Cation Contributions To Potentiometric Dye Responses In Human Platelets

The potentiometric dye, Di-S-C3(5) (0.9μM) was incubated with stirred suspensions of-3washed human platelets (5-6 × 104μl) in buffer containing 137mM NaCl, 2.7mM KCl, 0.2% dextrose, and 25mM Tris-HCl, pH 7.4 in an Ami neo-Bowman fluorometer, with excitation λ 620nm and emmision λ 680nm. Equilibrium levels of fluorescence (F) nearly doubled as external potassium concentration (K °) was increased from 2.7mM to 105.6mM. Addition of°2mM CaCl2 always produced immediate transient increases in F, regardless of K0 , in contrast to the increases or decreases produced by the potassium ionophore, valino- mycin (VAL) (5μM) in adherance to the electrochemical gradient determined by the choice of K°. Equilibrium F was also relatively insensitive to external calcium (Ca°) at either high or low levels of K . However, add?tion of the calcium ionophore, A23187 (10μM) produced immediate increases in F, with peak values (F-A23peak) increasing sharply with increasing Ca°. At a given Ca the F-A23peak was insensitve to K ° however, with the prior addition of VAL, F-A23peak became sensitive to K , particularly so at the lowest values of K° , where the F-A23peak was significantly lower in tne presence than in the absence of VAL. Replacement of sodium by choline had no significant effect on equilibrium F or on responses to VAL or to A23187- Platelet agglutination induced by ristocetin plus cryoprecipitate was not accompanied by similar increases in F.These findings suggest that Di – S–C3 F reflects a strong contribution of potassium permeability in the resting state, with relatively little contribution by calcium. Production of an activated state in platelets by A23187, in contrast, results in an increase in F that appears to reflect a major contribution by calcium.