INTEGRIN ALPHA(V)BETA(3) AND PHOSPHOLIPASE-C REGULATE PROSTACYCLIN FORMATION OF ENDOTHELIAL-CELLS CAUSED BY ANCROD-GENERATED FIBRIN

Ancrod-generated fibrin has been shown to stimulate prostacyclin synth esis of human umbilical vein endothelial cells (Chang et al., 1994, Bi ochem. Biophys. Res. Commun. 203, 1920). We further investigated its m echanism of action. The increment of 6-keto prostaglandin F-1 alpha, s timulated by ancrod-generated fibrin was almost completely inhibited w hen endothelial cells were either pretreated with 50 mu M -(N,N'-dieth ylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) or preloaded with 15 mu M ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). 6- Keto prostaglandin F-1 alpha production during 2 and 10 h incubation p eriod was also inhibited by 1.2 mM l-bis-(beta-aminoethylether)-N,N,N' ,N'-tetraacetic acid (EGTA) (41 +/- 12 and 53 +/- 17% inhibition, resp ectively). Further, ancrod-generated fibrin caused a rapid-onset incre ase in [H-3]inositol monophosphate (IP1) formation in endothelial cell s. This increase in IP1 was significantly inhibited by 1 mM Gly-Pro-Ar g-Pro, 1 mM neomycin or 100 ng/ml pertussis toxin. At the same time, n eomycin and pertussis toxin also significantly inhibited 6-keto prosta glandin F-1 alpha synthesis of endothelial cells stimulated by ancrod- generated fibrin. Additionally, the increment of IP1 production as wel l as prostacyclin production were significantly inhibited by monoclona l antibodies directed against alpha(v) beta(3) These results suggest t hat intra- and extra-cellular Ca2+ participate in prostacyclin synthes is stimulated by ancrod-generated fibrin. Ancrod-generated fibrin stim ulates pertussis toxin-sensitive G-protein regulated phosphoinositide breakdown, which is responsible for prostacyclin synthesis. This augme ntation in prostacyclin synthesis and phosphoinositide breakdown cause d by ancrod-generated fibrin are, at least in part, mediated by fibrin binding to integrin alpha(v) beta(3) on endothelial cells.