Wilson, Darren Jonathan; (1997) Studies of the seven transmembrane domain thrombin receptor on human platelets and megakaryocytic cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes a study of the role of the human seven transmembrane domain thrombin receptor (ThrR) in platelet activation and investigates the functional epitopes of this receptor as potential sites for antiplatelet therapy. Whole blood flow cytometry was used to study aggregation independent platelet activation and the effect of current antiplatelet therapy. Aspirin had no effect on platelet fibrinogen binding or P-selectin expression (degranulation), whilst GPIIbllla antagonists, (monoclonal antibodies, echistatin) totally inhibited platelet fibrinogen binding but had no effect on P-selectin expression. Synthetic peptide analogues of the N-terminus of the receptor were capable of activating platelets causing maximal fibrinogen binding and P-selectin expression, but at concentrations 1000 fold higher than thrombin. The most potent N-terminal peptide mimetic was TRAP1-6, SFLLRN, the minimal sequence required for activation was TRAP1-3, SFL. Polyclonal antibodies were raised to synthetic peptide analogues of the extracellular domains of the ThrR. Antibodies to N-terminal regions bound to the receptor on platelets and on megakaryocytic human erythroleukaemia cells (HEL). These antibodies inhibited thrombin-and TRAP1-6-induced platelet activation as assessed by flow cytometry and aggregation, but had no effect on ADP induced activation or aggregation. Antibodies raised to the 'cleaved peptide' region of the thrombin receptor showed strong binding to the immunising peptide, whilst antibodies to the three extracellular loops of the seven transmembrane domains reacted only weakly. None of these antibodies bound to the receptor, either on platelets or HEL cells. The N-terminal antibodies were used to demonstrate differences in post-activation events in nucleated cells and platelets; the ThrR on HEL cells was downregulated from the surface, whilst it remained on the surface of platelets, following activation by thrombin or TRAP1 -6. In an attempt to obtain ThrR cDNA, to allow further analysis of functional epitopes via expression and site directed mutagenesis studies, HEL cells were used to prepare RNA from which cDNA was synthesized. The coding region of the 3' end of the thrombin receptor from base 716-1599 (approximately 60% of the coding region) was amplified using PCR, cloned into the phagemid Bluescript and sequenced. Nested PCR, with additional primers to the 5' end of the receptor, and RT-PCR failed to amplify the 5' end of the receptor. Preparation of genomic DNA from both HEL cells and whole blood allowed reamplification of the 3' end, but was unsuccessful in amplifying the 5' end of the receptor gene.

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