Comparison of platelet receptors P2Y12, GPIIB/IIIA, GPVI, and GPIBα between the Cape chacma baboon and the human

[摘要] English: Background: Acute coronary syndrome is globally a major cause of morbidity andmortality. Treatment and prevention involve the use of an anti-platelet agent. Thecurrent available agents have either side-effects or are relatively ineffective.Therefore, there exists a need to develop safer and more effective agents. Plateletreceptors are a target for anti-platelet agents and new generation agents function ona molecular level. The Cape chacma baboon (Papio ursinus) has been a popularmodel for the pre-clinical evaluation of anti-platelet agents. However, limitedmolecular data are available for these animals, restricting its translational value. Theaim of this study was to characterize four common platelet receptors in the Capechacma baboon and compare the results to human data.Methods: The platelet receptors P2Y12, glycoprotein (GP) VI, GPIIb/IIIa and GPIbαwere selected for this study. Light transmission platelet aggregometry was performedto assess baboon platelet function; receptor number quantification was performed byflow cytometry; and Sanger sequencing was done on genomic baboon DNA. Allresults were compared to normal human data.Results: Baboon ADP-induced platelet aggregation results were significantlydifferent from normal human results, even at ADP levels four times (40 μM) thehighest human concentration of 10 μM. Baboon collagen-induced aggregationremained significantly different at twice (8 μg/ml) the highest human concentration of4 μg/ml. However, the differences in collagen-induced aggregation results were notclinically 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 humanconcentration for ristocetin (2.5 mg/ml) baboon platelets gave statistically similar results. At double the highest human concentration (1 mg/ml) arachidonic acidresults remained significantly different between baboons and human.Baboon quantification results showed a 37% increase in GPIIb, 27% increase inGPIIIa and 25.5% increase in GPIbα. GPVI quantification failed due to non-reactivemonoclonal antibodies. P2Y12 quantification was not possible, as no commercialmonoclonal 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 proteinsequence showed 95% similarity. It included a 14 amino acid difference and a threeamino acid deletion. One change was at a region where an amino acid change hasbeen implicated in reduced collagen-induced platelet aggregation in humans. Twodifferences were directly adjacent to a collagen-binding amino acid. The deletion waswithin the signalling region of GPVI. Exon 28 of GPIIb could not be sequenced. TheGPIIb protein sequence for exon 1-27 was 98.2% similar and for exons 29-30 therewas 98.3% similarity. There was an 18 amino acid difference. One amino acidchange 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 signalpeptide, VWF-binding-, PEST / macroglycoprotein-, transmembrane- andcytoplasmic 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 forbaboon platelet aggregation may be attributed to the increase in surface receptornumbers. However, receptor-number, optimal agonist concentrations and potentiallyinhibiting amino acid changes should be noted for future studies. Non-reactiveantibodies and changes in critical amino acids caused the baboon GPVI to be notcomparable to humans. The Cape chacma baboon (Papio ursinus) is therefore,deemed a suitable animal model for the evaluation of human-targeted anti-plateletagents directed against the receptors P2Y12, GPIIb/IIIa and GPIbα, but not for theevaluation of human-targeted anti-GPVI agents.

[发布机构] University of the Free State