Changes in both calcium pool size and morphology of human platelets incubated in various concentrations of calcium ion. Calcium-specific bleb formation on platelet-membrane surface.
In this study, the response of gel-filtered human platelets to extracellular Ca2+ at Ca2+ concentrations [Ca2+]o of 1-10 mM was investigated. The distribution of Ca2+ among various pools was studied using: (1) quin2, to estimate the cytosolic free Ca2+ concentration ([Ca2+]i); and (2) 45CaCl2 plus EGTA, to quantitate the sizes of the EGTA-releasable, EGTA-nonreleasable and surface-bound Ca2+ pools. The morphological changes were revealed by scanning electron-microscopy (scanning EM), and the effect on thrombin-stimulated aggregation was examined using an aggregometer. Platelets continuously sequestered Ca2+ into both EGTA-releasable and EGTA-nonreleasable pools to maintain a low [Ca2+]i level. The rate of sequestration to the EGTA-releasable pool was independent of [Ca2+]o, while that of the EGTA-nonreleasable pool exhibited first-order kinetics. The cell morphology changed gradually from discoid to the tadpole-like type, and finally to irregular forms. This morphological change correlated with the gradual increase in [Ca2+]i. The EGTA-nonreleasable pool saturated at about 3000 pmol/10(8) cells. This saturation resulted in a drastic increase in the EGTA-releasable pool size, and the cell was lysed concomitantly. The maximum safety capacity of the EGTA-releasable pool was estimated to be 1100 pmol/10(8) cells. The contribution of the cellular compartments to these two pool sizes is extensively discussed. The surface-bound pool size also increased continuously. When two different capacities were reached, i.e., 160 and 600 pmol/10(8) cells, the binding rate increased above the initial rate by 7- and 11-fold, respectively. Hence, the surface-binding capacity might be a critical factor which alters the membrane structure and exposes more binding sites. The cell surface appeared to have blebs, after the binding size had reached more than 600 pmol/10(8) cells. Bleb formation resulted in the inhibition of platelet function. Divalent cations, such as Mg2+, Sr2+ and Ba2+ did not cause bleb formation, which could mean that this formation is a Ca2+-specific phenomenon.