Investigation of the role of platelet turnover on platelet inhibition and thrombus formation with regard to antiplatelet therapy

Abstract

Aspirin is often prescribed in patients with acute coronary syndromes together with an ADP-P2Y12 inhibitor such as clopidogrel or prasugrel, an established treatment protocol called dual antiplatelet therapy. Although short lived, these drugs act irreversibly upon their targets and so are used as once-a-day treatments. The daily platelet turnover in healthy humans is approximately ten to fifteen per cent but can be considerably increased in disease conditions such as diabetes or chronic kidney disease. This leads to the daily emergence of an uninhibited subpopulation among the larger population of inhibited platelets. The aim of this thesis was the investigation of the role and contribution of this minority of uninhibited platelets in platelet aggregation and thrombus formation. Investigations found a nonlinear increase in arachidonic acid (AA)-induced aggregation in PRP containing rising proportions of uninhibited platelets mixed with aspirin-treated platelets. In contrast, stimulation of PRP containing mixed proportions of prasugrel active metabolite (PAM)-treated and uninhibited platelets by ADP showed a linear relationship between aggregatory responses and proportions of uninhibited platelets. This indicated that only uninhibited platelets would contribute to aggregate formation. However, confocal images of prelabelled platelets allowing the differentiation between inhibited and uninhibited platelets, revealed clustering of uninhibited platelets in the centre of aggregates surrounded by PAM-inhibited platelets. In contrast confocal images of uninhibited platelets combined with aspirin-treated platelets showed random, intermingled platelet distribution when stimulated by AA. Further in depth analyses by confocal microscopy and flow cytometry found the recruitment of PAM-inhibited platelets to be an active αIIbβ3-mediated process, independent of thromboxane A2 release. Whereas clustering of uninhibited platelets was not detected under flow conditions, an increase of platelet deposition with rising proportions of aspirin and/or PAM-free platelets was observed. These experiments clearly demonstrate that a general population of platelets can contain subpopulations that respond differently to overall stimulation of the population.