Characterization of the calcium-sequestering process associated with human platelet intracellular membranes isolated by free-flow electrophoresis

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Abstract
By using density-gradient fractionation and high-voltage free-flow electrophoresis, human platelet membranes were separated into highly purified subfractions of surface (SM) and intracellular (IM) origin. Associated exclusively with the IM fraction is an ATP-dependent Ca2+ uptake that, in the absence of oxalate, reaches steady-state levels in 5-10 min. When Ca2+-EGTA [ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid] buffers were used to control the external Ca2+ concentrations (range 0.1-50 .mu.M) there was an increase in the intravesicle steady-state level of Ca2+ up to 10 .mu.M external Ca2+ concentration. Above this level the intravesicle space becomes saturated at a concentration between 10 and 20 nmol of Ca2+ .cntdot.(mg of protein)-1. The ionophore A23187 promotes a rapid and almost total release of the sequestered Ca2+ (> 90%, t1/2 [half-time] 1-2 min). The presence of oxalate in the external medium greatly enhances the Ca2+ accumulation to levels as high as 200 nmol .cntdot.(mg of protein-1), but the uptake process is more variable and rarely reaches steady-state level even after 2 h incubation. Moreover, accumulation in the presence of oxalate effects ionophore release with less than 80% depletion in 45-60 min. These findings, taken together with the known presence in the platelet of a wide variety of functional and metabolic processes triggered by this cation, suggest that the platelet IM has a key role in controlling cytosolic Ca2+ concentrations.