Arachidonic acid (AA) is liberated from platelet membrane phospholipids during stimulation
and promotes cellular aggregation through the synthesis of thromboxane A2. Two pathways; phospholipase A2 (PLA2) or phospholipase C (PLC) followed by the action of acylglycerolipases, are thought
to be activated during platelet stimulation and supply the necessary AA. We have reported
that mono (2-ethylhexyl)phthalate (MEHP), a physiological metabolite of the plasticizer
di(2-ethylhexyl)-phthalate (DEHP), commonly used in a variety of medical devices and
storage containers, inhibits PLA2, but not PLC in platelet lysates. The effects of MEHP on intact platelets were studied.
PLA2 activity in intact platelets or lysates was assayed by incubating them with 2-14C-arachidonyl-phosphatidylcholine and measuring formation of free 14C-arachidonic acid in 10 min. Platelet lysates hydrolyzed 10% of the substrate while
2.6% was hydrolyzed by intact platelets. The amount of MEHP needed to inhibit 14C-AA liberation was 0.35 mM for platelet lysates and 0.7 mM for intact platelets.
Platelet aggregation induced by collagen was inhibited by MEHP (1 mM), although responses
to adenosine diphosphate, AA and ionophore were unaffected. Identical effects on platelet
aggregation were found when indomethacin (0.1 mM) was added. Higher concentrations
of MEHP blocked platelet aggregation induced by adenosine diphosphate or AA but not
ionophore or synergistic pairs of these stimuli, indicating a more generalized membrane
disruption at higher MEHP concentrations. These results suggest that MEHP acts in
a similar manner to indomethacin to block PLA2-mediated liberation of arachidonate during platelet aggregation
(supported by MRC and NHRDP, Canada)
