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Title: Investigations into the interactions of coagulation FXI with its binding partners
Author: Wong, Szu Shen
ISNI:       0000 0004 6423 3954
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2017
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In this body of work, the interactions of coagulation factor XI with high molecular weight kininogen and laminin-111 were investigated using various biophysical methods such as surface plasmon resonance technology and protein x-ray crystallography. These techniques were utilised to identify and characterize key binding sites. Prior to the start of this PhD study, a series of FXI binding peptides were developed by Novo Nordisk A/S (Denmark) with the aim of using them for FXI purification. Sequence alignment of these peptides revealed the shared tripeptide motif 'DFP' that is the main focus of this PhD investigation. This motif is also found in HMWK that circulates bound to FXI in plasma and in extracellular matrix laminin-111. Initially, the expression levels of human recombinant coagulation FXI is investigated. Following this, the successful co-crystallization of coagulation FXI with two 'DFP' binding peptides lead to the identification of a key hydrophobic pocket in the underside of the Apple 2 domain of FXI. The stoichiometry of the FXI-HMWK complex was also investigated using size exclusion chromatography and Edman degradation. Our data suggests that 1 FXI dimer circulates bound to 1 HMWK dimer. Surface plasmon resonance technology was utilised to probe the equilibrium binding constants of coagulation FXI interaction with full length HMWK and laminin. This study revealed multiple binding sites are involved in the protein complex formation. The 'DFP' motif is the shared binding motif between HMWK and laminin to FXI. The motif 'SDDDWIPDIQID' which is found N-terminus to the 'DFP' motif on HMWK was also identified to be important for HMWK binding to FXI. This part of the study also revealed that the peptides, domain 6 of HMWK and Laminin-111's G-like domain bound selectively to the apple 2 domain of FXI when compared with the apple 3 domain of FXI. Finally, coagulation assays were performed using platelet poor human plasma using activated partial thromboplastin time and calibrated automated thrombogram assays. We found that the 'DFP' peptides were able to alter coagulation parameters such as delaying the start of thrombin generation and reducing total thrombin generated in the absence of tissue factor. We also found that these `DFP' peptides did not interfere with the extrinsic pathway of coagulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available