New Fluorogenic Substrates for α-Thrombin, Factor Xa, Kallikreins, and Urokinase1

Abstract

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.

Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin

“…2a). The P2-Gly was most effective as previously reported [7,24,26,35,361, resulting in the increase of the k,,, value. In addition, the Gly and Ala residues contributed to increase the l/Km value.…”

“…The importance of apolar residues adjacent to the susceptible bond, especially a Pro residue at the P2 site [7], has also been reported by using peptide p-nitroanilides [23 - [30, 311. This specificity of a-thrombin is presumed to be the result from an apolar binding site adjacent to the catalytic site in a-thrombin [32].…”

“…The substrate specificity of plasma kallikrein has been studied with peptide-p-nitroanilides [26] and peptide-NH-Mec [7]. Plasma kallikrein exhibits a very strict specificity towards a Phe residue at the P2 site [7,261.…”

“…Bovine trypsin effectively hydrolyzed peptide-NH-Mec substrates containing Ala and Pro at the P2 site. The most reactive substrate was Boc-Gln-Ala-Arg-NH-Mec (k,,, = 120 sK1, K, = 6.0 pM, k,,,/K, = 20000000 M-' s-').Peptide amides of 7-amino-4-methylcoumarine (Mec) originally developed for the sensitive assay for a-chymotrypsin [l] [7], and horseshoe crab clotting factors [12]. Most of these substrates were designed based on the information from the COOH-terminal sequences of the 'activation peptides', which are liberated during the conversions of plasma serine protease zymogens to their active enzymes.…”

Protein‐Templated Formation of an Inhibitor of the Blood Coagulation Factor Xa through a Background‐Free Amidation Reaction

“…4‐Aminomethylbenzamidine ( 14 ) was employed as the nucleophile and typical S1‐binding fragment of trypsin‐like serine proteases 17. The inhibition of factor Xa was determined in an enzyme activity assay using the fluorogenic substrate 7‐( N ‐Boc‐leucinyl‐glycinyl‐arginyl)‐7‐amino‐4‐methylcoumarin ( K M =76 μ m ) for detection 18. Carboxylic acid 1 was found to be a moderately active competitive inhibitor of factor Xa with a binding affinity ( K I ) of 5.5 m m , and the K I  value of 14 was 0.68 m m .…”