Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: Differential inhibition by PGE 1, local anesthetics and a serine-protease inhibitor

Abstract

The formation of malondialdehyde (MDA) and rabbit aorta contracting substance (RCS) induced by treatment of platelets with thrombin and collagen, but not that produced from exogenous arachidonic acid, is inhibited by prostaglandin E 1 (10 −8 − 10 −7M), the local anesthetics tetracaine, SKF 525-A and dibucaine (1 mM), and the serine-protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The burst in oxygen consumption which accompanies platelet stimulation by thrombin and collagen in the presence of antimycin A, known to be due to the oxidation of endogenous arachidonate, is also markedly suppressed by PGE 1, tetracaine and PMSF. The inhibitory effect of PGE 1 is strongly potentiated by theophylline (1.0 mM). Addition of the Ca 2+ ionophore A23187 to platelet suspensions overcomes PGE 1 and PMSF inhibition of MDA and RCS formation, and induces a vigorous increase in O 2 consumption. Tetracaine and dibucaine, however, block the responses to A23187. Formation of MDA and RCS (a mixture of PG endoperoxides and TXA 2) due to stimulation by thrombin and collagen depends upon activation of Ca 2+-dependent phospholipase A 2 (PLA 2) to supply free arachidonate from specific membrane phospholipids. These experiments therefore indicate that increased cellular cAMP, induced by PGE 1, antagonizes the mobilization of the Ca 2+ which is normally required for PLA 2 activity. Thrombin-stimulated platelets exhibit enhanced 45Ca uptake which probably reflects exchange of extracellular Ca 2+ with an increased available pool of exchangeable intracellular Ca 2+. PGE 1 strongly suppresses this 45Ca uptake, providing more direct evidence supporting the view that cAMP prevents the rise in free cytoplasmic Ca 2+ induced by thrombin. Under conditions which make sufficient free cytoplasmic Ca 2+ available (i.e., A23187), despite high cellular cAMP, formation of RCS and MDA, and O 2 uptake are nearly normal indicating that activation of PLA 2 can occur. Local anesthetics on the other hand since they abolish the response to A23187 as well, appear to directly antagonize the ability of Ca 2+ to activate PLA 2. The effect of PMSF suggests that stimulus-specific proteases may be involved in the thrombin and collagen-induced activation of PLA 2 activity.