The renin-angiotensin system (RAS) is widely known as an important regulator of systemic blood pressure, but it also plays an important role in the pathophysiology of thrombosis. Among the pathways of RAS, the ACE2/Ang-(1-7)/MAS axis counter regulates the Ang-II/AT1 axis and relays on metabolites which are biologically active through the whole body. Preliminary work from our group has demonstrated the presence of MAS and MrgD1 receptors on human platelets, but its potential effects on platelet function have yet to be confirmed. In this study, we describe the effects of different RAS compounds (Ang-I, Ang-II, Ang-(1-7), Ang-(1-9), Alamandine) on the function of healthy human’s platelets in vitro, focusing on platelet aggregation and adhesion.
Platelet function was measured in a HTS (high-troughput screening) format using platelet-rich plasma (PRP) and washed platelets (WP) obtained from healthy donors. For platelet aggregation, PRP and WP were incubated with agonists, and the plate was shaken for 15 min at 1200 rpm at room temperature. Immediately afterwards, platelet aggregation was assessed by measuring ab- sorbance (= light transmission aggregometry). For platelet adhesion, the plate was first incubated with platelet-stimulating agonists, then platelets and the corresponding RAS-agonist were added and incubated. Only adherent platelets were retained and the liquid was discarded. After incubation with BCECF.AM, platelet adhesion was assessed recording fluorescence with a spectrophotometer.
Our aggregation assay showed one significant result for WP activated with Collagen-I (20 μg/mL) and incubated with Ang-(1-9) (0.1 nM), which induces an inhibited aggregation (-42%±21).
Our adhesion assay showed some significant deviations of which the following were in a physiological range : platelets with Ang-(1-7) (1 μM - 10 μM) and Collagen-III (8 μg/mL), showing an inhibition of adhesion (1 nM : -44%±11, 10 nM : -53%±12). Furthermore, a number of significant deviations were found at extra-physiological concentrations. Platelets with Ang-I (1 mM) show a significant change if triggered by Collagen-I at 8 μg/mL (+250%±71), CRP at 10 μg/mL (+178%±27) and Fibrinogen at 1 μg/mL (-40%±9). Platelets incubated with Ang-(1-9) (1 mM) and Laminin 411 (15 μg/mL) show an inhibited adhesion (-64%±14). Platelets with Alamandine (100 μM) and Collagen-III (8 μg/mL) also showed inhibited adhesion (-33%±8).
As almost all our findings occur in an extraphysiological range, we conclude that the RAS com- pounds have no effect on platelet adhesion or aggregation of platelets from healthy donors, except maybe an inhibition of adhesion with Ang-(1-7) with Collagen-III. Further studies should investigate the effect of the RAS compounds on treated and hypertensive patients and platelets.