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|Title:||A structure-function relationship study involving the first epidermal growth factor-like domain of human coagulation factor VII|
|Authors:||Leonard, John Normand Blair|
|Advisor:||Blajchman, Morris A.|
|Keywords:||Medical Sciences;Medical Sciences|
|Abstract:||<p>The interaction of circulating coagulation factor VII with cell surface associated tissue factor (TF) is critical for the initiation of blood coagulation, and appears to play a significant role in several other biological processes. Thus, an understanding of the mechanism of this interaction should provide valuable insight into the development of these important processes. The first epidermal growth factor-like domain (EGF-1) of factor VII is the principle contact site with TF and possesses the structural elements characteristic of the ubiquitous EGF-like motif. The aim of this thesis was to investigate the relationship between the conformational structure of the EGF-1 domain in factor VII and its function in the initiator complex. Recombinant generation and analysis of a naturally occurring factor VII EGF-1 mutant (N57D) indicated a protein deficient in its cellular secretion, as well as possessing compromised procoagulant activity and TF binding capability. Using structural investigative techniques, this mutation was found to compromise the appropriate conformation of the EGF-1 domain via the loss of a previously undescribed, structurally-important intermolecular hydrogen bond, highlighting the delicate balance between conformation and function in this domain. Once developed, these structural investigative techniques allowed further investigation, indicating the EGF-1 domain adopted a conformation specific to the "active state" of factor VIIa and requiring the presence of calcium and the adjacent Gla domain. Once activated, occupation of the active site of factor VIIa with substrate-like inhibitors resulted in further conformational changes to the EGF-1 domain, indicating the presence of a novel active site occupant-specific allosteric linkage between the EGF-1 domain and the active site. This allosteric link also appeared to affect the functional processes of TF binding and enzymatic activity of the factor VIIa molecule. Overall, this study has contributed new knowledge on the relation of structural elements of the factor VII EGF-1 domain to the important factor VIIa functional processes of TF binding and enzymatic activity. As well, the study indicates a novel role for the EGF-1 domain in the unique co-factor mediated specificity observed for factor VIIa.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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