Heparin promotes proteolytic inactivation by thrombin of a reactive site mutant (L444R) of recombinant heparin cofactor II

Abstract

A heparin cofactor II (HCII) mutant with an Arg substituted for Leu444 at the P1 position (L444R-rHCII) was previously found to have altered proteinase specificity (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 5623-5628). The present study characterizes the effect of glycosaminoglycans on the substrate versus inhibitor activity of L444R-rHCII. Heparin increased the stoichiometry of inhibition of L444R-rHCII with α-thrombin (compared with minus glycosaminoglycan) but decreased it with R93A, R97A, R101A-thrombin, a mutant thrombin that does not bind glycosaminoglycans. Dermatan sulfate decreased the stoichiometry of inhibition of L444R-rHCII with both proteinases. SDS- polyacrylamide gel electrophoresis showed no proteolysis of L444R-rHCII when incubated with R93A, R97A, R101A-thrombin in the absence or the presence of glycosaminoglycan or with α-thrombin and dermatan sulfate. In contrast, greater than 75% of the L444R-rHCII was converted to a lower molecular weight form when incubated with α-thrombin/heparin. A time course of α-thrombin inhibition by L444R-rHCII/heparin showed a rapid but transient inhibition with approximately 80% of the α-thrombin activity being regained after 6 h of incubation. In contrast, all other combinations of inhibitor, proteinase, and glycosaminoglycan resulted in complete and sustained inhibition of the proteinase. Heparin fragments of 8-20 polysaccharides in length rapidly accelerated L444R-rHCII inhibition of both α-thrombin and R93A, R97A, R101A- thrombin. After extended incubations, R93A, R97A, R101A-thrombin was completely inhibited by L444R-rHCII with all the heparin fragments, but approximately 30-50% of α-thrombin activity remained with fragments long enough to bridge HCII-thrombin. These results collectively indicate that ternary complex formation, mediated by heparin, increases L444R-rHCII inactivation by α-thrombin.