Date of Award

6-2015

Document type

Thesis

Degree Name

MSc by research (Master of Science by research)

First Supervisor

Dr Marian Brennan

Second Supervisor

Dr Dermot Cox

Funder/Sponsor

Lei Pharmaceuticals Ltd, Hong Kong

Keywords

Anfibatide, GPIb, Antagonist, Flow Cytometry, Aggregation

Abstract

Thrombosis is central to the pathogenesis of many life-threatening diseases, including stroke and myocardial infarction. Current treatments for thrombosis, particularly anti-platelet agents that target GPIIb/IIIa, can cause serious bleeding complications. Unlike GPIIb/IIIa, GPIb initiates thrombus formation at the high shear stress that is prominent in atherosclerotic vessels. Therefore, this target could prevent pathological thrombosis while leaving normal haemostasis at low shear rates relatively unaffected. Anfibatide is a novel GPIb antagonist isolated from snake venom in China. Anfibatide will enter phase Ib-IIa trials in early 2015 and therefore, it is necessary to develop an assay to measure its efficacy in patients. We have developed a flow cytometry based receptor occupancy assay. In this assay, total GPIb receptor numbers are measured using an anti-GPIb monoclonal antibody, and the percentage of GPIb receptor occupancy by anfibatide is measured. Receptor occupancy by anfibatide correlated with dose-dependent inhibition of ristocetin-induced aggregation in whole blood by anfibatide. Functional studies were carried out using anfibatide. Anfibatide did not inhibit binding of two anti-GPIIb/IIIa antibodies. Anfibatide partially inhibited the anti-GPIb antibodies VM16d, and SZ 2, indicating that it binds to GPIbα between the thrombin-binding site and the sulfated tyrosine motif. Anfibatide did not cause platelet activation or loss of single platelets in vitro. Anfibatide had no effect on aggregation induced by low dose or high dose thrombin, ADP or arachidonic acid. Anfibatide inhibited Streptococcus sanguinis 133-79 induced aggregation by 50% in half of healthy donors, but had no effect on Staphylococcus aureus Newman aggregation. The receptor occupancy assay is optimised and available for use in Phase Ib-IIa clinical trials, and in future clinical use of anfibatide.

Creative Commons License

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

File Size

1.52 MB

Comments

A thesis submitted for the degree of Master of Science from the Royal College of Surgeons in Ireland in 2015.

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