Reassessment of the evidence for the role of secreted ADP in biphasic platelet aggregation. Mechanism of inhibition by creatine phosphate plus creatine phosphokinase.

The observation that platelet secretion occurs in parallel with the second wave of platelet aggregation necessitates reassessment of the generally accepted concept that secreted ADP is the cause of second-wave aggregation. The major evidence for an involvement of secreted ADP is inhibition of second-wave aggregation by enzyme-catalyzed removal of ADP, interpreted as removal of secreted ADP. An alternative hypotheses, that the observed inhibition is due to a decrease in potentiation by extracellular ADP present prior to addition of stimulus, has been tested with the enzyme system CP plus CPK (CP/CPK). Low levels of CP/CPK inhibited gamma-thrombin-induced second-wave aggregation only after preincubation. When the second wave of gamma-thrombin- or epinephrine-induced aggregation was inhibited by very high levels of CP/CPK, the inhibition was overcome by an increase in the level of stimulus. These results are inconsistent with the idea that CP/CPK blocks second-wave aggregation by removing secreted ADP, but they are consistent with a decreased potentiation of the platelets due to a lower level of extracellular ADP prior to stimulation. Freshly prepared platelet-rich plasma contained a mean (n = 16) of 12 nM extracellular ADP. These data demonstrate that inhibition by CP/CPK cannot be taken as evidence for the involvement of secreted ADP.