen sind die Phosphatidylinositol-3-Kinase, AKT, Phospholipase Cγ2 und Proteinkinase C sowie die p42/44-MAP-Kinase, die p38 MAP-Kinase und die Phospholipase A2. Für die Aktivierung von PLCγ2, AKT und der p42/44-MPAK konnte eine Abhängigkeit von der Tyrosinkinase JAK2 gezeigt werden. Für diese intrazellulären Mediatoren konnte ebenfalls unter Verwendung von verschiedenen Tyrosinkinase-Inhibitoren die Bedeutung bei der Thrombozytenadhäsion an immobilisiertem Fibrinogen bzw. der Aggregation nachgewiesen werden. Die in dieser Studie aufgedeckten Kaskaden zeigen somit biologisch relevante Leptin-vermittelte Signalwege in Thrombozyten auf und sollen zu einem besseren Verständnis des Zusammenhangs von Hyperleptinämie und erhöhtem Thromboserisiko bei Adipositas beitragen.

Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, in response to thrombin, were unaffected. Arf6-/- mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets. Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase-activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent-insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets.

Thrombosis is the pathological formation of platelet aggregates that cause stroke and heart attack\textemdash the leading causes of death in developed nations. Determining effective dosages for platelet therapies (e.g. aspirin, Integrilin, and Plavix) to prevent thrombosis is a persistent medical challenge (studies estimate up to 45% of patients exhibit insufficient responses to these drugs) and recent studies have implicated pathological flow conditions of high shear rates and stenosis morphology as primary factors. However, there are currently no diagnostic instruments able to recapitulate a range of such pathological flow conditions for evaluating thrombosis with and without these drugs. In this work, a microfluidic device and associated optical system were designed and fabricated for simultaneous measurement of platelet aggregation at multiple initial wall shear rates within multiple stenotic channels in label-free whole blood and used to characterize thrombosis at varying dosages of two platelet therapies: acetyl-salicylic acid (aspirin) and eptifibatide (Integrilin). Results from our studies show the effects of pathologically high shear rates on enhancing platelet thrombosis and demonstrate the widely varied, shear-dependent efficacy of each therapy. This study lays the foundation for the future development of a medical diagnostic for optimizing the type and dosage of patient platelet therapy and to better understand their mechanisms of action.

No description available.

Platelets have been shown to be a main contributor to thrombus formation in stenotic arteries leading to acute coronary syndromes. It is thought that increased activation and adhesion of platelets under variable shear and complex flow conditions contribute to thrombosis. The objective of this work was to evaluate the relationship between asymmetric stenosis hemodynamics and platelet adhesion using in-vitro models developed to properly simulate physiological conditions. In this study, platelet rich plasma was circulated through stenotic and straight coronary artery models. Adhesion results were obtained by post-perfusion fluorescent labelling and imaging of adhered platelets. Analysis of platelet area coverage has shown maximum adhesion occurs in the distal region of the stenosis. Most likely this is due to increased exposure time of platelets to the wall of the recirculation zone following the stenosis and that exposure being directly after a period of high shear stress. This result gives us a better understanding of the importance of both shear and flow conditions in coronary artery thrombosis.

Copy of author's previously published article in pocket on back end-paper. / Includes bibliography (leaves 135-185). / 185, [94] leaves, [32] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to characterise the expression of PETA-3 (Platelet Endothelial Tetraspan Antigen-3), CD9, CD63 and ?gb?s1 integrins in normal human tissue ; to determine the subcellular localisation in endothilial cells and platelets ; to investigate protein-protein interactions involving PETA-3 ; and to examine the effects of anti-PETA-3 monoclonial antibodies on platelet and endothilial cell function. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1999

Copies of author's previously published articles inserted. / Bibliography: leaves 267-303. / xviii, 304 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Experiments described in the thesis address the anti-aggregatory effects and mechanism of action of the prophylactic anti-anginal agent perhexiline maleate. In particular, it was sought to examine if perhexiline had an anti-aggregatory effect which may contribute to its proven therapeutic efficacy. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 2000

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 5 uppsatser.