by Lee Shun Keung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 73-91.

The study of platelet messenger and micro-RNAs is of increasing interest owing to the fact that platelets contain the machinery to splice and translate mRNA into proteins in response to inhibitory or activating signals. However, the relatively small size (roughly 4000-5000 transcripts) and short half-life of the platelet transcriptome makes this a technically challenging aspect of platelet biology to investigate. The aims of these thesis investigations were therefore to optimise protocols for the isolation of platelets for downstream RNA analyses and function testing, to investigate the functional capabilities of platelet subpopulations rich in RNA, and to understand the functional and transcriptomic impact of gene mutations predicted to influence platelet function. I found that the optimal method for isolating platelets from whole blood is to use simple single step centrifugation to obtain platelet rich plasma. This method is as effective as more involved methods at reducing white blood cell contamination whilst causing minimal platelet activation. Using this method in combination with flow cytometric cell sorting techniques I was able to isolate the newly formed reticulated platelet sub-population and to confirm the link between reticulation status and increased RNA content. Furthermore, using a range of platelet function assays I demonstrated that reticulated platelets are more reactive than non-reticulated platelets. By obtaining blood samples from a patient with a PLA2G4A mutation I was able to show that loss of cPLA2α enzymatic activity alters both platelet function and the expression of certain mRNA transcripts. My investigations using samples from a range of patients with bleeding tendencies show the benefit of combining deep platelet phenotyping with next generation sequencing to understand the causation of bleeding disorders. Together these investigations highlight the utility of genomic DNA and platelet specific mRNA studies in providing novel insights in to pathways regulating platelet reactivity.

206 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--Dept. of Medicine, University of Adelaide, 1974

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 95-101).

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.

Platelets are small, anucleate cells that fulfill a central role in hemostasis through their ability to adhere to sites of vessel injury, where they change shape, secrete molecules, and aggregate to stop blood leakages. Their secretory organelles - alpha (α)-granules, dense (δ)-granules and lysosomes - are critical in mediating the formation of a hemostatic plug, and interference with their biogenesis leads to bleeding disorders. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Studies in our laboratory have identified the only proteins known to date to be relevant for α-granule biogenesis: the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B. Mutations in VPS33B have been associated with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. ARC platelets are pale and agranular in appearance, and display a complete absence of α-granule structures and contents. Using a yeast two-hybrid screen, mass spectrometry, co-immunoprecipitation, and bioinformatics studies, VPS16B was identified as a VPS33B-binding protein. Platelets from a patient with ARC syndrome containing mutations in C14orf133 encoding VPS16B recapitulate the phenotype observed in VPS33B-null platelets. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B co-localized with markers of the trans-Golgi network, late endosomes and α-granules. Depletion of VPS16B in platelet progenitor cells - human iii megakaryocytes - resulted in absent α-granules, though α-granule proteins were still synthesized. Taken together, these data identify VPS16B as a novel molecule required for platelet α-granule biogenesis.

Platelets are small, anucleate cells that fulfill a central role in hemostasis through their ability to adhere to sites of vessel injury, where they change shape, secrete molecules, and aggregate to stop blood leakages. Their secretory organelles - alpha (α)-granules, dense (δ)-granules and lysosomes - are critical in mediating the formation of a hemostatic plug, and interference with their biogenesis leads to bleeding disorders. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Studies in our laboratory have identified the only proteins known to date to be relevant for α-granule biogenesis: the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B. Mutations in VPS33B have been associated with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. ARC platelets are pale and agranular in appearance, and display a complete absence of α-granule structures and contents. Using a yeast two-hybrid screen, mass spectrometry, co-immunoprecipitation, and bioinformatics studies, VPS16B was identified as a VPS33B-binding protein. Platelets from a patient with ARC syndrome containing mutations in C14orf133 encoding VPS16B recapitulate the phenotype observed in VPS33B-null platelets. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B co-localized with markers of the trans-Golgi network, late endosomes and α-granules. Depletion of VPS16B in platelet progenitor cells - human iii megakaryocytes - resulted in absent α-granules, though α-granule proteins were still synthesized. Taken together, these data identify VPS16B as a novel molecule required for platelet α-granule biogenesis.

No description available.

The objective of this project was to study tree effect of physical conditioning on platelet function. Twelve Ball State University undergraduate males were tested for platelet retention and platelet aggregation with collagen and adenosine diphosphate before, during and after a controlled six and one half week physical training program consisting of calisthenics and jogging. A significant increase was found in the tendency of platelets to aggregate with collagen while no change was found in aggregation with ADP or in platelet retention. It was concluded that: i) more work is necessary to verify this training effect, and ii) further work should be done considering other variables in the blood coagulation and fibrinolytic pathways in order to gain a better overall understanding of the effect of exercise on hemostasis.

No description available.