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Volume 272, Number 2, Issue of January 10, 1997 pp. 888-893
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Heparin Promotes Proteolytic Inactivation by Thrombin of a Reactive Site Mutant (L444R) of Recombinant Heparin Cofactor II

(Received for publication, July 19, 1996, and in revised form, September 17, 1996)

Angelina V. Ciaccia , Annemieke J. Willemze ^¶ and Frank C. Church ^¶

From the Departments of  Pharmacology and  Pathology and Laboratory Medicine, ^¶ Center for Thrombosis and Hemostasis, The University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

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 -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 -thrombin and dermatan sulfate. In contrast, greater than 75% of the L444R-rHCII was converted to a lower molecular weight form when incubated with -thrombin/heparin. A time course of -thrombin inhibition by L444R-rHCII/heparin showed a rapid but transient inhibition with approximately 80% of the -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 -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 -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 -thrombin.

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