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

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dc.contributor Badenhorst, Philip N.
dc.contributor De Kock, André
dc.creator Janse van Rensburg, Walter James
dc.date 2016-01-12T09:43:19Z
dc.date 2016-01-12T09:43:19Z
dc.date 2015
dc.date.accessioned 2018-06-24T05:26:56Z
dc.date.available 2018-06-24T05:26:56Z
dc.identifier http://hdl.handle.net/11660/2147
dc.identifier.uri http://hdl.handle.net/11660/2147
dc.description 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.
dc.description Afrikaans: Agtergrond: Akute koronêre sindroom is wêreldwyd ‘n belangrike oorsaak van morbiditeit en mortaliteit. Anti-plaatjiemiddels word vir voorkoming en behandeling gebruik, maar die beskikbare produkte het newe-effekte en is nie ewe effektief nie. Daarom is daar 'n behoefte om veiliger en meer doeltreffende middels te ontwikkel. Plaatjieoppervlak-reseptore is 'n teiken vir anti-plaatjiemiddels en die nuwe generasie middels funksioneer op 'n molekulêre vlak. Die Kaapse bobbejaan (Papio ursinus) is 'n gesogte model vir die pre-kliniese evaluering van nuwe antiplaatjiemiddels. Die beskikbare molekulêre data vir hierdie diere is egter onvoldoende en beperk die oorplasingswaarde van die spesie. Die doel van hierdie studie was dus om vier algemene plaatjiereseptore in die Kaapse bobbejaan te karakteriseer en die resultate met beskikbare menslike data te vergelyk. Metodes: Die plaatjiereseptore P2Y12, glikoproteïen (GP) VI, GPIIb/IIIa en GPIbα is vir hierdie studie geselekteer. Lig-oordrag-plaatjie-aggregometrie is uitgevoer om bobbejaan-plaatjiefunksie te bepaal; reseptorkwantifisering is deur middel van vloeisitometrie gedoen; en Sanger DNS-volgordebepaling is op genomiese bobbejaan-DNS gedoen. Alle resultate is met normale menslike data vergelyk. Resultate: Bobbejaan-ADP-geïnduseerde plaatjieaggregasie resultate het beduidend van dié by die mens verskil, selfs met ADP-vlakke vier maal hoër as die hoogste menslike konsentrasie (40 mM). Bobbejaan-kollageen-geïnduseerde aggregasie het ook beduidend van die mens verskil teen twee keer die hoogste menslike konsentrasie (8 mg/ml). Die verskille was egter nie klinies relevant nie aangesien almal behalwe een bobbejaan se resultate (teen 8 mg/ml) binne die normale menslike verwysingsreikwydte geval het. Teen dubbel die hoogste menslike konsentrasie vir ristocetin (2.5 mg/ml) het bobbejaanplaatjies statisties soortgelyke agglutinasieresultate getoon. Teen dubbel die hoogste menslike konsentrasie vir aragidoonsuur (1 mg/ml) het resultate aanduidend verskillend gebly. Bobbejaan-kwantifiseringresultate het in vergelyking met mensplaatjies 'n 37% toename in GPIIb, ‘n 27% toename in GPIIIa en ‘n 25.5% toename in GPIbα getoon. GPVI kwantifisering het misluk as gevolg van nie-reaktiewe monoklonale teenliggaampies. P2Y12 kwantifisering was nie moontlik nie, aangesien daar geen kommersiële monoklonale teenliggaampies teen P2Y12 bestaan nie. Die P2Y12-aminosuurvolgorde was 98.8% soortgelyk. P2Y12 het met net vier aminosure verskil, waarvan geeneen as funksioneel noodsaaklik beskou word nie. Die GPVI-aminosuurvolgorde het 'n 95% ooreenstemming. Dit sluit 'n 14- aminosuurverskil en 'n drie-aminosuurdelesie in. Een verandering was in 'n gebied waar 'n aminosuurverandering in mense beskryf is wat tot verminderde kollageengeïnduseerde plaatjieaggregasie lei. Twee verskille was ook direk aangrensend aan ‘n kollageen-bindende aminosuur. Die delesie is geleë in die seinstreek van GPVI. Ekson 28 van GPIIb se volgordebepaling was onsuksesvol. Die GPIIbaminosuurvolgorde vir ekson 1-27 was 98.2%, en vir eksons 29-30, 98.3% ooreenstemmend met 'n 18-aminosuurverskil. Slegs een aminosuurverandering was in die ligandbindingstreek. Die GPIIIa-aminosuurvolgorde was 99.6% ooreenstemmend, met drie aminosuurveranderinge. Een verandering was in die ligandbindingstreek. 54-aminosuurveranderings is in die GPIbα opeenvolging gevind. Die aminosuurvolgorde van die seinpeptied, VWF-bindingsdomein, PEST / makroglikoproteïen-, transmembraan- en sitoplasmiese-domein het onderskeidelik 93.8%, 89.4%, 57.9%, 90.5% en 95.0% ooreenstemming getoon. 246 basisse van GPIbα se basisopeenvolgingbepaling het misluk. Bespreking en Gevolgtrekking: Op basisopeenvolging- en funksionele vlak is die bobbejaan P2Y12, GPIIb/IIIa en GPIbα vergelykbaar met mense. Die hoër agonisvlakke wat vir bobbejaan-plaatjieaggregasie benodig word kan aan ‘n toename in oppervlakreseptor toegeskryf word. Reseptor-hoeveelheid, die optimale agonis konsentrasies en moontlike inhiberende aminosuurverskille moet egter in gedagte gehou word met toekomstige studies. Nie-reaktiewe teenliggaampies en veranderinge in kritiese aminosure het veroorsaak dat GPVI nie vergelykbaar met mense is nie. Die Kaapse bobbejaan (Papio ursinus) word dus as ‘n geskikte dieremodel vir die evaluering van mensgerigte antiplaatjiiemiddels teen die reseptore P2Y12, GPIIb/IIIa en GPIbα beskou, maar nie vir die evaluering van mensgerigte anti-GPVI middels nie.
dc.language en
dc.publisher University of the Free State
dc.rights University of the Free State
dc.subject Acute coronary syndrome
dc.subject Anti-platelet agents
dc.subject Platelet receptors
dc.subject P2Y12
dc.subject Glycoprotein VI
dc.subject Glycoprotein IIb/IIIa
dc.subject Glycoprotein Ibα
dc.subject Cape chacma baboon
dc.subject Tromboses -- Drug therapy
dc.subject Coronory heart disease
dc.subject Chama baboon
dc.subject Thesis (Ph.D. (Haematology and Cell Biology))--University of the Free State, 2015
dc.title Comparison of platelet receptors P2Y12, GPIIB/IIIA, GPVI, and GPIBα between the Cape chacma baboon and the human
dc.type Thesis


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