Summary
Incubation of washed human platelets at pH 5.3 at 37° C induces a slow secretion of
stored compounds. The secretion is 60% inhibited by metabolic inhibitors and blocked
at 0° C.
The major changes in the metabolic adenine nucleotide levels in the platelets precede
the secretion, which is accompanied by a drop in ADP and an increase in inosine and
hypoxanthine. The incubation does not lead to significantly increased loss in cytoplasmic
compounds from the platelets.
Ultrastructural changes which accompany pH 5.3-induced secretion include a coalescence
of cytoplasmic material in the center and a decrease in densely stained material in
the periphery of the platelet. These changes are only seen by prolonged incubation
(60 min). In the presence of 2 mM fluoride similar changes are seen after 3 min incubation.
Other changes induced by fluoride, i. e. the formation of swollen areas in the periphery
of the platelet are not induced by incubation at pH 5.3 alone.
The metabolic changes seen by incubation at pH 5.3 are to a great degree reversible,
even when secretion has been induced. Although secretion cannot be induced by incubation
of platelets at pH 7.6 alone, pH 5.3-induced secretion continues at the higher pH.
Whereas incubation at pH 5.3 in the cold does not induce secretion, incubation with
2 mM fluoride under these conditions will result in at least 50% secretion of stored
compounds when pH and temperature are increased, indicating that fluoride has been
taken up by the platelet by passive diffusion. This concentration of fluoride is below
the threshold for induction of secretion at pH 7.6.
In contrast to secretion induced by thrombin, and in agreement with the findings with
fluoride, the calcium ionophore A 23187 is equally effective as inducer of secretion
at pH 5.3 and at pH 7.6. This also agrees with the findings that when secretion has
been “initiated” in the cold with thrombin at pH 7.4, the extrusion which takes place
at higher temperature has a shift in pH optimum towards the acid pH and is only partially
inhibited at pH 5.5, while the action of agents which effect platelet secretion from
the outside, including added Ca++, is blocked below pH 6, and the induction effect of thrombin below pH 6.5.
We propose that the agents which cause secretion at low pH have a direct intracellular
effect, probably on the level of cytoplasmic Ca++, while the agents which are inhibited by the low pH act indirectly via external membrane
sites.
