AGGREGATION RESPONSE OF PLATELETS DURING INHIBITION OF PHOSPHOLIPASE A₂

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)