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Structure and Functions of Canine Protein C

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Title: Structure and Functions of Canine Protein C
Author: Wong, Valerie M.
Department: Department of Pathobiology
Program: Veterinary Science
Advisor: Wood, Robert Darren
Abstract: The structure and biological properties of activated protein C (APC) in humans have been well documented. Although APC was first described to be an anticoagulant, recent studies demonstrated anti-inflammatory and cytoprotective effects of the human protein C pathway. Recombinant human APC (rhAPC) was previously used for treating severe sepsis in humans, and non-anticoagulant rhAPC was suggested to be effective for treatment of stroke. In contrast, little is known about canine protein C (CnPC). The first step to exploring the therapeutic potential of CnPC is to characterize its structure and functions. This thesis first describes purification of CnPC from plasma by salt precipitation followed by immunoaffinity chromatography using an anti-human protein C antibody. The purified protein was composed of a heavy chain (with three different glycoforms) and a glycosylated light chain. The tandem mass spectra of the peptides obtained following trypsin digestion and liquid chromatography showed that this protein was CnPC, as predicted from the CnPC nucleotide sequence. Upon activation by Protac, a specific activator of protein C, the heavy chain underwent the predicted reduction in molecular weight. Canine activated protein C (CnAPC) showed amidolytic, anti-factor V, and anti-factor VIII activities. Both CnPC and CnAPC inhibited canine neutrophil chemotaxis towards recombinant canine interleukin-8 and recombinant human complement component C5a in vitro. To further explore the mechanisms involved in anti-chemotactic effects of CnAPC, expression of endothelial protein C receptor (EPCR) on canine neutrophils was determined. Blockade of EPCR with a monoclonal antibody neutralized the anti-chemotactic effects of CnAPC. These results showed that the anti-chemotactic effect of CnPC and CnAPC was dependent on EPCR and independent of CnAPC’s anticoagulant properties. Additionally, the anti-apoptotic effects of CnPC were studied in cultured canine aortic endothelial cells. Apoptosis was induced by incubation with staurosporine. Compared to untreated cells, cells pretreated with CnPC and CnAPC showed significantly decreased annexin-V binding on flow cytometry and significantly decreased expression of caspase-3/7 activities as measured in a functional assay. These results suggested that CnPC and CnAPC had anti-apoptotic effects. In summary, CnPC was purified from plasma and showed biological effects beyond its major role as an endogenous anticoagulant. The therapeutic potential implicated by the anti-chemotactic and anti-apoptotic effects of CnPC warrants further investigation.
Date: 2014-01
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
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Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada