Arachidonate metabolism, 5-hydroxytryptamine release and aggregation in human platelets activated by palmitaldehyde acetal phosphatidic acid.
Palmitaldehyde acetal phosphatidic acid ( PGAP ) caused dose-dependent aggregation of human platelets resuspended in modified Tyrode medium, with a threshold concentration of 0.5-1 microM and an EC50 of 4 microM. Concentrations of PGAP which elicited biphasic irreversible aggregation concomitantly induced formation of 1.02 +/- 0.029 nmol (mean +/- s.e. mean) of malondialdehyde (MDA) per 10(9) platelets and caused release of 58 +/- 2.8% of platelet [14C]-5-hydroxytryptamine ([14C]-5-HT) from prelabelled platelets; no MDA formation or [14C]-5-HT release occurred at lower doses of PGAP which elicited only monophasic reversible aggregation. Adenosine 5'-pyrophosphate (ADP)-induced platelet activation resulted in formation of 0.344 +/- 0.004 nmol of MDA per 10(9) platelets in association with irreversible aggregation and 49.1 +/- 1% release of [14C]-5-HT. Mepacrine, a phospholipase A2 inhibitor, at 2.5 microM reduced PGAP -induced MDA formation and [14C]-5-HT release by the resuspended platelets without affecting irreversible aggregation; higher concentrations of mepacrine abolished all three responses. Chlorpromazine, a calmodulin antagonist, similarly inhibited PGAP -induced MDA formation and irreversible aggregation, and at 100 microM abolished monophasic aggregation. The cyclo-oxygenase inhibitor indomethacin caused a concentration-dependent reduction of PGAP -induced MDA formation by resuspended human platelets without significantly inhibiting [14C]-5-HT release or irreversible aggregation; concentrations (greater than or equal to 1.75 microM) which inhibited MDA formation by more than 94% abolished [14C]-5-HT release, and converted second phase irreversible aggregation to an extensive reversible response. 2-Methylthioadenosine 5'-phosphate (2 methylthio-AMP), an ADP antagonist, inhibited PGAP -induced MDA formation, [14C]-5-HT release and second phase aggregation in the human platelet suspensions in a parallel, concentration-dependent manner; at 9.4 microM 2-methylthio-AMP, both MDA formation and [14C]-5-HT release were abolished and monophasic, reversible aggregation remained. Albumin was required for aggregation of washed human platelets to PGAP . Irreversible PGAP -induced aggregation of washed [14C]-arachidonate-labelled platelets was accompanied by a low net loss of 14C from platelet phospholipids, an equivalent increase in 14C in free fatty acids, and the appearance of 14C in thromboxane (Tx)B2; mepacrine reduced the loss in 14C from phospholipids and inhibited aggregation and formation of [14C]-TxA2. Thrombin-induced aggregation was accompanied by substantial loss of 14C from phospholipids and equivalent gains of 14C in free fatty acids and TxB2; mepacrine pretreatment caused partial inhibition of thrombin-induced aggregation, halved the net 14C loss from phospholipids, but had little effect on the appearance of 14C in TxB2. 6 It is concluded that in human platelets PGAP-induced dense granule release and irreversible aggregation are dependent on the liberation of arachidonate and its metabolism via prostaglandin endoperoxides to thromboxane, that PGAP and thrombin elicit mobilization of arachidonate from different pools of membrane phospholipids, and that the mechanism of PGAP-activation of human platelets differs from those of thrombin- and ADP-activation.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007213:Indomethacin / Q000494:pharmacology; D008055:Lipids / Q000032:analysis; D008315:Malondialdehyde / Q000097:blood; D010974:Platelet Aggregation / Q000187:drug effects; D010976:Platelet Count; D011796:Quinacrine / Q000494:pharmacology; D012701:Serotonin / Q000097:blood* / Q000494:pharmacology*; D013873:Thionucleotides / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D001095:Arachidonic Acids; D008055:Lipids; D013873:Thionucleotides; D016718:Arachidonic Acid; D012701:Serotonin; C002480:methylthio-ADP; D008315:Malondialdehyde; D000244:Adenosine Diphosphate; D013917:Thrombin; D011796:Quinacrine; D002746:Chlorpromazine; D007213:Indomethacin