Effect of osmotic shock and urea on phosphatidylserine scrambling in thrombocyte cell membranes

Abstract

Blood passing the renal medulla enters a strongly hypertonic environment challenging functional properties and survival of blood cells. In erythrocytes, exposure to hyperosmotic shock stimulates Ca(2+) entry and ceramide formation with subsequent cell membrane scrambling, an effect partially reversed by high concentrations of Cl(-) or urea. Cell membrane scrambling with phosphatidylserine exposure is part of the procoagulant phenotype of platelets. Coagulation in the hypertonic renal medulla would jeopardize blood flow in the vasa recta. The present study thus explored whether hypertonic environment and urea modify phosphatidylserine exposure of human platelets. FACS analysis was employed to estimate cytosolic Ca(2+) activity with Fluo3 fluorescence, ceramide formation, P-selectin, and glycoprotein IIb/IIIa activation with fluorescent antibodies and phosphatidylserine exposure with annexin V-binding. The spontaneous platelet aggregation was measured by impedance aggregometry. Hyperosmotic shock (addition of 500 mM sucrose or 250 mM NaCl) significantly enhanced cytosolic Ca(2+) activity, ceramide formation, phosphatidylserine exposure, platelet degranulation, and aggregability. Addition of 500 mM urea to isotonic saline did not significantly modify cytosolic Ca(2+) activity, ceramide abundance, or annexin V-binding but significantly blunted the respective effects of hypertonic shock following addition of 500 mM sucrose. In isotonic solutions, both ceramide (20 microM) and Ca(2+) ionophore ionomycin (0.5 microM) increased annexin V-binding, effects again significantly blunted by 500 mM urea. Moreover, oxidative stress by addition of 0.5 mM peroxynitrite increased cytosolic Ca(2+) activity and triggered annexin V-binding, effects again blunted in the presence of 500 mM urea. The observations reveal that hyperosmotic shock and oxidative stress trigger a procoagulant platelet phenotype, an effect blunted by the presence of high urea concentrations.