Intracellular Regulation of the Metabolism of Arachidonic Acid in Human Platelets

Abstract

The synthesis of prostaglandins and thromboxanes by human platelets is limited by the availability of the fatty acid precursor, arachidonic acid. Although large amounts of arachidonic acid are esterified to platelet phospholipids, only the free acid can be utilized by the oxygenation pathways of platelets. Since there are only trace amounts of free arachidonic acid in platelets, the enzymatic liberation of this fatty acid from platelet phospholipids can be considered the initial and rate limiting step of these oxygenation pathways. This process is catalyzed by a phospholipase A2 whose role as the rate limiting enzyme makes it a prime target for the intracellular regulation of prostaglandin and thromboxane synthesis.A second mechanism for the regulation of prostaglandin synthesis has been hypothesized. Several commonly occurring unsaturated fatty acids, e.g., oleic and linoleic acids, are capable of inhibiting prostaglandin cyclooxygenase. If these fatty acids are liberated from phospholipids along with arachidonic acid, they could limit the production of prostaglandins and thromboxanes. Thus, the types and amounts of fatty acids released from platelet phospholipids could regulate the amounts of prostaglandins and thromboxanes produced.We have investigated these two types of intracellular regulation and have found that 1) intracellular cyclic nucleotides and divalent cations are involved in the regulation of the platelet phospholipase A2 activity stimulated by thrombin and 2) this phospholipase A2 activity catalyzes the specific release of arachidonic acid from phospholipids, thereby obviating the regulatory role of liberated oleic and linoleic acids.

Effect of endotoxin on arachidonic acid release and thromboxane B2 production by human platelets

“…A modification of the procedure of Bills et a1 [4] was used to evaluate the thrombinstimulated release of 14C-arachidonic acid from the prelabeled platelet phospholipids. After stirring for five minutes following addition of thrombin, endotoxin plus thrombin, or buffer, the samples were extracted with chloroform:methanol (1 :2), and then 4.8 ml of chloroform and 4.8 ml of 0.1 M EDTA were added.…”