Summary
Thrombin and other agonists that induce secretion and aggregation in human platelets
also activate phospholipase D (PLD), but the signaling cascade leading to activation
of PLD in human platelets is not yet clear. We have determined that apyrase, which
scavenges ADP secreted during platelet activation, is able to block or reduce the
PLD activation stimulated by low (0.1 U/ml or less) or high (0.3-1.0 U/ml) concentrations
of thrombin, respectively. Neither ADP (up to 100 μM) nor its more potent analogue
2-methylthio-ADP (up to 100 μM), however, are able to stimulate PLD alone, and even
the addition of fibrinogen, which results in platelet aggregation, is not sufficient
for PLD activation. In contrast, ADP is able to stimulate PLD in the presence of low
concentrations of thrombin that alone have little or no effect, suggesting ADP may
play an amplifying role in platelet PLD activation. This hypothesis is supported by
the finding that the purinergic receptor antagonist ARL 66096, an ATP analogue, reduces
in a concentration-dependent fashion the PLD response to thrombin (IC50 = 28 nM with 0.1 U/ml thrombin). ARL 66096 also abolishes the PLD activation by ADP
observed in the presence of low concentrations of thrombin, confirming that the antagonist
inhibits an ADP-dependent component of the response. In addition, the thromboxane
A2 receptor agonist U46619 activates PLD, and this response is markedly reduced by ARL
66096. Concomitantly, phosphorylation of the protein kinase C substrate pleckstrin
in response to thrombin or U46619 is partially or totally inhibited by ARL 66096,
respectively, consistent with ADP stimulation of protein kinase C being involved in
the PLD response to these agonists. Based on these findings, we conclude that ADP
secretion and activation of purinergic ADP receptors is an important amplification
mechanism in the signal transduction pathways leading to PLD activation in human platelets.
