Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 196-212). Also available in electronic version. Access restricted to campus users.

published_or_final_version / Pathology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-99) Also available in print.

In the past couple of decades, the zebrafish has been widely used to study hemostatic disorders. In this study, we generated a CRISPR/Cas9 mediated zebrafish mutant that contains a 55-nucleotide insertion in exon 29 of the von Willebrand factor (vwf) gene. The mutants had impaired ristocetin-mediated agglutination of whole blood, prolonged PTT and more bleeding in the lateral incision compared to wild-type fish. The bleeding phenotype observed here is similar to the phenotype observed in vwf knockout mice and patients with von Willebrand disease (VWD). The mutant model developed here can thus be used for exploring the role of Vwf in angiogenesis and for developing gene therapy. The deficiency of VWF causes VWD and the etiology remains unknown in 30% of Type 1 VWD cases. Previous studies have identified that the ABO blood group and ST3GAL4 (glycosyltransferases) are involved in the regulation of VWF levels. Since VWF is heavily glycosylated, we hypothesized that other glycosyltransferases may also be involved in regulating VWF. We performed a knockdown screen of 234 glycosyltransferase genes and identified 14 genes that altered Vwf levels. The sequencing of these genes in Type 1 VWD patients could help identify novel mutations to decipher the molecular basis for the unknown etiologies in Type 1 VWD. Moreover, therapeutic interventions could be designed in the future by modulation of these gene products to control bleeding or thrombosis.Zebrafish has three f9 genes, f9a, f9b, and f9l and the ortholog to human F9 is unknown. RNA analysis showed an age-dependent increase in expression of all three genes from larval stages to adults, comparable to those observed in mice and humans while mass spectrometry and immunohistochemistry confirmed the presence of all three proteins in the fish. Based on coagulation assays performed after individual gene knockdown and immunodepletion, we identified that zebrafish f9a has functional activity similar to human F9 and Fixl is functionally similar to Fx. Thus, the zebrafish could be used to identify factors controlling f9 gene expression with age and for modeling Hemophilia B in the quest to develop gene therapy protocols. In zebrafish, dilute plasma with exogenously added human fibrinogen was used for kinetic coagulation assays. Here, we developed a microkinetic assay using 25% zebrafish or 30% human plasma followed by the addition of coagulation activators and CaCl2. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established human kinetic curves. Moreover, clotting times derived from these kinetic curves were identical to human PT, PTT, and Russel's viper venom time. Thus, the microkinetic assay developed here could measure blood coagulation activity in small animal models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.