Inhibition of human thrombin assessed with different substrates and inhibitors. Characterization of fibrinopeptide binding interaction.
The activity of human thrombin has been assessed with fibrinogen, N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide, N-alpha-benzoyl-arginine-p-nitroanilide, N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and p-nitrophenylacetate: increased rates of hydrolysis were found for N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide compared to N-alpha-benzoyl-arginine-p-nitroanilide and p-nitrophenylacetate. Phenylmethyl sulfonyl fluoride and N-alpha-tosyl-L-lysine chloromethyl ketone inhibited, to the same degree, the activity toward each substrate. Inclusion of N-alpha-tosyl-arginine methyl ester in the phenylmethyl sulfonyl fluoride reaction mixtures protected the enzyme from inhibition as shown with N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. N-Acetylimidazole inhibited the activity towards fibrinogen, N-alphrosine-p-nitrophenyl ester to varying degrees. Inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide was completely reversible with neutral hydroxylamine, whereas coagulant activity towards fibrinogen was only partially regained. Human fibrinopeptide A inhibited activity toward N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. The mode of inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide by fibrinopeptide A was non=competitive (K1 = 3.02.10(-5) M), whereas N-alpha-toysyl-arginine methyl ester was a competitive inhibitor of this substrate (K1 = 2.6.10(-5) M). These studies demonstrate more than one binding domain for fibrinogen on human thrombin.
D001120:Arginine / Q000378:metabolism; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D001777:Blood Coagulation; D005340:Fibrinogen / Q000494:pharmacology*; D005344:Fibrinopeptide A / Q000494:pharmacology; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D010648:Phenylacetates / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D006898:Hydroxylamines; D007093:Imidazoles; D010648:Phenylacetates; D014107:Tosyllysine Chloromethyl Ketone; D005344:Fibrinopeptide A; D014443:Tyrosine; D010664:Phenylmethylsulfonyl Fluoride; D005340:Fibrinogen; D001586:Benzoylarginine Nitroanilide; D001120:Arginine; D013917:Thrombin