HUMAN ALPHA-THROMBIN 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 α-thrombin at the B-chain Trp148-Thr149 bond generating a new form, ζ-thrombin. While incubation of α-thrombin with cathepsin G at pH 7.4 and 37 °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 a-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 °C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 ± 0.60 vs 1.32 ± 0.18 µM and Kcat = 51.9 ± 2.9 vs 35.8 ± 6.4 s‒1 with α-thrombin vs chymotrypsin-prepared ζ-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 °C). Some 95% of the clotting activity was lost when ζ-thrombin was passed through trypsin-Sepharose 4B under conditions for converting α- to nonclotting β- and subsequently γ-thrombin. The resulting γ-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(me-thylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 °C. These elution peaks correspond to 240, 330, and 350 mM NaCl for γ-, α-, and ζ-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in γ-like thrombins but is intact in ζ-thrombin. Unlike α-thrombin, ζ-thrombin more rapidly loses clotting activity when incubated at pH 7.4 and 37 °C, where upon <90% behaves as denatured protein not retained on CG-50 resin. Thus, the ζ-cleavage destabilizes the protein but does not appreciably effect enzymic properties, such as clotting activity requiring both the catalytic site and adjacent regions, as well as the anion-binding exosite. © 1990, American Chemical Society. All rights reserved.