title: Structural and functional properties of human alpha-thrombin, phosphopyridoxylated alpha-thrombin, and gamma T-thrombin. Identification of lysyl residues in alpha-thrombin that are critical for heparin and fibrin(ogen) interactions.
abstract: alpha-Thrombin derivatives obtained either by site-specific modification at lysyl residues (phosphopyridoxylated) or by limited trypsinolysis (gamma T-thrombin) were compared to correlate structural modifications with the functional reactivity toward fibrin(ogen) and heparin. alpha-Thrombin phosphopyridoxylated in the absence of heparin (unprotected) showed approximately 2 mol of label incorporated/mol of thrombin, but only 1 mol of label incorporated/mol of proteinase when modified in the presence of added heparin (protected). In contrast to native alpha-thrombin, both phosphopyridoxylated alpha-thrombin derivatives failed to interact with a fibrin monomer-agarose column and had reduced fibrinogen clotting activity, which is very similar to gamma T-thrombin. Heparin accelerated the rate of antithrombin III inhibition of alpha-thrombin, heparin-protected modified-alpha-thrombin, and gamma T-thrombin in a manner consistent with a template mechanism but was without effect on unprotected modified alpha-thrombin. In a heparin-catalyzed antithrombin III inhibition assay of alpha-thrombin, we found that D-Phe-Pro-Arg chloromethyl ketone-active site-inactivated gamma T-thrombin competed for heparin binding. It has been shown that limited proteolysis/autolysis of the B-chain of alpha-thrombin in the area around Arg-B73 (in beta T/beta- and gamma T/gamma-thrombin), but not that around Lys-B154 (in gamma T/gamma-thrombin), diminishes specific interactions with fibrinogen (Hofsteenge, J., Braun, P. J., and Stone , S. R. (1988) Biochemistry 27, 2144-2151). In unprotected modified alpha-thrombin, lysyl residues B21, B65, B174, and B252 were phosphopyridoxylated. In heparin-protected modified alpha-thrombin, only lysyl residues B21 and B65 were phosphopyridoxylated. These observations suggest that lysyl residues 21/65 of the B-chain of alpha-thrombin are involved in fibrin(ogen) interactions, and lysyl residues 174/252 of the B-chain are important in heparin interactions.
authors.affiliation: Department of Pathology and Medicine, University of North Carolina School of Medicine, Chapel Hill 27599-7035.
authors.forename: F C
authors.initials: FC
authors.lastname: Church
authors.forename: C W
authors.initials: CW
authors.lastname: Pratt
authors.forename: C M
authors.initials: CM
authors.lastname: Noyes
authors.forename: T
authors.initials: T
authors.lastname: Kalayanamit
authors.forename: G B
authors.initials: GB
authors.lastname: Sherrill
authors.forename: R B
authors.initials: RB
authors.lastname: Tobin
authors.forename: J B
authors.initials: JB
authors.lastname: Meade
mesh_terms: D000911:Antibodies, Monoclonal; D000990:Antithrombin III / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D008239:Lysine; D010446:Peptide Fragments / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D000990:Antithrombin III; D005337:Fibrin; D005340:Fibrinogen; D006493:Heparin; D013917:Thrombin; D008239:Lysine; C024593:pyridoxine 5-phosphate