Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained.
Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Brezniak
D V
DV
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Brower
M S
MS
Witting
J I
JI
Walz
D A
DA
Fenton
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation; D056649:Cathepsin G; D002403:Cathepsins / Q000097:blood*; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
D046911:Macromolecular Substances; D009842:Oligopeptides; D011480:Protease Inhibitors; D005340:Fibrinogen; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; D013917:Thrombin