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Observations on the activity of nucleoside diphosphokinase on the external surface of washed rabbit platelets have suggested that phosphorylation of ADP at the expense of intracellular ATP occurs in association with aggregation. We have studied aggregation and shape change of washed human platelets labelled in vitro with 32Pi, using ADP or an analogue, 2-methylthio ADP, to induce aggregation in the presence of fibrinogen. EDTA was added during (30sec) or after (2min) aggregation, and the platelets were removed gently by centrifugation through silicone oil. Nucleotides were concentrated from the medium by absorption on, and elution from charcoal-coated beads, and were analysed by paper electrophoresis and HPLC. ATP (50-1OOuM) was added to some samples to trap any labelled ATP formed. This reduced the rate of aggregation but did not stop it. Less than 0.1% of the intracellular radioactivity was recovered as extracellular ATP and this amount was not signfleantly increased by ADP. Under these conditions less than 2000 molecules of ADP were phosphorylated for the aggregation of each platelet. This suggests that phosphorylation of ADP is not required for aggregation or for shape change.

Is Phosphorylation Of Adp Required For The Induction Of Shape Change And Aggregation?