Ultrastructure of the platelet release reaction in response to various aggregating agents and their inhibitors

Platelet aggregation is thought to be mediated by the secretion, or 'release', of calcium and nucleotides from platelets in response to various aggregating agents. The present study describes the pattern of morphologic changes that accompany specifically such nucleotide and Ca⁺⁺ release as isolated from other events in platelet function. Washed human platelets were incubated at 37° C. for 5 to 10 min with thrombin, collagen, ADP, or epinephrine and fixed in 3% glutaraldehyde and 1% OsO₄ for electron microscopy. The supernatants, and in some cases the platelet pellets as well, were assayed for nucleotides and calcium released. At 37° C., where maximal release of nucleotides and Ca⁺⁺ occurs in response to thrombin and collagen, α granules, thought to contain the substances released, almost completely disappear. A complex lamellar vacuolar system, continuous with the cell membrane and probably the pathway for outward movement of nucleotides, now dominates platelet ultrastructure. Wisps of electron dense material, comprising fibers 50 to 80 Å in diameter with an occasional 180 to 220 A periodicity, are conspicuous in the lamellar spaces. These wisps are morphologically similar to fibrin precipitated either in vivo or in vitro. Others have described how epinephrine and ADP are less effective in a washed platelet system in stimulating release; the above morphologic changes associated with release are not seen. Prostaglandin E₁ and theophylline, potent inhibitors of aggregation, inhibit thrombin induced nucleotide and Ca⁺⁺ release at 37° C.; many of the ultrastructural events described are also inhibited. Platelets incubated at 4° C. together with the releasing agents, or at 37° C. without them, secrete only minimal amounts of nucleotides and calcium and correspondingly undergo none of the morphologic changes that characterize maximal release. Different degrees of release are thus well correlated with a pattern of morphologic changes whose occurrence may well be integral in subsequent platelet aggregation, clot retraction, and hemostasis.