Platelets Serve as Circulating Mediators of Cardioprotection by Remote Ischemic Conditioning in Healthy Volunteers

Abstract

Background Brief cycles of occlusion and reperfusion in a tissue/organ remote from the heart protect the myocardium from sustained severe ischemia/reperfusion injury. Such remote ischemic conditioning (RIC) is operative in all species tested so far, including humans. Although unclear in its details, the underlying signal transfer involves humoral factors as evidenced by the transfer of cardioprotection via plasma or plasma-derivatives from one individual to another individual´s heart, even across species. We have recently demonstrated, that the platelet count increases in response to RIC in healthy volunteers within 60 min.1 Aim To study whether or not platelets serve as carriers for RIC's cardioprotective signalling. Methods Venous blood was collected from healthy volunteers (2 females/ 3 males, 32±7 years) before and 60 min after RIC (3 × 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/ 5 min deflation). Blood (80 mL) was drawn into polypropylene tubes containing sodium citrate (3.2 %), apyrase (0.1 U/ml) and prostaglandin E1 (1 µmol/L)and centrifuged (100 × g) for 15 min at room temperature. The obtained platelet-rich plasma was centrifuged again (1000 × g) for 12 min and the platelet pellet washed twice with buffer (pH 6.5) and finally re-suspended in buffer containing bovine serum albumin (3.5 mg/mL, pH 7.35). The platelet count was adjusted to 2.5 × 103 platelets/µL. This platelet suspension was either used for infusion experiments (washed platelets) or further processed by supplementing CaCl2 (1 mmol/L) followed by centrifugation (14.000 × g) at 4oC for degranulation; the supernatant was then retrieved (releasate). Functionality of washed platelets was assessed by light transmission aggregometry in response to stimulation by protease-activated receptor 1 activating peptide. Rat hearts were isolated and buffer-perfused at constant pressure (70 mmHg). Hearts were infused with washed platelets (25.000/mL × min) or with the releasate (final dilution 1:10) for 8 min followed by 2 min washout before being subjected to 30 min global zero-flow ischemia followed by 120 min reperfusion. Infarct size was demarcated by triphenyl tetrazolium chloride staining and calculated as percent of total ventricular mass. Results Infarct size was reduced by infusion of washed platelets retrieved after RIC in comparison to those retrieved before RIC (Figure A). Similar results were obtained by infusion of platelet-free releasates after RIC (Figure B). Conclusion In response to RIC in healthy volunteers, platelets serve as carriers for cardioprotective factors. The precise transfer of platelet protective factors from the circulation to the myocardium and the nature of the platelet-derived factors remain to be identified. 1H R Lieder et. al., P2589, Europ Heart J, 40 (1), 2019, ehz748.0915.