ANALYSIS OF TISSUE-PLASMINOGEN ACTIVATOR SPECIFICITY USING PEPTIDYL FLUOROGENIC SUBSTRATES

A series of 54 fluorogenic substrates have been synthesized and evalua ted for tissue-type plasminogen activator (tPA) hydrolysis in an attem pt to create efficient sensitive substrates for tPA and to investigate substrate structure-efficiency correlations. All substrates contain t he 6-amino-1-naphthalenesulfonamide (ANSN) leaving group, Arg in the P -1 position, various amino acids in the P-2 and P-3 positions, and var ious substituents in the sulfonamide moiety of the leaving group (P' p osition). The majority of substrates have relatively low K-M values (< 100 mu M), reaching as low as 2.6 mu M, and reasonably high k(cat) val ues (up to 3.6 s(-1)). These substrates have higher affinity, higher h ydrolysis rates, and higher efficiency for two-chain tPA than for the single-chain form of this enzyme. Analysis of the P-3 structure influe nce on substrate efficiency demonstrates that compounds which contain D-isomers of N-blocked bulky amino acids, such as Phe, Leu, and Val, i n this position are more efficient for tPA than substrates with N-unbl ocked small amino acids (Ser or Pro) in the P-3 position. The second-o rder rate constants and k(cat) values for substrate hydrolysis increas e with decreases in the P-2 amino acid hydrophobicity in the following manner: Leu < Val and Gly < Ser < Pro. Substrates which contain an AN SN leaving group had a higher affinity for tPA than substrates with p- nitroaniline or 7-amino-4-methylcoumarin leaving groups. Analyses of s ubstrate hydrolysis dependence on the substrate P' structure show that the k(cat) and the second-order rate constants increased with an incr ease in the size of monoalkyl substituent in the sulfonamide moiety, w hereas substrates which contain either glycine methyl ester or a dialk yl group displayed the lowest efficiency for tPA. The substrate Boc-(p -F)Phe-Pro-Arg-ANSNHC(2)H(5) allowed quantitation of tPA at a concentr ation as low as 1 pM, a concentration significantly lower than the pla sma concentration of this protein. Evaluation of the activation of sin gle-chain tPA by factor Xa demonstrates that prothrombinase is approxi mately 3-fold more efficient in activating sc-tPA than factor Xa alone , increasing the initial rate of activation from 0.0055 nM/s per 1 nM of factor Xa to 0.017 nM/s per 1 nM.