PARTIAL-PURIFICATION OF A TRYPSIN-LIKE PROTEINASE IN PLATELETS

Among various fluorogenic substrates for trypsin-like proteinases, ter t-butyloxycarbonyl-L-valyl-L-prolyl-L-arginine 4-methylcoumarin-7-amid e was strongly hydrolyzed by a crude extract of rabbit platlets. The p roteinase was partially purified (92-fold) from rabbit platelets by su ccessive chromatographic separations on phenyl-Sepharose CL-4B, L-argi nine-Sepharose 4B and Sephadex C-200 columns. Its molecular mass was f ound to be greater than 200 kDa by analytical gel filtration and its o ptimal pH was approximately 9. The proteinase activity was strongly in hibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, tosyl-L-lysine chrolomethyl ketone, leupeptin, p-nitrophenyl-p-guanid inobenzoate, and also by the 2,4-dimethylphenyl ester of amidinopiperi dine-4-propionic acid and the 4-tert-butylphenhl ester of trans-4-guan idinomethylcyclohexanecarboxylic acid which strongly inhibit platelet aggregation induced by various stimuli.