The role of ADP in endotoxin-induced equine platelet activation

We have shown previously that endotoxin induces platelet aggregation in equine heparinised whole blood in a platelet-activating factor (PAF; 1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine) dependent manner. ADP is an agonist of platelets and is present in platelet dense granules with ATP in high concentrations. An investigation was carried out to establish whether endotoxin-induced platelet activation was associated with release of platelet ATP and ADP. ADP-scavenging enzyme systems significantly inhibited endotoxin-induced aggregation. Plasma levels of adenine nucleotides were measured using a luminometric assay following incubation of heparinised equine whole blood with endotoxin (300 ng/ml). After addition of endotoxin, ATP and ADP were released from the platelets and then subsequently degraded to AMP. WEB2086 (4-{3-[4-( o-chlorophenyl)-9-methyl-6 H-thieno[3,2,- f]- s-triazolo[4,3- a][1,4]diazepin-2-yl]proprionyl}-morpholine) (100 nM), a competitive PAF receptor antagonist, inhibited endotoxin-induced aggregation and also inhibited the release of adenine nucleotides from the platelets. It is concluded that endotoxin-induced aggregation is dependent upon ADP released from platelet dense granules.