Haematology and Cell Biology

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Browsing Haematology and Cell Biology by Subject "Acute coronary syndrome"

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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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    The evaluation of tirofiban hydrochloride in a high shear rate arterial thrombosis model in baboons
    (University of the Free State, 2009-11) Janse van Rensburg, Walter James; Meiring, S. M.; Roodt, J. P.
    English: Background: Acute coronary syndrome (ACS) is a major cause of mortality and morbidity world-wide, and is responsible for roughly 2.5 million hospital admissions world-wide annually. ACS is commonly associated with platelet thrombus formation on disrupted atherosclerotic plaques, therefore effective and safe anti-platelet drugs are needed to help treat and prevent ACS. The current most popular anti-platelet drugs are associated with increased bleeding risk and reduced efficacy, thus drugs with a wider therapeutic window (more efficacy with less bleeding) need to be developed. Tirofiban hydrochloride is a small, short half-life molecule that inhibits platelet aggregation by antagonising the glycoprotein IIb/IIIa receptor on platelets preventing fibrinogen and von Willebrand factor to cross-link platelets, thereby inhibiting the final pathway of platelet aggregation. Tirofiban hydrochloride was believed to be a very promising drug due to its short half-life, as an antidote strategy is not needed to reverse adverse bleeding events, but it soon fell out of favour when it was found not to be as effective as for example abciximab in preventing ischaemic events. This was possibly due to the recommended dose being suboptimal. Methods and Results: We studied the efficacy of tirofiban hydrochloride to inhibit platelet thrombus formation on an injured and partially occluded artery by evaluating the effect of escalating doses on cyclic flow reduction (CFR) formation in a high shear arterial thrombosis model in baboons, and also evaluated its safety in two different bleeding models. We then compared our results to results found in the same model using clopidogrel. A significant effect on the number of CFRs was only observed after injection of three times (30 μg/kg bolus plus 0.45 μg/kg/min infusion) the therapeutic dose tirofiban, but it was a weak inhibitor at this dose. Only after injection of nine times (90 μg/kg bolus plus 1.35 μg/kg/min infusion) the recommended therapeutic dose, a strong complete inhibition was observed. A further dose of 27 times (270 μg/kg bolus plus 4.05 μg/kg/min infusion) the recommended therapeutic dose was given to evaluate the effect of an overdose on the bleeding tendency. A significant prolongation in bleeding time (3.05 minutes to 11.90 minutes) was observed after injection of nine times the therapeutic dose, an average 2.7 ± 2.44 fold increase in blood loss was also observed at this dose. A maximum increase in blood loss of an average of 3.4 ± 1.77 fold was seen after injection of 27 times the therapeutic dose. The efficacy of tirofiban hydrochloride was comparable to that of clopidogrel found in earlier studies, but the blood loss was much less when compared to the average 4.3 ± 2.6 fold increase with clopidogrel at 2.5 mg/kg and 8.0 ± 5.0 fold increase at 5 mg/kg. Conclusion: Tirofiban hydrochloride is an effective anti-platelet drug, but only offers adequate protection against arterial thrombosis at a dose between three and nine times the recommended therapeutic dose. However, it still remains safer in terms of bleeding than the most common anti-platelet drugs used today. We recommend that further in vivo studies be done to determine the optimal dose for tirofiban hydrochloride treatment, and that new clinical trials be done with higher dose tirofiban hydrochloride.