Date of Award

11-2015

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Niamh Moran

Funder/Sponsor

National Bioinformatics Imaging Platform Ireland (NBIP)

Keywords

Platelet Activation, Proteins

Abstract

The integrin αIIbβ3 is a heterodimeric protein that exists in an inactive, resting state within the membrane of quiescent platelets. In response to platelet activation within a pro-thrombotic environment, the integrin αIIbβ3 becomes active, serves as a receptor for a number of ligands including fibrinogen, and orientates platelet – platelet cohesion. The αIIb and β3 subunits maintain the integrin in a resting conformational state by forming a tight clasp at their cytoplasmic tails (CT’s). Disruption of this clasp by platelet activation initiates the transition of the integrin from an inactive conformation to an active ligand binding state. The highly conserved αIIb membrane proximal motif (989KVGFFKR995) resides at the binding interface of the αIIb and β3 CT contacts and acts as a docking site for integrin binding proteins. These binding partners regulate the association of the integrin αIIb and β3 CT’s and hence, the activation state of the integrin. Therefore, since αIIb and β3 CT seperation is a fundamental feature of integrin activation, much research has emphasized on the dynamic relationship between the conserved motif of the αIIb CT and its binding partners. RNF181 was previously demonstrated to be a potential integrin binding protein as it had an affinity for the 989KVGFFKR995 motif. Given the preliminary nature of this earlier work, this thesis focused on exploring a possible, functional relationship between RNF181 and αIIbβ3. Using a variety of experimental approaches, strong evidence for a functional co-association of integrin αIIbβ3 and RNF181 was revealed. Using confocal microscopy, co-localization between the two proteins in platelets and integrin αIIbβ3 expressing CHO cells was demonstrated. Using CO-IP’s, Thermophoresis and ITC, a direct affinity between RNF181 and αIIbβ3 was demonstrated. The interaction was observed to have a moderate and transient affinity. Using bioinformatics and peptide arrays, the precise binding sites that allowed RNF181 to interact with the integrin αIIb cytoplasmic tail was explored. In particular, short linear peptide sequences that mediate their binding capacity were identified. Using platelet function assays, it was demonstrated that palmitoylated RNF181 derived peptides potently and specifically inhibited platelet / integrin αIIbβ3 activation. Finally, through a preliminary study using siRNA, a role for RNF181 as being a positive regulator of integrin αIIbβ3 activation was suggested.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

6.19 MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2015.

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