Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets.

F Bartoli,H.K Lin,F Ghomashchi,M.H Gelb,M.K Jain,R Apitz-Castro

doi:10.1016/s0021-9258(17)40727-7

Abstract

An analogue of arachidonic acid in which the COOH group is replaced by a trifluoromethyl ketone group (COCF3) has recently been shown to be a tight binding inhibitor of the 85-kDa cytosolic phospholipase A2 that is found in platelets and other cells (Street, I. P., Lin, H.-K., Laliberté, F., Ghomashchi, F. G., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). This trifluoromethyl ketone inhibits most of the arachidonate release from the phospholipid pool in thrombin-stimulated human platelets at concentrations of 0-40 microM with 4 x 10(8) platelets/ml. A structure-function analysis of related compounds reveals a good correlation between the inhibition of the purified phospholipase A2 and the blockage of arachidonate release in platelets. A number of recently described potent inhibitors of the 14-kDa phospholipase A2 that is secreted from activated platelets have no effect on the level of free arachidonate production. Furthermore, the addition of a large amount of recombinant 14-kDa phospholipase A2 to platelets does not produce free arachidonate, and it does not alter the amount of arachidonate released following platelet activation with thrombin. These studies provide strong pharmacological evidence for the role of the cytosolic phospholipase A2 in producing most, if not all, of the liberated arachidonate in thrombin-stimulated human platelets, and they show that tight binding membrane-residing inhibitors of the cytosolic phospholipase A2 can block the eicosanoid cascade in living cells.

Highlights

An analogue of arachidonic acid in which theCOOH that operate at the membranelaqueous phase interface can group is replaced by atrifluoromethylketonegroup be addressed because many aspeocftsinterfacial catalysis have (COCF,) has recently been shown to be a tight binding been described in a quantitative fashion, andspecific competiinhibitor of the 85-kDa cytosolic phosphoAli,ptahsaet is tive inhibitors for several classeosf phospholipases have been found in platelets and othcerlls
This trifluoromethyl selectively cleaves this ester linkage, has been considered as a ketone inhibits most of the arachidonate release from prime candidate to catalyze the releoafstehis polyunsaturated the phospholipid pool in thrombin-stimulated human platelets at concentrations of 0-40
Ambiguities in the definition of arachidonate liberation persist because the pathway for of the lack of a of arachidonate release in platelets

Summary

NI NI

AACH(OHN)ICF, AACNOICH, AACOCF,Cl 18:3-COCF3 MJ3N3 I MJ45 HK42 HK61 y-LNinIolenoyl amide NILinoleoyl amide. The amounoft platelet arachidonate released was obtained as the inhibitors for 1min, and thrombin (0.5 unitdm final concentra- moles of internal standard addedto the samples multipliedby the ratio tion) was added. If not all, of the with microsomes for 5 min, and the reaction was initiatbeyd the thrombin-induced release of arachidonate isblocked at concenaddition of 25 pl of substrate stock solution containingl-O-[aLkyl-1',2'3Hlsn-glycero-3-phosphocholine(0.1pCi/tube, 50 Ci/mmol, DuPont NEN),unlabeled 1-0-hexadecyl-sn-glycero-3-phosphocholi(nBeiomol, to give a final concentration in theassay of.1VM),and 0.25 mg/ml fatty acid-poor bovine serum albumin (Fraction V, Calbiochem). Usingcells labeled with [,H]arachidonate to monitoragonist-inducedrelease of this fatty acid can be misleading This is because not all pools of cellular arachidonate may be radiolabeled to the same extent, and, the and Dyer lipid extraction(441, and aliquotsof the chloroform-rich layers release of L3H1arachidonate may not reflect the total mass of were resolved by thin layer chromatography on silica gel plates using liberated arachidonate [6]. Under the conditions emchlorofodmethanovacetic aciuwater(50:25:8:4, v/v/v/v)(R, values for ployed in thisstudy, all major phospholipid classes are substanp1h-aolskpyhlo-cshno-lgilnyecaerreo-03.-2p4haosnpdho0c.6h4o,lirenseapnecdtivle-lya)l.kAylf-te2r-avciysul-aslinz-agtiloyncebryo-3-tiallyradiolabeled with exogenously supplied arachidonate, radioscanning (Bioscan), the produc3t,H-labeled 1-alkyl-2-acyl-sn-glyc- and thespecific radioactivity of the arachidonate in phosphatiero-3-phosphocholine, was scraped and submitted to liquid scintillationdylinositides is essentially the same as that of the cellular counting

RESULTS

DISCUSSION

Findings

All of the arachidonic acid analogues tested in this study