Doctoral Degrees (Haematology and Cell Biology)

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Browsing Doctoral Degrees (Haematology and Cell Biology) by Subject "Anti-platelet agents"

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    Comparison of platelet receptors P2Y12, GPIIB/IIIA, GPVI, and GPIBα between the Cape chacma baboon and the human
    (University of the Free State, 2015) Janse van Rensburg, Walter James; Badenhorst, Philip N.; De Kock, André
    English: Background: Acute coronary syndrome is globally a major cause of morbidity and mortality. Treatment and prevention involve the use of an anti-platelet agent. The current available agents have either side-effects or are relatively ineffective. Therefore, there exists a need to develop safer and more effective agents. Platelet receptors are a target for anti-platelet agents and new generation agents function on a molecular level. The Cape chacma baboon (Papio ursinus) has been a popular model for the pre-clinical evaluation of anti-platelet agents. However, limited molecular data are available for these animals, restricting its translational value. The aim of this study was to characterize four common platelet receptors in the Cape chacma baboon and compare the results to human data. Methods: The platelet receptors P2Y12, glycoprotein (GP) VI, GPIIb/IIIa and GPIbα were selected for this study. Light transmission platelet aggregometry was performed to assess baboon platelet function; receptor number quantification was performed by flow cytometry; and Sanger sequencing was done on genomic baboon DNA. All results were compared to normal human data. Results: Baboon ADP-induced platelet aggregation results were significantly different from normal human results, even at ADP levels four times (40 μM) the highest human concentration of 10 μM. Baboon collagen-induced aggregation remained significantly different at twice (8 μg/ml) the highest human concentration of 4 μg/ml. However, the differences in collagen-induced aggregation results were not clinically relevant from the human results, because all except one result (at 8 μg/ml) fell within the normal human reference range. At double the highest human concentration for ristocetin (2.5 mg/ml) baboon platelets gave statistically similar results. At double the highest human concentration (1 mg/ml) arachidonic acid results remained significantly different between baboons and human. Baboon quantification results showed a 37% increase in GPIIb, 27% increase in GPIIIa and 25.5% increase in GPIbα. GPVI quantification failed due to non-reactive monoclonal antibodies. P2Y12 quantification was not possible, as no commercial monoclonal antibodies exist for it. The P2Y12 protein sequence was 98.8% similar. It differed by only four amino acids, none of which have been described as functionally essential. The GPVI protein sequence showed 95% similarity. It included a 14 amino acid difference and a three amino acid deletion. One change was at a region where an amino acid change has been implicated in reduced collagen-induced platelet aggregation in humans. Two differences were directly adjacent to a collagen-binding amino acid. The deletion was within the signalling region of GPVI. Exon 28 of GPIIb could not be sequenced. The GPIIb protein sequence for exon 1-27 was 98.2% similar and for exons 29-30 there was 98.3% similarity. There was an 18 amino acid difference. One amino acid change was in the ligand-binding region. The GPIIIa protein sequence was 99.6% similar, with three amino acid changes. One change was in the ligand-binding region. 54 amino acid changes were found in GPIbα. The protein sequences of the signal peptide, VWF-binding-, PEST / macroglycoprotein-, transmembrane- and cytoplasmic domains showed 93.8%, 89.4%, 57.9%, 90.5% and 95.0% similarity, respectively. 246 bases of GPIbα failed to sequence. Discussion and Conclusion: Sequentially and functionally baboon P2Y12, GPIIb/IIIa and GPIbα is comparable to humans. The higher agonist-levels needed for baboon platelet aggregation may be attributed to the increase in surface receptor numbers. However, receptor-number, optimal agonist concentrations and potentially inhibiting amino acid changes should be noted for future studies. Non-reactive antibodies and changes in critical amino acids caused the baboon GPVI to be not comparable to humans. The Cape chacma baboon (Papio ursinus) is therefore, deemed a suitable animal model for the evaluation of human-targeted anti-platelet agents directed against the receptors P2Y12, GPIIb/IIIa and GPIbα, but not for the evaluation of human-targeted anti-GPVI agents.