At the site of vascular injury platelet aggregation serves to stem blood flow, initiate the inflammatory response, and stimulate wound healing. Platelets become stimulated, release their granule contents, and become adherent to one another. Platelet granules contain important clotting factors and regulators of aggregation. Disabled-2 (Dab2) is a negative regulator of platelet aggregation released from platelet α-granules. Dab2 binds to the αIIbβ3 integrin, through the PTB domain, and blocks fibrin binding to the integrin which serves as the major cause of platelet-platelet interactions. Dab2 is also capable of binding to sulfatides, through the N-PTB region, expressed on the outer leaflet of adjacent cells. Dab2-sulfatide binding decreases Dab2's ability to interact with the αIIbβ3 integrin, however sulfatides activate and stimulate platelet-platelet and platelet-leukocyte aggregation. Sulfatide addition to platelets stimulates increased αIIbβ3 integrin and P-selectin expression through stimulation of continued platelet degranulation, and these surface receptors mediate platelet heterotypic and homotypic aggregation. Here, we show that Dab2 N-PTB binding of sulfatides serves to increase the inhibitory affect of Dab2. Sulfatide stimulation of platelet degranulation can be blocked by the addition of N-PTB. Inhibition of sulfatide induced αIIbβ3 integrin and P-selectin expression result in decreased platelet-platelet aggregation under flow. N-PTB also blocks sulfatide induced platelet-leukocyte interactions and aggregation. Experimental data supports the hypothesis that Dab2-sulfatide binding serves to increase the inhibition of platelet aggregation. / Master of Science

Thrombosis, the major cause of heart attack and strokes,1 is triggered by localized clotting of the blood as the result of deregulated platelet aggregation. During the repair of vascular injury, clotting usually occurs when platelets adhere to each other at the site of vascular injury in order to stop bleeding.2 Distinct protein receptors and adhesive ligands together with the blood flow conditions govern this process. One of the negative regulators in platelet aggregation is Disabled-2 (Dab2), a modular protein that is released upon platelet activation to the extracellular platelet surface.3 Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for ï ¡IIï ¢3 integrin binding on the activated platelet surface.3 Sulfatides are also found on the platelet surface,4 interacting with adhesive and coagulation proteins5-7 and, thus, they are thought to play a major role in haemostasis and thrombogenesis. Here, we show that the Dab2 PTB domain specifically interacts with sulfatides through two conserved basic motifs. The sulfatide-binding site overlaps with that of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) in the PTB domain. Whereas sulfatides recruit the Dab2 PTB domain to the platelet surface, thus sequestering the protein from thrombin-mediated platelet aggregation, the phosphoinositide mediates its internalization. Experimental data support the hypothesis that two pools of Dab2 co-exist at the platelet surface and that the balance between them controls the extent of the clotting response. / Master of Science

Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk with skin flora, such as Staphylococcus epidermidis and Staphylococcus capitis, being the predominant contaminants. These bacteria are able to form surface-attached aggregates or biofilms, which are present in the skin of healthy blood donors and can subsequently be isolated from contaminated PCs. Disinfection of the venipuncture area before donation with a combination of 2% chlorhexidine-gluconate and 70% isopropanol is used at Canadian Blood Services. However, not all bacteria are eliminated during skin disinfection since contaminated PCs are still captured during routine PC screening. In this thesis, the ability of biofilm-forming S. epidermidis and S. capitis to resist the currently used disinfectants was explored. It was demonstrated that although a combination of chlorhexidine and isopropanol has a bactericidal effect, it is unable to completely eradicate skin flora biofilms. Several countries have implemented Pathogen Inactivation Technologies (PITs) as a measure to help control transfusing bacterially-contaminated PCs by exposing PC units to ultra violet light. However, no investigations have been done to evaluate the ability of PITs against bacterial biofilms, which was one of the objectives of this thesis. Data revealed that the efficacy of a currently used PIT, the Mirasol® system, is similar for S. epidermidis present in PCs produced from whole blood inoculated with biofilm or non-biofilm cells. However, treatment effectiveness was strain dependent. In conclusion, further investigation to improve donor skin disinfection and PITs should be considered. Surveillance at Canadian Blood Services shows that contamination rates in single-donor apheresis PCs (Aph-PCs) is generally higher than in four-donor buffy coat platelet pools (BC-PCs). This study investigated whether the BC-PC production method contributes to this observation as BC-PCs are derived from WB that is left to rest overnight while Aph-PCs are collected directly from the donor. Data showed that WB hold during BC-PC production does not have a broad-spectrum bactericidal effect and therefore other factors contribute to low rates of contamination in BC-PCs. The work presented in this thesis provides an insight to bacterial residence and persistence during blood product manufacturing and makes suggestions for PC safety improvements.

No description available.

No description available.

Platelets play an essential role in haemostasis by adhering to the damaged vessel wall and forming a platelet plug to arrest bleeding. Although platelets are traditionally thought of as pro-coagulant, they possess the ability to harbour functional proteins that are key to fibrinolysis, the breakdown of the blood clot, on their surface. They are therefore substantially well equipped to regulate local fibrinolysis. This thesis aims to further define the role of platelets in fibrinolysis, in particular platelet-derived plasminogen activator inhibitor 1 (PAI-1) and plasminogen. PAI-1 is the principal physiological inhibitor of tissue-type plasminogen activator (tPA), and plasminogen is the zymogen for plasmin. In Chapter 3, we show that platelet-derived PAI-1 is released from platelet α-granules by an αIIbβ3 and fibrin dependent mechanism. We found that a significant portion of α-granular PAI1 is retained on the surface of highly activated PS-positive platelets, and activity analysis revealed the majority of PAI-1 on the platelet surface was in its active form. The functional role of platelet PAI-1 was investigated by analysis of tPA-mediated lysis of Chandler model thrombi. Our data revealed a striking dependence for platelet PAI-1 in stabilising platelet-rich thrombi against degradation. Chapter 4 characterises the expression of a novel transmembrane receptor, Plg-RKT, on the surface of human and mouse platelets. This revealed that plasminogen and Plg-RKT augment one another's binding to the platelet surface. Furthermore, analysis of plasminogen binding to the platelet surface revealed two distinct binding sites: 1) via Plg-RKT and 2) via a fibrin and αIIbβ3 dependent mechanism. Finally, Chapter 5 of this thesis discusses the optimisation of a system that monitors thrombus formation and fibrinolysis under flow. Use of this model will help to further elucidate the complex role that platelets play in controlling the balance between coagulation and fibrinolysis.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As plaquetas sÃo um grupo de cÃlulas que tÃm participaÃÃo fundamental nos processos hemostÃticos. Durantes estes eventos, tais cÃlulas passam por uma drÃstica transformaÃÃo de sua estrutura, o que inclui alteraÃÃo de forma, saindo de um perfil discÃide para um completamente amorfo. ConseqÃentemente, este fenÃmeno de reestruturaÃÃo da arquitetura celular està intimamente ligado a um rearranjo do citoesqueleto plaquetÃrio, estrutura localizada abaixo da membrana celular responsÃvel pela forma, estabilidade, maleabilidade, dentre outras caracterÃsticas mecÃnicas e fisiolÃgicas da membrana plasmÃtica. Neste contexto, a investigaÃÃo das caracterÃsticas elÃsticas de plaquetas ativadas, como sÃo denominadas ao iniciar o processo acima citado, apresenta-se como uma alternativa viÃvel ao aprofundamento no conhecimento da funÃÃo plaquetÃria no organismo. Para tanto, a microscopia de forÃa atÃmica (AFM), surge como uma tÃcnica de grande utilidade a este tipo de investigaÃÃo. Capaz de tocar a superfÃcie celular com forÃas da ordem de piconewtons, o microscÃpio de forÃa atÃmica pode ser utilizado como um nanoindentador, ou seja, de posse da geometria da sonda e da constante de mola do cantilever empregado, grandezas como elasticidade e viscosidade da amostra podem ser determinadas. Neste trabalho, duas subtÃcnicas de AFM foram empregadas no estudo de plaquetas humanas sadias ativadas, tais foram: Force Plot e Force Volume. A primeira fornece um grÃfico chamado de curva de forÃa, obtida com uma Ãnica indentaÃÃo na superfÃcie celular. Jà a segunda reÃne todas estas curvas numa imagem conhecida como imagem de volume de forÃa. Tais ferramentas provÃem informaÃÃes que, trabalhadas com um modelo matemÃtico adequado, no nosso caso o modelo de Hertz, tornam possÃveis a determinaÃÃo, nÃo apenas pontual, mas em todo o corpo da cÃlula, das propriedades mecÃnicas plaquetÃrias.

Bibliography: leaves 300-326. / 326 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Evaluates an in vitro experimental system which was designed to assess functions of platelets and cultured endothelial cells when they were incubated either independently or in combination. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995

Thesis (M. D. S.)--University of Hong Kong, 2003. / Title proper from title frame. Also available in printed format.

Copy of author's previously published article in pocket on back end-paper. Includes bibliography (leaves 135-185). 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.