Print version
Click to downloadSearch Pub Med
Ex vivo assay to investigate the endogenous inhibitory influence on platelets of endothelial derived vascular mediators Constant exposure of platelets within the circulation to the anti-thrombotic mediators prostaglandin I2 (PGI2) and nitric oxide (NO), derived from the vascular endothelium, stimulates production of the intraplatelet cyclic nucleotides cAMP and cGMP and so maintains an inhibitory tone. Critical to the dynamic regulation of platelet tone is the rapid interplay between the rate of cyclic nucleotide synthesis and the rate of cyclic nucleotide degradation by phosphodiesterase enzymes (PDEs). As PDEs act extremely rapidly, ex vivo and in vitro studies of platelet function are commonly conducted in the absence of this physiological inhibitory tone. We have recently developed a miniaturized whole blood aggregation assay (1) that, coupled with our ‘Optimul’ dried-agonist plates (2), permit platelet studies to be initiated as soon as blood is drawn. Here we sought to determine whether this approach may allow for ex vivo detection of endothelial regulation of platelet function. Aged but healthy male volunteers (66yo – 75yo) were recruited according to an inclusion criteria of non-smokers, no medication, no cardiovascular disease or other chronic diseases, and BMI < 30. The study was approved by the Ethics Committee of Copenhagen and Frederiksberg communities (H-3-2012-176) and conducted in accordance with the guidelines of the Declaration of Helsinki. Catheters (20G) were placed in both femoral arteries, and the right femoral vein from where blood was obtained after an infusion with acetylcholine chloride (i.a, Michol 100μg min-1(kg leg mass)-1, 5mins) prior to and post (1 hour) single dose sildenafil (Sildenafil Actavis,100mg, p.o.). Femoral arterial blood flow was measured with ultrasound Doppler (Logic E9, GE Healthcare). Platelet aggregation was performed by immediately (<60secs) placing 45µl aliquots of citrated whole blood (0.32% w/v) into the individual wells of a 96-well plate, with or without agonist, and vigorously mixing (1200rpm, 37°C, 5min). At the end of this period samples were removed, diluted and labelled using a platelet specific antibody to permit single platelet counts by flow cytometry. The reductions in single platelet counts of each sample relative to vehicle stimulation were calculated and concentration response curves for platelet aggregation were constructed. Infusion of acetylcholine led to an increase in femoral blood flow from 0.2 ± 0.1L/min to 1.8 ± 0.3L/min. Sildenafil had no significant effect on blood flow changes (post sildenafil flow: 0.4 ± 0.1L/min and 2.0 ± 0.2L/min, p>0.05 by paired t test respectively). Following treatment of volunteers with sildenafil the (log) concentration of collagen causing 25% aggregation was increased from -5.7±0.2 to -5.0±0.3 g/ml, representing a 5-fold reduction in potency. This loss of platelet reactivity was reflected in a reduction in the maximally achieved platelet aggregation in response to collagen from 53±10% to 27±8% (p<0.05 by two-way ANOVA, n=4 for all). The rapid loss of cyclic nucleotide tone within the platelet upon venepuncture and the inherent delays in existing protocols means that an important regulator of platelet reactivity is largely absent during routine ex vivo testing. Our results suggest that immediate testing of platelets within whole blood may provide a method that is sensitive to the levels of circulating platelet cyclic nucleotide tone. Such an approach could allow for closer investigation of the critical role endothelial function plays in regulating platelet reactivity in vivo in humans. 1. Proceedings of the British Pharmacological Society at http://www.pa2online.org/abstracts/Vol11Issue3abst206P.pdf 2. Chan et al, Platelets 22:485-494, 2011.
|