Haematology and Cell Biology

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Browsing Haematology and Cell Biology by Advisor "De Kock, André"

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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.
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    DNA characterization of the FGA locus in the human genome
    (University of the Free State, 2002-11) Asfaw, Estifanos Kebede; De Kock, André; Pretorious, G. H. J.
    English: The human alpha fibrinogen (FGA) short tandem repeat locus is found in the long arm of chromosome 4. It is located in the third intron of the alpha fibrinogen gene. This complex highly polymorphic tetranucleotide repeat locus together with other STR DNA markers is extensively used in personal identification in medical and forensic sciences. STRs are also used to study genetic variation in distinct ethnic groups and in disease diagnosis. More than 80 alleles have been reported for this locus from various population frequency studies. A few sequence studies have also reported 11 sequence variants to date. The FGA locus was found to have high heterozygosity and power of discrimination. The aim of this study was to characterise the sequence of microvariant and off-ladder complete and microvariant alleles of the FGA locus that were observed during routine paternity analyses. The characterization of the sequence of as many as possible of alleles observed in our study population would also be attempted. A total of 62 DNA specimens were selected and sequence characterized either for one or both alleles. The DNA specimens were 52 from Negroid, 5 mixed ancestry, 4 Caucasian and 1 SAN origin. The PCR reaction was used to amplify the selected alleles. The band of interest was cut from the gel and purified in consecutive PCR and purification steps till separate single bands were obtained. The purified single bands were sequenced using a BigDye terminator ready reaction kit in both forward and reverse reactions separately. These products were precipitated with ethanol acetate and subjected to capillary electrophoresis on an ABIPrism 310 Genetic Analyser using POP6 polymer. The results were analysed using the "Sequence Analysis Software version 2.1". The data obtained were checked, printed and compared with STR analysis results and FGA sequence reports. From the selected 62 specimens a total of 76 complete and microvariant alleles, the size of which ranged between 16.1 (224bp) to 44.2 (337bp) were found. These represent 27 different alleles (13 complete and 14 interalleles). In this study 2 novel (previously undescribed) alleles (40.2 and 41.2) were found. Three sequence variants (26, 28 and 43.2) with two variants each were observed. Two alleles 43.2 and 44.2 that had reported sequence variants were found to have different sequence structures from the published sequences. Forty-nine of the 76 sequenced alleles were within ladder and the remaining 29 were off-ladder. Only 8.41% (4/49) of the within ladder alleles had been wrongly assigned allelic numbers with routine STR analysis. The difference between the routine assignment and the sequencing of these alleles was only 1 or 2 bp. In contrast, all of the 29 off-ladder alleles were wrongly assigned. In this instance the difference was 2 or more base pairs. Although this study was conducted on conveniently selected DNA samples, it had significant results. Three sequence variants, 2 newalleles and, 1 allele, which had been reported, but sequence had not been described was found. Additionally, two other alleles with reported sequences were found, but their sequence structure differed from the published sequences. The samples in this investigation were not representative of the population groups that are found in the Free State province and we suggest further population-based studies of STR loci that are commonly used in paternity and forensic investigation. The information obtained from such studies will disclose the frequency of sequence variant alleles.
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    "Killer-cell immunoglobulin-like receptor haplotype diversity in three Free State population groups"
    (University of the Free State, 2006-11) Louw, Marius; De Kock, André; Louw, Vernon; Coetzee, Marius
    English: In the foregoing project, an investigation was made into the relative KIR gene frequencies of three South African cohorts. Playing an important part in innate immunity, KIR fill a vital gap between viral onsets and cell mediated humeral immunity. Being able to sense when cells are abnormal, NK cells possess the ability to destroy cells which show altered HLA molecules during KIR/HLA interaction. Ethnic cohorts that were investigated included African black, mixed ancestry and the Caucasian populations. From these individuals DNA material was extracted using a “salting out” method before SSP-PCR genotyping. Seventeen primer pairs were used in the identification of individual KIR genes. PCR products were electrophoresed against a molecular weight marker in order to verify the correct fragment size. Products were viewed on a UV light where observations were noted, and indicated as present or absent. Data was recorded onto a spreadsheet indicating the absence or presence of each particular gene. Tabulated results were used in the construction of graphs as well as χ2 calculations. These graphs were used in the critical analysis of linkage disequilibrium as well as comparative analysis between the ethnic cohorts. Findings indicate that all framework genes are present in all cohorts. The Black African and mixed ancestry cohorts have not been genotyped for the KIR genes before. Investigation within non-framework genes revealed the identification of several new haplotypes, with the majority observed within the mixed ancestry cohort. Positive linkage disequilibrium was detected between 2DL2-2DS2 and 2DL5B-2DS5 for both the black African and Caucasian cohorts while 2DL1-2DL2 and 2DL5B- 2DS5 linkages were found in the mixed ancestry population. No negative linkages were observed for any of the three cohorts.