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PubMed1988

Activation of heparin cofactor II by heparin oligosaccharides.

Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required.

Animals↗

PubMed1997

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

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

Heparin↗

PubMed1987

Carboxylate polyanions accelerate inhibition of thrombin by heparin cofactor II.

The heparin cofactor II (HCII)/thrombin inhibition reaction is enhanced by various carboxylate polyanions. In the presence of polyaspartic acid, the HCII/thrombin reaction is accelerated more than 1000-fold with the second-order rate constant increasing from 3.2 x 10(4) M-1 min-1 (in the absence of polyAsp) to 3.6 x 10(7) M-1 min-1 as the polyAsp concentration is increased from 1 to 250 micrograms/ml. This accelerating effect was observed for HCII/thrombin, though to varying degrees, with other carboxylate polyanions. In contrast to HCII, the rate of antithrombin III inhibition of thrombin was decreased in the presence of polyAsp. The HCII/thrombin complex is rapidly formed in the presence of 10 micrograms/ml polyAsp when 125I-labeled-thrombin is incubated with plasma. It is possible that at physiological sites rich in carboxylate polyanions, thrombin may be preferentially inhibited by HCII.

Anions↗

PubMed2002

Contribution of basic residues of the A helix of heparin cofactor II to heparin- or dermatan sulfate-mediated thrombin inhibition.

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated 1000-fold by heparin or dermatan sulfate. To investigate the contribution of basic residues of the A helix of HCII to this activation, we constructed amino acid substitutions (K101Q, R103L, and R106L) by site-directed mutagenesis. K101Q greatly reduced heparin cofactor activity and required a more than 10-fold higher concentration of dermatan sulfate to accelerate thrombin inhibition compared with wild-type recombinant HCII. Thrombin inhibition by R106L was not significantly stimulated by dermatan sulfate. These results provide evidence that basic residues of the A helix of HCII (Lys(101) and Arg(106)) are necessary for heparin- or dermatan sulfate-accelerated thrombin inhibition.

Amino Acid Sequence↗

PubMed1993

Mutagenesis of thrombin selectively modulates inhibition by serpins heparin cofactor II and antithrombin III. Interaction with the anion-binding exosite determines heparin cofactor II specificity.

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Thrombin is inhibited by the serpins antithrombin III and heparin cofactor II in a reaction that is dramatically accelerated by glycosaminoglycans. The structural basis of the interaction with these inhibitors was investigated by introducing single amino acid substitutions into the anion-binding exosite (R68E, R70E) and unique insertion loops (K52E, K154A) of thrombin. The rate of inhibition of these recombinant thrombins by antithrombin III and heparin cofactor II was determined in the absence and presence of glycosaminoglycan. The second order rate constant (k2) for inhibition by antithrombin III without heparin was 3.7 x 10(5) M-1 min-1 for wild-type thrombin; rates for the mutant thrombins varied less than 2-fold. For inhibition by antithrombin III with heparin, the rate constant was 4.5 x 10(8) M-1 min-1 for wild-type thrombin with no significant differences between any of the recombinant thrombins. In contrast, the rate constant for inhibition by heparin cofactor II without glycosaminoglycan was 4.3 x 10(4) M-1 min-1 for wild-type thrombin; rates were 10-fold slower for thrombin K52E and 2- to 3-fold slower for thrombins R68E and R70E. The rate constants for inhibition of wild-type thrombin by HCII in the presence of heparin or dermatan sulfate were 9.2 x 10(8) M-1 min-1 and 9.0 x 10(8) M-1 min-1, respectively. Compared to wild-type thrombin, the rate of inhibition by HCII with glycosaminoglycan was 5- to 15-fold slower for thrombins K52E and R70E and 50- to over 100-fold slower for thrombin R68E. Thrombin K154A was inhibited by heparin cofactor II with rates similar to wild-type thrombin in all assays. These results suggest that heparin cofactor II interacts with residue Lys-52 in the proposed S1' subsite and with residues Arg-68 and Arg-70 in the anion-binding exosite of thrombin, and that these interactions contribute to the molecular basis of heparin cofactor II specificity for thrombin.

Antithrombin III↗

PubMed1985

Reactive site peptide structural similarity between heparin cofactor II and antithrombin III.

Heparin cofactor II (Mr = 65,600) was purified 1800-fold from human plasma to further characterize the structural and functional properties of the protein as they compare to antithrombin III (Mr = 56,600). Heparin cofactor II and antithrombin III are functionally similar in that both proteins have been shown to inhibit thrombin at accelerated rates in the presence of heparin. There was little evidence for structural homology between heparin cofactor II and antithrombin III when high performance liquid chromatography-tryptic peptide maps and NH2-terminal sequences were compared. A partially degraded form of heparin cofactor II was also obtained in which a significant portion (Mr = 8,000) of the NH2 terminus was missing. The rates of thrombin inhibition (+/- heparin) by native and partially degraded-heparin cofactor II were not significantly different, suggesting that the NH2-terminal region of the protein is not essential either for heparin binding or for thrombin inhibition. A significant degree of similarity was found in the COOH-terminal regions of the proteins when the primary structures of the reactive site peptides, i.e. the peptides which are COOH-terminal to the reactive site peptide bonds cleaved by thrombin, were compared. Of the 36 residues identified, 19 residues in the reactive site peptide sequence of heparin cofactor II could be aligned with residues in the reactive site peptide from antithrombin III. While the similarities in primary structure suggest that heparin cofactor II may be an additional member of the superfamily of proteins consisting of antithrombin III, alpha 1-antitrypsin, alpha 1-antichymotrypsin and ovalbumin, the differences in structure could account for differences in protease specificity and reactivity toward thrombin. In particular, a disulfide bond which links the COOH-terminal (reactive site) region of antithrombin III to the remainder of the molecule and is important for the heparin-induced conformational change in the protein and high affinity binding of heparin does not appear to exist in heparin cofactor II. This observation provides an initial indication that while the reported kinetic mechanisms of action of heparin in accelerating the heparin cofactor II/thrombin and antithrombin III/thrombin reactions are similar, the mechanisms and effects of heparin binding to the two inhibitors may be different.

Amino Acid Sequence↗

PubMed1997

Sulfated polysaccharide from the leaves of Artemisia Princeps activates heparin cofactor II independently of the Lys173 and Arg189 residues of heparin cofactor II.

A sulfated polysaccharide (AFE-HCD) purified from the leaves of Artemisia princeps Pamp selectively accelerated the rate of thrombin inhibition by heparin cofactor II (HCII). By using plasma derived HCII and bacterial expressed recombinant HCII molecules, the interaction between each HCII molecule and AFE-HCD was analyzed. AFE-HCD accelerated thrombin inhibition by plasma derived HCII or bacterial expressed wild type HCII to the same extent (IC50: 0.056 micrograms/ml for plasma derived HCII and 0.066 micrograms/ml for recombinant HCII under the experimental condition). The recombinant HCII (rHCII) molecule with Lys173-->Leu or Arg189-->His substitution, which is defective in interactions with heparin and dermatan sulfate, respectively, is activated by AFE-HCD to inhibit thrombin in a manner similar to wild type rHCII. These results suggested that activation of HCII was independent of its Lys173 or Arg189 residue. Although AFE-HCD is a selective activator of HCII like dermatan sulfate, the amino acid residue required for the activation of HCII was distinct form that of dermatan sulfate as well as heparin.

Arginine↗

PubMed1998

Ligand binding to thrombin exosite II induces dissociation of the thrombin-heparin cofactor II(L444R) complex.

Heparin cofactor II (HCII) inhibits thrombin rapidly in the presence of heparin or dermatan sulfate. The product of the inhibition reaction is a kinetically stable, 1:1 complex between the two proteins. We recently observed that heparin induces dissociation of complexes containing thrombin and the reactive site mutant HCII(L444R) to yield active thrombin and cleaved inhibitor (Han, J. -H., Van Deerlin, V. M. D., and Tollefsen, D. M. (1997) J. Biol. Chem. 272, 8243-8249). In the current study, we have investigated the mechanism by which heparin induces dissociation of the thrombin-HCII(L444R) complex. Heparin oligosaccharides >/=6 sugars in length induce dissociation, which suggests that dissociation does not depend on binding of a heparin molecule simultaneously to both proteins in the complex. Binding of heparin to HCII(L444R) in the complex also does not appear to be required, since the heparin dose response is unaltered for complexes containing the double mutant HCII(L444R/K173Q), which has decreased affinity for heparin. By contrast, binding of heparin to thrombin appears to be necessary and sufficient to induce dissociation. First, heparin fails to induce dissociation of complexes that contain thrombin(K236E), a variant with decreased heparin affinity. Second, a monoclonal IgG that interacts with the heparin-binding site of thrombin mimicks heparin in its ability to induce dissociation of the thrombin-HCII(L444R) complex. Finally, the complex of HCII(L444R) with thrombin(desPPW), which binds normally to heparin but lacks Pro60BPro60CTrp60D in an insertion loop ("60-loop") between the heparin-binding site and the catalytic site, does not dissociate in the presence of heparin. These results suggest that binding of heparin to thrombin induces an allosteric effect causing destabilization of the thrombin-HCII(L444R) complex and that the allosteric effect may be mediated by the 60-loop.

Arginine↗

PubMed1999

Enhancement of heparin cofactor II anticoagulant activity.

Heparin cofactor II (HCII) is a serpin whose thrombin inhibition activity is accelerated by glycosaminoglycans. We describe the novel properties of a carboxyl-terminal histidine-tagged recombinant HCII (rHCII-CHis(6)). Thrombin inhibition by rHCII-CHis(6) was increased >2-fold at approximately 5 microgram/ml heparin compared with wild-type recombinant HCII (wt-rHCII) at 50-100 microgram/ml heparin. Enhanced activity of rHCII-CHis(6) was reversed by treatment with carboxypeptidase A. We assessed the role of the HCII acidic domain by constructing amino-terminal deletion mutants (Delta1-52, Delta1-68, and Delta1-75) in wt-rHCII and rHCII-CHis(6). Without glycosaminoglycan, unlike wt-rHCII deletion mutants, the rHCII-CHis(6) deletion mutants were less active compared with full-length rHCII-CHis(6). With glycosaminoglycans, Delta1-68 and Delta1-75 rHCIIs were all less active. We assessed the character of the tag by comparing rHCII-CHis(6), rHCII-CAla(6), and rHCII-CLys(6) to wt-rHCII. Only rHCII-CHis(6) had increased activity with heparin, whereas all three mutants have increased heparin binding. We generated a carboxyl-terminal histidine-tagged recombinant antithrombin III to study the tag on another serpin. Interestingly, this mutant antithrombin III had reduced heparin cofactor activity compared with wild-type protein. In a plasma-based assay, the glycosaminoglycan-dependent inhibition of thrombin by rHCII-CHis(6) was significantly greater compared with wt-rHCII. Thus, HCII variants with increased function, such as rHCII-CHis(6), may offer novel reagents for clinical application.

Alanine↗

PubMed2002

Inhibition of a thrombin anion-binding exosite-2 mutant by the glycosaminoglycan-dependent serpins protein C inhibitor and heparin cofactor II.

Antithrombin (ATIII), heparin cofactor II (HCII) and protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) are serine protease inhibitors (serpins) whose thrombin inhibition activity is accelerated in the presence of glycosaminoglycans. We compared the inhibition properties of PCI and HCII to ATIII using R93A/R97A/R101A thrombin, an anion-binding exosite-2 (exosite-2) mutant that has greatly reduced heparin-binding properties. Heparin-enhanced PCI inhibition of R93A/R97A/R101A thrombin was only approximately 2-fold compared to 40-fold enhancement with wild-type recombinant thrombin. Thrombomodulin (TM) (with or without the chondroitin sulfate moiety) accelerated PCI inhibition of both wild-type and R93A/R97A/R101A thrombins. HCII achieved the same maximum activity in the presence of heparin with both wild-type and R93A/R97A/R101A thrombins; however, the optimum heparin concentration was 20 times greater than the reaction with wild-type thrombin, indicative of a decrease in heparin affinity. Dermatan sulfate (DSO4)-catalyzed HCII thrombin inhibition was unchanged in R93A/R97A/R101A thrombin compared to wild-type recombinant thrombin. These results suggest that PCI is similar to ATIII and depends upon ternary complex formation with heparin and these specific thrombin exosite-2 residues to accelerate thrombin inhibition. In contrast, HCII does not require Arg(93), Arg(97) and Arg(101) of thrombin exosite-2 and further supports the hypothesis that HCII uses an allosteric process following glycosaminoglycan binding to inhibit thrombin.

Allosteric Regulation↗

PubMed1988

Modulation of heparin cofactor II function by S protein (vitronectin) and formation of a ternary S protein-thrombin-heparin cofactor II complex.

The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin as well as factor Xa against fast inactivation by antithrombin III. The interference of S protein with glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in these studies. S protein significantly counteracted the anticoagulant activity of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of 0.3 micrograms/ml heparin, 0.5 micrograms/ml pentosan polysulfate, or 2 micrograms/ml dermatan sulfate, S protein induced a concentration-dependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin), or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and carboxymethylation of the protein increased the neutralizing activity of S protein towards heparin and pentosan polysulfate. The results of these functional experiments correlated well with the demonstration of direct binding of S protein to both polysaccharides but not to dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective in neutralizing other dermatan sulfate compounds with varying degree of sulfation, a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding domain of S protein, counteracted their anticoagulant activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombins↗

PubMed1985

Turnover study of heparin cofactor II in healthy man.

Heparin cofactor II (HC II) is a heparin-dependent inhibitor of thrombin, distinct from antithrombin III (AT III). This study was designed to evaluate its metabolism in healthy subjects. Purified HC II was labelled with 125I by the lactoperoxidase-glucose oxidase technique. The biological activity of the HC II was unchanged after labelling as was its migratory pattern by crossed immunoelectrophoresis in the presence of heparin or dermatan sulfate. Three healthy volunteers were injected with 10 microCi and the plasma radioactivity was measured daily. The data were approximated by a sum of two exponential terms and the metabolism of HC II was described by a two compartment mamillary system. The mean values of fractional catabolic rate, intravascular fraction and half-life of the elimination phase were respectively: 0.44 d-1, 0.60 and 2.53 d. These parameters are of the same order of magnitude as those reported in the literature for AT III. The plasma HC II concentration in the 3 subjects ranged from 61 to 82 micrograms/ml as estimated using our purified preparation. Accordingly, the absolute catabolic rate ranged from 1.17 to 1.36 mg X kg-1 X d-1.

Adult↗

PubMed1996

Active site for heparin cofactor II in low molecular mass dermatan sulfate. Contribution to the antithrombotic activity of fractions with high affinity for heparin cofactor II.

Dermatan sulfate (DS) is currently under clinical investigation as new antithrombotic agent. Unlike heparin, DS does not act through Antithrombin III (ATIII) but primarily through thrombin on Heparin Cofactor II (HCII). HCII is activated by the oversulfated sequence (IdoA2SO3-GalNAc4SO3)4 or by both the sequences (IdoA2SO3-GalNAc4SO3)n and (IdoA-GalNAc-4,6SO3)n, [n > or = 2]. A Low Molecular Mass Dermatan Sulfate (LMM-DS), endowed with a bioavailability three-four times higher than DS, by subcutaneous route, was obtained by chemical depolymerization of DS. The LMM-DS was fractionated by anion exchange and size exclusion chromatography. Fractions with high and low charge densities, high and low molecular masses, and high (2.66) and low (0.07) potencies on HCII were isolated. A relationship between the in vitro HCII-mediated inhibition of thrombin and the chain length of DS fractions containing oversulfated sequences was found [by a multiple regression test]. The in vivo activity increased until it reached a plateau. The important influence on the HCII activity of natural IdoA-GalNAc-4,6SO3 disaccharide was confirmed by investigation on oversulfated DS obtained by a limited and selective chemical 6-O-sulfation in GalNAc4SO3 units of DS.

Animals↗

PubMed1990

Effect of low molecular weight heparin preparations on the inhibition of thrombin by heparin cofactor II.

1. Heparin molecules approximately 24 to 30 residues in length are required to catalyze the thrombin-HC II reaction. The requirement for heparin molecules of this length is consistent with a model for catalysis in which heparin binds HC II and thrombin simultaneously to form a ternary complex in a manner similar to that proposed for the thrombin-AT III reaction. Smaller molecules (18 or more monosaccharide units in length) are required to catalyze the thrombin-AT III reaction. 2. The specific AT III-binding pentasaccharide containing 3-O-sulfated glucosamine is not required for activity with HC II. 3. Some low molecular weight heparin preparations have significant activity with HC II (approximately 10 to 20% that of standard heparin). This is probably related to the presence of species with molecular weights greater than 6000 to 7500 (24 to 30 monosaccharide units) in these preparations.

Antithrombin III↗

PubMed1986

Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides.

The in vitro anticoagulant effects of standard heparin (SH) and of seven other sulphated polysaccharides (SPS) were investigated by measuring activated partial thromboplastin time (APTT) prolongation of normal plasma and of plasmas selectively depleted of antithrombin III (AT III), of heparin cofactor II (HC II) and of both heparin cofactors. This allowed the determination of the relative contribution of each of the two heparin cofactors to the SPS anticoagulant effect. The SPS varied in their relative activities as catalysts of thrombin inhibition by purified AT III or HC II. The anticoagulant activities of heparin and dermatan sulphate were primarily attributable to their ability to enhance thrombin inhibition by AT III and HC II respectively. Heparin had an additional minor anticoagulant activity which was independent of both AT III and HC II. Pentosan polysulphate, high molecular weight dextran sulphate, heparin with low affinity for AT III and a sulphated heparin derivative had weaker anticoagulant activities in normal plasma than standard heparin. The anticoagulant activities of these last four SPS in plasma depleted of both AT III and HC II were similar to their respective activities in normal plasma. This suggests that these SPS act by directly preventing thrombin generation rather than by enhancing thrombin inhibition.

Antithrombin III↗

PubMed1988

Heparin cofactor II assay. Elimination of heparin and antithrombin-III effects.

Functional assays for heparin cofactor II (HC-II) are based on the inactivation of thrombin by HC-II in the presence of dermatan sulfate (DS). Residual thrombin is measured in a chromogenic assay. Interference by the antithrombin-III (AT-III)/heparin complex, which also rapidly inactivates thrombin, must be eliminated from the HC-II test system. Commercial DS is contaminated with heparin, while plasma specimens to be tested contain AT-III. After NaNO2/acetic acid treatment of DS (to inactivate heparin), there was enough residual heparin to cause AT-III interference. Treatment of plasma with commercially available anti-AT-III antiserum largely, but not completely, removed AT-III interference from the HC-II assay. With commercially available reagents, both NaNO2/acetic acid treatment of DS and anti-AT-III treatment of plasma were needed to eliminate heparin/AT-III interference. Protamine sulfate inactivated DS as well as heparin and could not be used to reduce AT-III/heparin interference with the HC-II assay.

Acetates↗

PubMed1991

Purification of heparin cofactor II from human plasma.

Heparin cofactor II (HCII) is an inhibitor of thrombin in human plasma whose activity is enhanced by heparin and dermatan sulphate. HCII was purified to homogeneity from normal human plasma with an overall yield of 7.5%. After treatment with barium chloride, precipitation with 50% saturated ammonium sulphate and dialysis of the resuspended precipitate against 0.02 M Tris-HCl (pH 7.4), the sample was chromatographed on a heparin-Sepharose CL 6B affinity column, DEAE-Sepharose CL 6B ion-exchange gel and an AcA 34 gel permeation column. For the final steps, a high-performance liquid chromatographic system was used which included ion-exchange chromatography on a Mono-Q column and gel permeation using a Superose column. The purified protein was homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The specific activity of purified HCII was 12.2 U/mg. The HCII activity was evaluated as antithrombin dermatan sulphate cofactor activity. A specific antiserum against HCII was raised in the rabbit.

Chromatography, Affinity↗

PubMed2000

Activation of heparin cofactor II by calcium spirulan.

Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose ability to inhibit alpha-thrombin is accelerated by a variety of sulfated polysaccharides in addition to heparin and dermatan sulfate. Previous investigations have indicated that calcium spirulan (Ca-SP), a novel sulfated polysaccharide, enhanced the rate of inhibition of alpha-thrombin by HCII. In this study, we investigated the mechanism of the activation of HCII by Ca-SP. Interestingly, in the presence of Ca-SP, an N-terminal deletion mutant of HCII (rHCII-Delta74) inhibited alpha-thrombin, as native recombinant HCII (native rHCII) did. The second-order rate constant for the inhibition of alpha-thrombin by rHCII-Delta74 was 2.0 x 10(8) M(-1) min(-1) in the presence of 50 microgram/ml Ca-SP and 10, 000-fold higher than in the absence of Ca-SP. The rates of native rHCII and rHCII-Delta74 for the inhibition of gamma-thrombin were increased only 80- and 120-fold, respectively. Our results suggested that the anion-binding exosite I of alpha-thrombin was essential for the rapid inhibition reaction by HCII in the presence of Ca-SP and that the N-terminal acidic domain of HCII was not required. Therefore, we proposed a mechanism by which HCII was activated allosterically by Ca-SP and could interact with the anion-binding exosite I of thrombin not through the N-terminal acidic domain of HCII. The Arg(103) --> Leu mutant bound to Ca-SP-Toyopearl with normal affinity and inhibited alpha-thrombin in a manner similar to native rHCII. These results indicate that Arg(103) in HCII molecule is not critical for the interaction with Ca-SP.

Binding Sites↗