Human .alpha.- to .zeta.-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Abstract

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.degree. 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.degree. C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 .+-. 0.60 vs 1.32 .+-. 0.18 .mu.M 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.degree. C). Some the 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.degree. C. These elution peaks correspond to 240, 330, and 350 mM NaCl for .gamma.-, .alpha.-, and .zeta.-thrombin, respectfully, implying that anion-binding exosite is partially destroyed in .gamma.-like thrombin but is intact in .zeta.-thrombin. Unlike .alpha.-thrombin, .zeta.-thrombin more rapidly loses clotting activity when incubated at pH 7.4 and 37.degree. C, where upon > 90% behaves as denatured protein not retained on CG-50 resin. Thus, the .zeta.-cleavage destabilizes the protein but does not appreciably effect enzymic properties, such as clotting properties, such as clotting activity requiring both the catalytic site and adjacent regions, as well as the anion-binding exosite.