Identification of regions of .alpha.-thrombin involved in its interaction with hirudin

Abstract

The contributions of various regions of human .alpha.-thrombin to the formation of the tight complex with hirudin have been assessed by using derivatives of thrombin. .alpha.-Thrombin in which the active-site serine was modified with diisopropyl fluorophosphate was able to bind hirudin, but its affinity for hirudin was decreased by 103-fold compared to unmodified .alpha.-thrombin. Modification of the active-site histidine with D-Phe-Pro-Arg-CH2Cl resulted in a form of thrombin with a 106-fold reduced affinity for hirudin. .gamma.-Thrombin is produced by proteolytic cleavage of .alpha.-thrombin in two surface loops corresponding to residues 65-83 and 146-150 in .alpha.-chymotrypsin [Berliner, L. J. (1984) Mol. Cell. Biochem. 61, 159-172; Birktoft, J. J., and Blow, D. M. (1972) J. Mol. Biol. 68. 187-240]. The .gamma.-thrombin-hirudin complex had a dissociation constant that was 106-fold higher than that of .alpha.-thrombin. Treatment of .alpha.-thrombin with pancreatic elastase resulted in a form of thrombin only cleaved in the loop corresponding to residues 146-150 in .alpha.-chymotrypsin, and this form, of thrombin had only a slightly reduced affinity for hirudin. By using limited proteolysis with trypsin, it was possible to isolate .beta.-thrombin which contained a single cleavage in the loop corresponding to residues 65-83 in .alpha.-chymotrypsin. This form of thrombin had a 100-fold decreased affinity for hirudin. Kinetic analysis of the binding of hirudin to .beta.-thrombin indicated that the 100-fold decrease in affinity was predominantly due to a decrease in the rate of association of the two molecules. The results are discussed with respect to the contributions of various regions of thrombin to its tight-binding interaction with hirudin.