Enhancement of Heparin Cofactor II Anticoagulant Activity (1999)

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-CHis6). Thrombin inhibition by rHCII-CHis6 was increased >2-fold at ∼5 μg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) at 50–100 μg/ml heparin. Enhanced activity of rHCII-CHis6 was reversed by treatment with carboxypeptidase A. We assessed the role of the HCII acidic domain by constructing amino-terminal deletion mutants (Δ1–52, Δ1–68, and Δ1–75) in wt-rHCII and rHCII-CHis6. Without glycosaminoglycan, unlike wt-rHCII deletion mutants, the rHCII-CHis6 deletion mutants were less active compared with full-length rHCII-CHis6. With glycosaminoglycans, Δ1–68 and Δ1–75 rHCIIs were all less active. We assessed the character of the tag by comparing rHCII-CHis6, rHCII-CAla6, and rHCII-CLys6 to wt-rHCII. Only rHCII-CHis6 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-CHis6 was significantly greater compared with wt-rHCII. Thus, HCII variants with increased function, such as rHCII-CHis6, may offer novel reagents for clinical application. 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-CHis6). Thrombin inhibition by rHCII-CHis6 was increased >2-fold at ∼5 μg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) at 50–100 μg/ml heparin. Enhanced activity of rHCII-CHis6 was reversed by treatment with carboxypeptidase A. We assessed the role of the HCII acidic domain by constructing amino-terminal deletion mutants (Δ1–52, Δ1–68, and Δ1–75) in wt-rHCII and rHCII-CHis6. Without glycosaminoglycan, unlike wt-rHCII deletion mutants, the rHCII-CHis6 deletion mutants were less active compared with full-length rHCII-CHis6. With glycosaminoglycans, Δ1–68 and Δ1–75 rHCIIs were all less active. We assessed the character of the tag by comparing rHCII-CHis6, rHCII-CAla6, and rHCII-CLys6 to wt-rHCII. Only rHCII-CHis6 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-CHis6 was significantly greater compared with wt-rHCII. Thus, HCII variants with increased function, such as rHCII-CHis6, may offer novel reagents for clinical application. serine protease inhibitor heparin cofactor II antithrombin III wild type recombinant carboxyl-terminal hexahistidine tag carboxyl-terminal pentahistidine-proline tag carboxyl-terminal hexa-alanine tag carboxyl-terminal hexalysine tag Δ1–68, and Δ1–75, deletion of residues 1–52, 1–68, and 1–75 in recombinant HCII, respectively anion-binding exosite-1 bovine serum albumin carboxypeptidase A polyethylene glycol 8000 Polybrene-hexadimethrine bromide tosyl-Gly-Pro-Arg-p-nitroanilide succinyl-Ala-Ala-Pro-Phe-p-nitroanilide normal hemostasis reference plasma human antithrombin III-deficient plasma carboxypeptidase A Serine protease inhibitors (serpins)1 are a class of highly conserved proteins whose prototypical member is α1-proteinase inhibitor (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar). Serpins function primarily to inhibit serine proteases that are involved in many normal biological processes including coagulation, fibrinolysis, inflammation, wound healing, and tissue repair as well as some pathological processes such as atherosclerosis and cancer metastasis (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar). Within the serpin superfamily is a subclass of (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar, F.C. PubMed Scopus Google Scholar). antithrombin III heparin cofactor II protease and (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar). by heparin, and with as inhibit proteases by as (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar). Serpins Within the of the serpin is the A. PubMed Scopus Google Scholar). protease this and as a the serpin and protease are in a in the protease is M. PubMed Google Scholar). is F.C. PubMed Scopus Google Scholar, M. R. PubMed Google Scholar, F.C. PubMed Scopus Google cofactor II is a whose activity is thrombin and Tollefsen D.M. PubMed Google Scholar, F.C. A. PubMed Scopus Google Scholar). the thrombin inhibitor R. PubMed Google Scholar, M. PubMed Scopus Google HCII inhibition of thrombin is accelerated by heparin and D.M. PubMed Google Scholar, F.C. PubMed Google Scholar). of thrombin inhibition by HCII are in the of many are that HCII is thrombin inhibitor Tollefsen D.M. PubMed Google Scholar, 1989; PubMed Scopus Google Scholar, F.C. PubMed Google cofactor II is in that is F.C. Biochemistry. PubMed Scopus Google Scholar, Tollefsen D.M. Biochemistry. PubMed Scopus Google Scholar). In the of glycosaminoglycan, HCII thrombin (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar, F.C. PubMed Google Scholar, PubMed Google Scholar, PubMed Google Scholar, Tollefsen D.M. PubMed Google Scholar, D.M. PubMed Scopus Google Scholar, F.C. 1997; Scholar, F.C. D. Stone S.R. PubMed Scopus Google Scholar). Heparin cofactor II a amino-terminal that is highly acidic and to as the In the of glycosaminoglycan, the acidic domain is to with the is highly is involved in binding. is that the acidic domain is acidic domain is to with the anion-binding exosite-1 of to HCII to D.M. PubMed Google Scholar, F.C. PubMed Google Scholar). a of the HCII of by of of by heparin to to this have a of residues as D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). this to a the with a A. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, F.C. PubMed Scopus Google Scholar). had as D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). that the tag is a to proteins to was D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google this the carboxyl-terminal recombinant HCII antithrombin and heparin cofactor and increased compared with wild-type recombinant HCII a the of HCII may with the carboxyl-terminal hexahistidine of carboxyl-terminal recombinant antithrombin III have compared with wild-type recombinant and the heparin of in a plasma-based thrombin inhibition that rHCII-CHis6 a novel have HCII mutant that is a significantly inhibitor of thrombin the wild-type rHCII-CHis6, is a carboxyl-terminal heparin cofactor In the of a in of thrombin In the of heparin, rHCII-CHis6 activity that to of the thrombin inhibitor of the hexahistidine tag to HCII the of this for heparin. In the activity of rHCII-CHis6 is with that the activity is a of the of the carboxyl-terminal tag and that rHCII-CHis6 to inhibit thrombin the as is highly on of of heparin cofactor activity in rHCII-CHis6 is by We another the activity is to We that the of a carboxyl-terminal hexahistidine tag to with the of to inhibit of serine thrombin and and on the heparin of the in activity HCII and with a carboxyl-terminal hexahistidine tag is are in and in (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, F.C. PubMed Google Scholar). the in antithrombin activity is a for that the hexahistidine tag is the properties of the D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). have of proteins in the was the at in the HCII is to inhibit thrombin in the of (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google F.C. PubMed Google Scholar, PubMed Google Scholar, PubMed Google Scholar, Tollefsen D.M. PubMed Google Scholar, D.M. PubMed Scopus Google Scholar, F.C. 1997; Scholar, F.C. D. Stone S.R. PubMed Scopus Google Scholar). the activity of the hexahistidine tag was with the serpin HCII the the that the domain is in antithrombin activity of rHCII-CHis6 that the acidic domain may in to thrombin inhibition in the of a of the carboxyl-terminal of that of thrombin is in the of thrombin inhibition by rHCII-CHis6. is by inhibition of a of thrombin at the increased activity of rHCII-CHis6 that residues of by by in involved in this inhibition increased of rHCII-CHis6 to compared with wt-rHCII to the that of the is to heparin reduced heparin for activity for rHCII-CHis6 is to and is with that for heparin and activity for F.C. PubMed Scopus Google Scholar, F.C. PubMed Google Scholar). in heparin of rHCII-CHis6 that the of HCII and for heparin and and with of HCII in the D.M. PubMed Scopus Google Scholar, F.C. 1997; Scholar). inhibition of wt-rHCII and rHCII-CHis6 with the acidic domain of HCII for that the to the domain in the increased activity of is that to the of a some of the hexahistidine on the of and Biochemistry. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Google have on the function of as a is for that heparin in the of at of were at and in the of is of the proteins are with and activity and heparin binding. of on the heparin of the of a in HCII to the hexahistidine tag the of hexahistidine and HCII, on the of the tag and the of the amino-terminal acidic domain of the of the amino-terminal acidic domain of HCII to the activity of rHCII-CHis6 by the wt-rHCII rHCII-CHis6. In and Tollefsen Tollefsen D.M. PubMed Google amino-terminal deletion mutants of for of wt-rHCII are in with function to the residues and are acidic and residues are in and and the of HCII is that is to to of thrombin Tollefsen D.M. PubMed Google Scholar). of on this of the of 1–75 the of HCII with in the of the of glycosaminoglycan, the deletion mutants of wt-rHCII had of protease inhibition on the the amino-terminal of is significantly involved in the inhibition of In all three in rHCII-CHis6 to of thrombin and in inhibition to the of the hexahistidine tag in rHCII-CHis6. the tag in the deletion mutants a in the of HCII inhibition is on this that the amino-terminal acidic domain the the hexahistidine tag with the hexahistidine tag to the tag the the of glycosaminoglycan, the thrombin inhibition are by deletion of the of wt-rHCII. In the of heparin the of residues 1–75 to in with the of the of thrombin inhibition by the rHCII-CHis6 are heparin cofactor activity of rHCII-CHis6 is with the deletion of the the and 1–75 a of activity with heparin and of activity that the the amino-terminal of HCII on the hexahistidine tag is residues and We for the and of HCII and amino-terminal acidic domain is of HCII, the that the may in to the compared rHCII-CHis6, rHCII-CAla6, and rHCII-CLys6 to the character of the carboxyl-terminal We that the activity of rHCII-CHis6 was a of the of on the a hexa-alanine a hexalysine tag to this We that the hexahistidine hexa-alanine tag increased the of thrombin inhibition in the of inhibition of another serine is by the of that the with the hexahistidine tag are a of on the in thrombin inhibition that the acidic is the of heparin a in the of thrombin inhibition by rHCII-CHis6 and a to a heparin In with and a in In the of increased of thrombin inhibition comparing rHCII-CHis6 to wt-rHCII. hexa-alanine and hexalysine are to the inhibition of thrombin in the of in and heparin by rHCII-CLys6 that this may have less the properties of rHCII-CHis6. that the the of to the of HCII is for increased heparin binding. that the in of inhibition with heparin to to the hexahistidine heparin, heparin, and such as such as and Google Scholar, 1997; PubMed Scopus Google Scholar, of D. Scholar). protease inhibitors and proteases are as 1997; PubMed Scopus Google Scholar, Google Scholar, Biochemistry. 1997; PubMed Scopus Google Scholar, A. M. 1997; PubMed Scopus Google Scholar, Carrell R.W. Stone S.R. PubMed Scopus Google Scholar, F.C. Biochemistry. PubMed Scopus Google Scholar, Stone S.R. 1997; PubMed Scopus Google Scholar). with have Biochemistry. 1997; PubMed Scopus Google Scholar, A. M. 1997; PubMed Scopus Google Scholar, Carrell R.W. Stone S.R. PubMed Scopus Google Scholar, F.C. Biochemistry. PubMed Scopus Google Scholar, Stone S.R. 1997; PubMed Scopus Google Scholar). offer some In the of heparin, the activity of rHCII-CHis6 is to that of the inhibitor histidine-tagged HCII at a significantly heparin wild-type In a plasma-based assay, increased activity of rHCII-CHis6 with heparin and HCII is a with activity to thrombin in the A activity in plasma M. A. PubMed Scopus Google and to for the of rHCII-CHis6 to a less active as a thrombin In increased antithrombin at heparin is to HCII mutant a of the offer a novel to Serine protease inhibitors (serpins)1 are a class of highly conserved proteins whose prototypical member is α1-proteinase inhibitor (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar). Serpins function primarily to inhibit serine proteases that are involved in many normal biological processes including coagulation, fibrinolysis, inflammation, wound healing, and tissue repair as well as some pathological processes such as atherosclerosis and cancer metastasis (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar). Within the serpin superfamily is a subclass of (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar, F.C. PubMed Scopus Google Scholar). antithrombin III heparin cofactor II protease and (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar). by heparin, and with as Serpins inhibit proteases by as (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar). Serpins Within the of the serpin is the A. PubMed Scopus Google Scholar). protease this and as a the serpin and protease are in a in the protease is M. PubMed Google Scholar). is F.C. PubMed Scopus Google Scholar, M. R. PubMed Google Scholar, F.C. PubMed Scopus Google Scholar). Heparin cofactor II is a whose activity is thrombin and Tollefsen D.M. PubMed Google Scholar, F.C. A. PubMed Scopus Google Scholar). the thrombin inhibitor R. PubMed Google Scholar, M. PubMed Scopus Google HCII inhibition of thrombin is accelerated by heparin and D.M. PubMed Google Scholar, F.C. PubMed Google Scholar). of thrombin inhibition by HCII are in the of many are that HCII is thrombin inhibitor Tollefsen D.M. PubMed Google Scholar, 1989; PubMed Scopus Google Scholar, F.C. PubMed Google Scholar). Heparin cofactor II is in that is F.C. Biochemistry. PubMed Scopus Google Scholar, Tollefsen D.M. Biochemistry. PubMed Scopus Google Scholar). In the of glycosaminoglycan, HCII thrombin (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google Scholar, F.C. PubMed Google Scholar, PubMed Google Scholar, PubMed Google Scholar, Tollefsen D.M. PubMed Google Scholar, D.M. PubMed Scopus Google Scholar, F.C. 1997; Scholar, F.C. D. Stone S.R. PubMed Scopus Google Scholar). Heparin cofactor II a amino-terminal that is highly acidic and to as the In the of glycosaminoglycan, the acidic domain is to with the is highly is involved in binding. is that the acidic domain is acidic domain is to with the anion-binding exosite-1 of to HCII to D.M. PubMed Google Scholar, F.C. PubMed Google Scholar). a of the HCII of by of of by heparin to to this have a of residues as D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). this to a the with a A. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, F.C. PubMed Scopus Google Scholar). had as D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). that the tag is a to proteins to was D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). In this the carboxyl-terminal recombinant HCII antithrombin and heparin cofactor and increased compared with wild-type recombinant HCII a the of HCII may with the carboxyl-terminal hexahistidine of carboxyl-terminal recombinant antithrombin III have compared with wild-type recombinant and the heparin of in a plasma-based thrombin inhibition that rHCII-CHis6 a novel have HCII mutant that is a significantly inhibitor of thrombin the wild-type rHCII-CHis6, is a carboxyl-terminal heparin cofactor In the of a in of thrombin In the of heparin, rHCII-CHis6 activity that to of the thrombin inhibitor of the hexahistidine tag to HCII the of this for heparin. In the activity of rHCII-CHis6 is with that the activity is a of the of the carboxyl-terminal tag and that rHCII-CHis6 to inhibit thrombin the as is highly on of of heparin cofactor activity in rHCII-CHis6 is by We another the activity is to We that the of a carboxyl-terminal hexahistidine tag to with the of to inhibit of serine thrombin and and on the heparin of the in activity HCII and with a carboxyl-terminal hexahistidine tag is are in and in (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, F.C. PubMed Google Scholar). the in antithrombin activity is a for that the hexahistidine tag is the properties of the D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). have of proteins in the was the at in the HCII is to inhibit thrombin in the of (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google F.C. PubMed Google Scholar, PubMed Google Scholar, PubMed Google Scholar, Tollefsen D.M. PubMed Google Scholar, D.M. PubMed Scopus Google Scholar, F.C. 1997; Scholar, F.C. D. Stone S.R. PubMed Scopus Google Scholar). the activity of the hexahistidine tag was with the serpin HCII the the that the domain is in antithrombin activity of rHCII-CHis6 that the acidic domain may in to thrombin inhibition in the of a of the carboxyl-terminal of that of thrombin is in the of thrombin inhibition by rHCII-CHis6. is by inhibition of a of thrombin at the increased activity of rHCII-CHis6 that residues of by by in involved in this inhibition increased of rHCII-CHis6 to compared with wt-rHCII to the that of the is to heparin reduced heparin for activity for rHCII-CHis6 is to and is with that for heparin and activity for F.C. PubMed Scopus Google Scholar, F.C. PubMed Google Scholar). in heparin of rHCII-CHis6 that the of HCII and for heparin and and with of HCII in the D.M. PubMed Scopus Google Scholar, F.C. 1997; Scholar). inhibition of wt-rHCII and rHCII-CHis6 with the acidic domain of HCII for that the to the domain in the increased activity of is that to the of a some of the hexahistidine on the of and Biochemistry. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Google have on the function of as a is for that heparin in the of at of were at and in the of is of the proteins are with and activity and heparin binding. of on the heparin of the of a in HCII to the hexahistidine tag the of hexahistidine and HCII, on the of the tag and the of the amino-terminal acidic domain of the of the amino-terminal acidic domain of HCII to the activity of rHCII-CHis6 by the wt-rHCII rHCII-CHis6. In and Tollefsen Tollefsen D.M. PubMed Google amino-terminal deletion mutants of for of wt-rHCII are in with function to the residues and are acidic and residues are in and and the of HCII is that is to to of thrombin Tollefsen D.M. PubMed Google Scholar). of on this of the of 1–75 the of HCII with in the of the of glycosaminoglycan, the deletion mutants of wt-rHCII had of protease inhibition on the the amino-terminal of is significantly involved in the inhibition of In all three in rHCII-CHis6 to of thrombin and in inhibition to the of the hexahistidine tag in rHCII-CHis6. the tag in the deletion mutants a in the of HCII inhibition is on this that the amino-terminal acidic domain the the hexahistidine tag with the hexahistidine tag to the tag the the of glycosaminoglycan, the thrombin inhibition are by deletion of the of wt-rHCII. In the of heparin the of residues 1–75 to in with the of the of thrombin inhibition by the rHCII-CHis6 are heparin cofactor activity of rHCII-CHis6 is with the deletion of the the and 1–75 a of activity with heparin and of activity that the the amino-terminal of HCII on the hexahistidine tag is residues and We for the and of HCII and amino-terminal acidic domain is of HCII, the that the may in to the compared rHCII-CHis6, rHCII-CAla6, and rHCII-CLys6 to the character of the carboxyl-terminal We that the activity of rHCII-CHis6 was a of the of on the a hexa-alanine a hexalysine tag to this We that the hexahistidine hexa-alanine tag increased the of thrombin inhibition in the of inhibition of another serine is by the of that the with the hexahistidine tag are a of on the in thrombin inhibition that the acidic is the of heparin a in the of thrombin inhibition by rHCII-CHis6 and a to a heparin In with and a in In the of increased of thrombin inhibition comparing rHCII-CHis6 to wt-rHCII. hexa-alanine and hexalysine are to the inhibition of thrombin in the of in and heparin by rHCII-CLys6 that this may have less the properties of rHCII-CHis6. that the the of to the of HCII is for increased heparin binding. that the in of inhibition with heparin to to the hexahistidine heparin, heparin, and such as such as and Google Scholar, 1997; PubMed Scopus Google Scholar, of D. Scholar). protease inhibitors and proteases are as 1997; PubMed Scopus Google Scholar, Google Scholar, Biochemistry. 1997; PubMed Scopus Google Scholar, A. M. 1997; PubMed Scopus Google Scholar, Carrell R.W. Stone S.R. PubMed Scopus Google Scholar, F.C. Biochemistry. PubMed Scopus Google Scholar, Stone S.R. 1997; PubMed Scopus Google Scholar). with have Biochemistry. 1997; PubMed Scopus Google Scholar, A. M. 1997; PubMed Scopus Google Scholar, Carrell R.W. Stone S.R. PubMed Scopus Google Scholar, F.C. Biochemistry. PubMed Scopus Google Scholar, Stone S.R. 1997; PubMed Scopus Google Scholar). offer some In the of heparin, the activity of rHCII-CHis6 is to that of the inhibitor histidine-tagged HCII at a significantly heparin wild-type In a plasma-based assay, increased activity of rHCII-CHis6 with heparin and HCII is a with activity to thrombin in the A activity in plasma M. A. PubMed Scopus Google and to for the of rHCII-CHis6 to a less active as a thrombin In increased antithrombin at heparin is to HCII mutant a of the offer a novel to We have HCII mutant that is a significantly inhibitor of thrombin the wild-type rHCII-CHis6, is a carboxyl-terminal heparin cofactor In the of a in of thrombin In the of heparin, rHCII-CHis6 activity that to of the thrombin inhibitor of the hexahistidine tag to HCII the of this for heparin. In the activity of rHCII-CHis6 is with that the activity is a of the of the carboxyl-terminal tag and that rHCII-CHis6 to inhibit thrombin the as is highly on of of heparin cofactor activity in rHCII-CHis6 is by We another the activity is to We that the of a carboxyl-terminal hexahistidine tag to with the of to inhibit of serine thrombin and and on the heparin of the in activity HCII and with a carboxyl-terminal hexahistidine tag is are in and in (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, F.C. PubMed Google Scholar). the in antithrombin activity is a for that the hexahistidine tag is the properties of the D. R. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). have of proteins in the was the at in the HCII is to inhibit thrombin in the of (2Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Advances in Experimental Medicine and Biology. 1997; Crossref PubMed Google F.C. PubMed Google Scholar, PubMed Google Scholar, PubMed Google Scholar, Tollefsen D.M. PubMed Google Scholar, D.M. PubMed Scopus Google Scholar, F.C. 1997; Scholar, F.C. D. Stone S.R. PubMed Scopus Google Scholar). the activity of the hexahistidine tag was with the serpin HCII the the that the domain is in antithrombin activity of rHCII-CHis6 that the acidic domain may in to thrombin inhibition in the of a of the carboxyl-terminal of that of thrombin is in the of thrombin inhibition by rHCII-CHis6. is by inhibition of a of thrombin at the increased activity of rHCII-CHis6 that residues of by by in involved in this inhibition increased of rHCII-CHis6 to compared with wt-rHCII to the that of the is to heparin reduced heparin for activity for rHCII-CHis6 is to and is with that for heparin and activity for F.C. PubMed Scopus Google Scholar, F.C. PubMed Google Scholar). in heparin of rHCII-CHis6 that the of HCII and for heparin and and with of HCII in the D.M. PubMed Scopus Google Scholar, F.C. 1997; Scholar). inhibition of wt-rHCII and rHCII-CHis6 with the acidic domain of HCII for that the to the domain in the increased activity of rHCII-CHis6. is that to the of a some of the hexahistidine on the of and Biochemistry. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Google have on the function of as a is for that heparin in the of at of were at and in the of is of the proteins are with and activity and heparin binding. of on the heparin of the of a in HCII to the hexahistidine tag the of hexahistidine and HCII, on the of the tag and the of the amino-terminal acidic domain of We the of the amino-terminal acidic domain of HCII to the activity of rHCII-CHis6 by the wt-rHCII rHCII-CHis6. In and Tollefsen Tollefsen D.M. PubMed Google amino-terminal deletion mutants of for of wt-rHCII are in with function to the residues and are acidic and residues are in and and the of HCII is that is to to of thrombin Tollefsen D.M. PubMed Google Scholar). of on this of the of 1–75 the of HCII with in the of glycosaminoglycans. In the of glycosaminoglycan, the deletion mutants of wt-rHCII had of protease inhibition on the the amino-terminal of is significantly involved in the inhibition of In all three in rHCII-CHis6 to of thrombin and in inhibition to the of the hexahistidine tag in rHCII-CHis6. the tag in the deletion mutants a in the of HCII inhibition is on this that the amino-terminal acidic domain the the hexahistidine tag with the hexahistidine tag to the tag the In the of glycosaminoglycan, the thrombin inhibition are by deletion of the of wt-rHCII. In the of heparin the of residues 1–75 to in with the of the of thrombin inhibition by the rHCII-CHis6 are heparin cofactor activity of rHCII-CHis6 is with the deletion of the the and 1–75 a of activity with heparin and of activity that the the amino-terminal of HCII on the hexahistidine tag is residues and We for the and of HCII and amino-terminal acidic domain is of HCII, the that the may in to the We compared rHCII-CHis6, rHCII-CAla6, and rHCII-CLys6 to the character of the carboxyl-terminal We that the activity of rHCII-CHis6 was a of the of on the a hexa-alanine a hexalysine tag to this We that the hexahistidine hexa-alanine tag increased the of thrombin inhibition in the of inhibition of another serine is by the of that the with the hexahistidine tag are a of on the in thrombin inhibition that the acidic is In the of heparin a in the of thrombin inhibition by rHCII-CHis6 and a to a heparin In with and a in In the of increased of thrombin inhibition comparing rHCII-CHis6 to wt-rHCII. hexa-alanine and hexalysine are to the inhibition of thrombin in the of in and heparin by rHCII-CLys6 that this may have less the properties of rHCII-CHis6. that the the of to the of HCII is for increased heparin binding. that the in of inhibition with heparin to to the hexahistidine heparin, heparin, and such as such as and Google Scholar, 1997; PubMed Scopus Google Scholar, of D. Scholar). protease inhibitors and proteases are as 1997; PubMed Scopus Google Scholar, Google Scholar, Biochemistry. 1997; PubMed Scopus Google Scholar, A. M. 1997; PubMed Scopus Google Scholar, Carrell R.W. Stone S.R. PubMed Scopus Google Scholar, F.C. Biochemistry. PubMed Scopus Google Scholar, Stone S.R. 1997; PubMed Scopus Google Scholar). with have Biochemistry. 1997; PubMed Scopus Google Scholar, A. M. 1997; PubMed Scopus Google Scholar, Carrell R.W. Stone S.R. PubMed Scopus Google Scholar, F.C. Biochemistry. PubMed Scopus Google Scholar, Stone S.R. 1997; PubMed Scopus Google Scholar). offer some In the of heparin, the activity of rHCII-CHis6 is to that of the inhibitor histidine-tagged HCII at a significantly heparin wild-type In a plasma-based assay, increased activity of rHCII-CHis6 with heparin and HCII is a with activity to thrombin in the A activity in plasma M. A. PubMed Scopus Google and to for the of rHCII-CHis6 to a less active as a thrombin In increased antithrombin at heparin is to HCII mutant a of the offer a novel to We and for in the of the mutants We for in the of this We and for of this and for this