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 Leu 444 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. Serine proteinase inhibitors (serpins)1 are a highly conserved family of proteins whose primary role is to regulate the activity of a wide variety of serine proteinases involved in processes such as blood coagulation, fibrinolysis, inflammation, and cancer metastasis (1-3). Serpins inhibit their target proteinase by functioning as suicide substrates. The serpin contains a peptide bond, termed P1-P1Ј by the nomenclature of Schechter and Berger (4), within an exposed region, called the reactive site loop, that is recognized as a substrate by the proteinase (2). The initial binding of the serpin and proteinase results in the formation of a Michaelis complex, which can then partition between a kinetically stable intermediate, resulting in proteinase inhibition, and a cleaved inactive inhibitor (3, 5, 6). Although the sequence of the amino acids in the reactive site loop and on the P1-P1Ј residues in particular determines in part the proteinase specificity of the serpin, they also determine the partitioning of the serpin into an inhibitor versus substrate pathway with a given proteinase. Heparin cofactor II (HCII), protein C inhibitor, pr...

Aspartic Acid Residues 72 and 75 and Tyrosine-sulfate 73 of Heparin Cofactor II Promote Intramolecular Interactions during Glycosaminoglycan Binding and Thrombin Inhibition

“…Protease Inhibition-Protease inhibition rates were determined as described previously (32,42,43). All assays were performed at room temperature in 96-well microtiter plates previously coated with 2 mg/ml BSA.…”

“…Assays were performed at least in triplicate on two or more recombinant protein preparations. Second order inhibition rate constants were calculated as described (32,42,43).…”

Enhancement of Heparin Cofactor II Anticoagulant Activity

“…Mutagenesis and Expression of Recombinant Proteins-To facilitate our studies of HCII, site-directed mutagenesis (35) was performed on full-length HCII cDNA subcloned into the pBlueScript SKϩ mutagenesis and cloning vector (Stratagene, La Jolla, CA) (36) at two sites to encode the identical amino acid sequence but with two nucleotide changes (at base pairs 595 and 1255) that create unique restriction sites (NheI and AflII) in the cDNA. DNA sequencing using a Sequenase version 2.0 kit (Amersham Pharmacia Biotech) identified positive clones.…”

“…DNA sequencing using a Sequenase version 2.0 kit (Amersham Pharmacia Biotech) identified positive clones. Full-length HCII cDNA containing these unique restriction sites was then subcloned into the baculoviral transfer vector pVL1392 (PharMingen, La Jolla, CA) via flanking EcoRI sites as described previously (36).…”

Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism

“…In general, the single most important residue in determining the inhibitor's activity and specificity is its P1 residue, which is directly recognized by the active site of protease (8,59). Most serpins select a target protease via the use of preferred residues at the P1 position for efficient recognition and inhibition.…”