Structural and Functional Properties of Human a-Thrombin, Phosphopyridoxylated a-Thrombin, and yT-Thrombin IDENTIFICATION OF LYSYL RESIDUES IN a-THROMBIN THAT ARE CRITICAL FOR HEPARIN AND FIBRIN(0GEN) INTERACTIONS*

a-Thrombin derivatives obtained either by site-specific modification at lysyl residues (phosphopyridoxylated) or by limited trypsinolysis (yT-thrombin) were compared to correlate structural modifications with the functional reactivity toward fibrin(ogen) and heparin. a-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 a-thrombin, both phosphopyridoxylated a-thrombin derivatives failed to interact with a fibrin monomer-agarose column and had reduced fibrinogen clotting activity, which is very similar to yT-thrombin. Heparin accelerated the rate of antithrombin I11 inhibition of a-thrombin, heparin-protected modified-athrombin, and yT-thrombin in a manner consistent with a template mechanism but was without effect on unprotected modified a-thrombin. In a heparin-catalyzed antithrombin I11 inhibition assay of a-thrombin, we found that D-Phe-Pro-Arg chloromethyl ketone-active site-inactivated yT-thrombin competed for heparin binding. It has been shown that limited proteolysis/ autolysis of the B-chain of a-thrombin in the area around Arg-B73 (in and yT/y-thrombin), but not that around Lys-B154 (in yT/y-thrombin), diminishes specific interactions with fibrinogen (Hofsteenge, J., Braun, P. J., and Stone, S. R. (1988) Biochemistry 27, 2144-2151). In unprotected modified a-thrombin, lysyl residues B21, B65, B174, and B252 were phosphopyridoxylated. In heparin-protected modified athrombin, only lysyl residues B2 1 and B65 were phosphopyridoxylated. These observations suggest hat lysyl residues 21/65 of the B-chain of a-thrombin are involved in fibrin(ogen) interactions, and lysyl residues 1741252 of the B-chain are important in heparin interactions.