Partial purification of a trypsin-like proteinase in platelets.

Among various fluorogenic substrates for trypsin-like proteinases, tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginine 4-methylcoumarin-7-amide was strongly hydrolyzed by a crude extract of rabbit platelets. The proteinase was partially purified (92-fold) from rabbit platelets by successive chromatographic separations on phenyl-Sepharose CL-4B, L-arginine-Sepharose 4B and Sephadex G-200 columns. Its molecular mass was found to be greater than 200 kDa by analytical gel filtration and its optimal pH was approximately 9. The proteinase activity was strongly inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, tosyl-L-lysine chrolomethyl ketone, leupeptin, p-nitrophenyl-p-guanidinobenzoate, and also by the 2,4-dimethylphenyl ester of amidinopiperidine-4-propionic acid and the 4-tert-butylphenyl ester of trans-4-guanidinomethylcyclohexanecarboxylic acid which strongly inhibit platelet aggregation induced by various stimuli.