Enhancement of Heparin Cofactor II Anticoagulant Activity
1999; Elsevier BV; Volume: 274; Issue: 49 Linguagem: Inglês
10.1074/jbc.274.49.34556
ISSN1083-351X
AutoresSusannah J. Bauman, Frank Church,
Tópico(s)Venomous Animal Envenomation and Studies
ResumoHeparin 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 glycosaminoglycan-binding serpins (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, 3Pratt C.W. Church F.C. Blood Coagul. & Fibrinolysis. 1993; 4: 479-490Crossref PubMed Scopus (56) Google Scholar). This group includes antithrombin III (ATIII), heparin cofactor II (HCII), protein C inhibitor, protease nexin-1, and plasminogen activator inhibitor-1 (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). Glycosaminoglycans bound by these serpins include heparin, chondroitin sulfates, dermatan sulfate, and proteoglycans with these molecules as side chains.Serpins inhibit their target proteases by acting as suicide substrates (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 contain an exposed reactive site loop. Within the reactive site loop of the serpin is the P1–P1′ bond (4Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4727) Google Scholar). The target protease will recognize this reactive site bond and attack it as a substrate. Once attacked, the serpin and protease are caught in a 1:1 covalent complex in which the protease is rendered inactive (5Gettins P. Patston P.A. Schapira M. Hematol. Oncol. Clin. N. Am. 1992; 6: 1393-1408Abstract Full Text PDF PubMed Google Scholar). The complex is then cleared via receptor-mediated endocytosis (6Pratt C.W. Church F.C. Pizzo S.V. Arch. Biochem. Biophys. 1988; 262: 111-117Crossref PubMed Scopus (28) Google Scholar, 7Hoxie J.A. Ahuja M. Belmonte E. Pizarro S. Parton R. Brass L.F. J. Biol. Chem. 1993; 268: 13756-13763Abstract Full Text PDF PubMed Google Scholar, 8Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar).Heparin cofactor II is a 65.5-kDa glycoprotein whose inhibitory activity is directed toward thrombin and chymotrypsin (9Parker K.A. Tollefsen D.M. J. Biol. Chem. 1985; 260: 3501-3505Abstract Full Text PDF PubMed Google Scholar, 10Church F.C. Noyes C.M. Griffith M.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6431-6434Crossref PubMed Scopus (50) Google Scholar). Unlike the physiologic thrombin inhibitor ATIII (11Olson S.T. Bjork I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar, 12Desai U.R. Petitou M. Björk I. Olson S.T. J. Biol. Chem. 1998; 273: 7478-7487Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), HCII inhibition of thrombin is accelerated by both heparin and dermatan sulfate (13Tollefsen D.M. Pestka C.A. Monafo W.J. J. Biol. Chem. 1983; 258: 6713-6716Abstract Full Text PDF PubMed Google Scholar, 14Rogers S.J. Pratt C.W. Whinna H.C. Church F.C. J. Biol. Chem. 1992; 267: 3613-3617Abstract Full Text PDF PubMed Google Scholar). Maximal rates of thrombin inhibition by HCII are seen in the presence of dermatan sulfate. As many dermatan sulfate-containing proteoglycans are located extravascularly it has been speculated that HCII is an extravascular thrombin inhibitor (15McGuire E.A. Tollefsen D.M. J. Biol. Chem. 1987; 262: 169-175Abstract Full Text PDF PubMed Google Scholar, 16Roughley P.J. White R.J. Biochem. J. 1989; 262: 823-827Crossref PubMed Scopus (78) Google Scholar, 17Whinna H.C. Choi H.U. Rosenberg L.C. Church F.C. J. Biol. Chem. 1993; 268: 3920-3924Abstract Full Text PDF PubMed Google Scholar).Heparin cofactor II is unusual in that its reactive site bond is Leu-Ser (18Griffith M.J. Noyes C.M. Tyndall J.A. Church F.C. Biochemistry. 1985; 24: 6777-6782Crossref PubMed Scopus (39) Google Scholar, 19Blinder M.A. Marasa J.C. Reynolds C.H. Deaven L.L. Tollefsen D.M. Biochemistry. 1988; 27: 752-759Crossref PubMed Scopus (72) Google Scholar). In the presence of glycosaminoglycan, HCII inhibits thrombin through an unusual mechanism (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, 14Rogers S.J. Pratt C.W. Whinna H.C. Church F.C. J. Biol. Chem. 1992; 267: 3613-3617Abstract Full Text PDF PubMed Google Scholar, 20Ragg H. Ulshofer T. Gerewitz J. J. Biol. Chem. 1990; 265: 5211-5218Abstract Full Text PDF PubMed Google Scholar, 21Ragg H. Ulshofer T. Gerewitz J. J. Biol. Chem. 1990; 265: 22386-22391Abstract Full Text PDF PubMed Google Scholar, 22van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Abstract Full Text PDF PubMed Google Scholar, 23Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar, 24Ciaccia A.V. Church F.C. Protein Pept. Lett. 1997; 4: 215-224Google Scholar, 25Myles T. Church F.C. Whinna H.C. Monard D. Stone S.R. J. Biol. Chem. 1998; 273: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Heparin cofactor II contains a unique amino-terminal region that is highly acidic and thus referred to as the “acidic domain.” In the absence of glycosaminoglycan, the acidic domain is believed to interact with the D-helix region, which is highly basic. The D-helix region is involved in glycosaminoglycan binding. When glycosaminoglycan is present, it has been suggested that the acidic domain is displaced. The displaced acidic domain is then able to interact with the anion-binding exosite-1 of thrombin.Standard procedures to purify HCII involve binding protein to heparin-Sepharose (26Tollefsen D.M. Majerus D.W. Blank M.K. J. Biol. Chem. 1982; 257: 2162-2169Abstract Full Text PDF PubMed Google Scholar, 27Griffith M.J. Noyes C.M. Church F.C. J. Biol. Chem. 1985; 260: 2218-2225Abstract Full Text PDF PubMed Google Scholar). However, a further investigation of the HCII mechanism of action by mutagenesis of its glycosaminoglycan-binding region would disrupt purification of protein by heparin affinity. Therefore, we began to derive alternative purification protocols to avert this problem. Many researchers have used a sequence consisting of six histidine residues as an affinity ligand (28Peters D. Frank R. Hengstenberg W. Eur. J. Biochem. 1995; 228: 798-804Crossref PubMed Scopus (13) Google Scholar, 29Robeva A.S. Woodard R. Luthin D.R. Taylor H.E. Linden J. Biochem. Pharmacol. 1996; 51: 545-555Crossref PubMed Scopus (70) Google Scholar, 30Kashlev M. Nudler E. Severinov K. Borukhov S. Komissarova N. Goldfarb A. Methods Enzymol. 1996; 274: 326-334Crossref PubMed Scopus (78) Google Scholar). By attaching this sequence to a protein, either the amino or carboxyl terminus, protein can be purified with a specialized Ni2+ matrix (31Hochuli E. Dobeli H. Schacher A. J. Chromatogr. 1987; 411: 177-184Crossref PubMed Scopus (971) Google Scholar, 32Teien A.N. Abildgaard U. Höök M. Thromb. Res. 1976; 8: 859-867Abstract Full Text PDF PubMed Scopus (161) Google Scholar, 33Church F.C. Whinna H.C. Anal. Biochem. 1986; 157: 77-83Crossref PubMed Scopus (62) Google Scholar). This method had been used successfully as an affinity purification ligand (28Peters D. Frank R. Hengstenberg W. Eur. J. Biochem. 1995; 228: 798-804Crossref PubMed Scopus (13) Google Scholar, 29Robeva A.S. Woodard R. Luthin D.R. Taylor H.E. Linden J. Biochem. Pharmacol. 1996; 51: 545-555Crossref PubMed Scopus (70) Google Scholar, 30Kashlev M. Nudler E. Severinov K. Borukhov S. Komissarova N. Goldfarb A. Methods Enzymol. 1996; 274: 326-334Crossref PubMed Scopus (78) Google Scholar). These data suggest that the histidine tag is a benign addition to proteins to which it was attached (28Peters D. Frank R. Hengstenberg W. Eur. J. Biochem. 1995; 228: 798-804Crossref PubMed Scopus (13) Google Scholar, 29Robeva A.S. Woodard R. Luthin D.R. Taylor H.E. Linden J. Biochem. Pharmacol. 1996; 51: 545-555Crossref PubMed Scopus (70) Google Scholar, 30Kashlev M. Nudler E. Severinov K. Borukhov S. Komissarova N. Goldfarb A. Methods Enzymol. 1996; 274: 326-334Crossref PubMed Scopus (78) Google Scholar).In this report we show the following: (a) carboxyl-terminal hexahistidine-tagged recombinant HCII (rHCII-CHis6) has enhanced progressive antithrombin and heparin cofactor activities and increased heparin-Sepharose binding compared with wild-type recombinant HCII (wt-rHCII); (b) a region within the amino terminus of HCII may interact with the carboxyl-terminal hexahistidine of rHCII-CHis6; (c) carboxyl-terminal hexahistidine-tagged recombinant antithrombin III (rATIII-CHis6) does not have these enhanced activities compared with wild-type recombinant ATIII (wt-rATIII); and (d) the enhanced heparin effect of rHCII-CHis6is maintained in a plasma-based thrombin inhibition assay. Collectively, these data suggest that rHCII-CHis6 could be a novel anticoagulant therapy.DISCUSSIONWe have “serpendipitously” constructed an HCII mutant that is a significantly better inhibitor of thrombin than the wild-type molecule. This mutant, rHCII-CHis6, is a carboxyl-terminal hexahistidine-tagged heparin cofactor II. In the absence of glycosaminoglycan we see a small increase in rates of thrombin inhibition. In the presence of heparin, rHCII-CHis6 has antithrombotic activity that reaches rates comparable to those of the physiologic thrombin inhibitor ATIII. Addition of the hexahistidine tag to HCII also increases the affinity of this molecule for heparin. In contrast, the enhanced activity of rHCII-CHis6 is not seen with other sulfated polysaccharides like dermatan sulfate, desmin, or fucoidan. Our results demonstrate that the activity is solely a result of the addition of the carboxyl-terminal histidine tag and that rHCII-CHis6 functions to inhibit thrombin through the same mechanism as wt-rHCII, which is highly dependent on ABE-1 of thrombin. Augmentation of heparin cofactor activity in rHCII-CHis6 is reversible by CPA proteolysis. We also presented another mutant, rHCII-CHis5Pro, which retains the enhanced activity but is resistant to CPA. We also showed that the addition of a carboxyl-terminal hexahistidine tag to ATIII actually interferes with the ability of ATIII to inhibit two of its target serine proteases, thrombin and Factor Xa, and has no influence on the heparin binding of the molecule. This contrast in activity between HCII and ATIII with a carboxyl-terminal hexahistidine tag is especially notable since their reactive site loops are very similar in sequence and in length (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 27Griffith M.J. Noyes C.M. Church F.C. J. Biol. Chem. 1985; 260: 2218-2225Abstract Full Text PDF PubMed Google Scholar). Therefore, the increase in antithrombin activity is not a general phenomenon for glycosaminoglycan-binding serpins.Previous work has indicated that when the hexahistidine tag is left attached, it rarely affects the properties of the native protein (28Peters D. Frank R. Hengstenberg W. Eur. J. Biochem. 1995; 228: 798-804Crossref PubMed Scopus (13) Google Scholar, 29Robeva A.S. Woodard R. Luthin D.R. Taylor H.E. Linden J. Biochem. Pharmacol. 1996; 51: 545-555Crossref PubMed Scopus (70) Google Scholar, 30Kashlev M. Nudler E. Severinov K. Borukhov S. Komissarova N. Goldfarb A. Methods Enzymol. 1996; 274: 326-334Crossref PubMed Scopus (78) Google Scholar). However, we have not found any examples of heparin-binding proteins being hexahistidine-tagged anywhere in the literature. This was the point at which we made our serendipitous finding.As described in the Introduction, HCII is believed to inhibit thrombin through an unusual mechanism in the presence of glycosaminoglycan (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,14Rogers S.J. Pratt C.W. Whinna H.C. Church F.C. J. Biol. Chem. 1992; 267: 3613-3617Abstract Full Text PDF PubMed Google Scholar, 20Ragg H. Ulshofer T. Gerewitz J. J. Biol. Chem. 1990; 265: 5211-5218Abstract Full Text PDF PubMed Google Scholar, 21Ragg H. Ulshofer T. Gerewitz J. J. Biol. Chem. 1990; 265: 22386-22391Abstract Full Text PDF PubMed Google Scholar, 22van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Abstract Full Text PDF PubMed Google Scholar, 23Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar, 24Ciaccia A.V. Church F.C. Protein Pept. Lett. 1997; 4: 215-224Google Scholar, 25Myles T. Church F.C. Whinna H.C. Monard D. Stone S.R. J. Biol. Chem. 1998; 273: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Since the enhanced activity of the hexahistidine tag was only seen with the glycosaminoglycan-binding serpin HCII but not ATIII, the data presented support the concept that the D-helix region-acidic domain interaction is altered. The increase in antithrombin activity of rHCII-CHis6 suggests that the acidic domain may be in an altered conformation to more easily encounter thrombin ABE-1. Data presented describing inhibition in the presence of hirugen, a peptide of the carboxyl-terminal region of hirudin, indicate that ABE-1 of thrombin is still very important in the mechanism of thrombin inhibition by rHCII-CHis6. This is further supported by inhibition of γT-thrombin, a proteolyzed form of thrombin defective at ABE-1. However, the slight residual increased activity of rHCII-CHis6 toward γT-thrombin might imply that other residues of ABE-1 not perturbed by proteolysis (or blocked by hirugen in α-thrombin) might be involved in this inhibition reaction. The increased binding of rHCII-CHis6 to heparin-Sepharose compared with wt-rHCII also lends support to the notion that part of the d-helix region is more accessible to heparin interaction. The reduced heparin concentration needed for peak activity for rHCII-CHis6 is related to heparin-Sepharose affinity and is consistent with that seen previously for heparin binding characteristics and activity for heparin-binding serpins (3Pratt C.W. Church F.C. Blood Coagul. & Fibrinolysis. 1993; 4: 479-490Crossref PubMed Scopus (56) Google Scholar, 42Pratt C.W. Whinna H.C. Church F.C. J. Biol. Chem. 1992; 267: 8795-8801Abstract Full Text PDF PubMed Google Scholar). The change in heparin but not dermatan sulfate binding of rHCII-CHis6 further implies that the glycosaminoglycan-binding site of HCII has both distinct and overlapping structural elements for heparin and dermatan sulfate interactions and agrees with previous variants/mutants of HCII altered in the D-helix region (23Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar, 24Ciaccia A.V. Church F.C. Protein Pept. Lett. 1997; 4: 215-224Google Scholar). The comparable inhibition rates of wt-rHCII and rHCII-CHis6 with chymotrypsin (which does not use either the acidic domain of HCII or glycosaminoglycans for inhibition) indicate that the reactive site loop has not been altered to an “activated” conformation, further implicating an alteration between the D-helix/acidic domain regions in the increased activity of rHCII-CHis6.It is also possible that these results could be due to the addition/exposure of a secondary heparin-binding site or some other effect of the hexahistidine on the conformation of HCII. Morgan and co-workers (43Borza D.-B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Crossref PubMed Scopus (76) Google Scholar, 44Borza D.-B. Morgan W.T. J. Biol. Chem. 1998; 273: 5493-5499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 45Peterson C.B. Morgan W.T. Blackburn M.N. J. Biol. Chem. 1987; 262: 7567-7574Abstract Full Text PDF PubMed Google Scholar) have published extensively on the function of histidine-proline-rich glycoprotein as a heparin-binding molecule. This protein is notable for its tandem histidine-rich repeats that bind heparin either in the presence of divalent cations or at sub-physiologic pH values. All of our experiments were carried out at physiologic pH and in the absence of added divalent cations. Trace metal contamination is an unlikely source of our results since the control CPA-digested proteins are treated with EDTA and still maintain enhanced activity and heparin binding. The lack of effect on the heparin binding ability of ATIII also argues against the addition of a new heparin-binding site in either ATIII or HCII due to the hexahistidine tag itself. To address further the mechanism of hexahistidine and HCII, we then focused our work both on the composition of the tag and the influence of the amino-terminal acidic domain region of HCII.We explored the contribution of the amino-terminal acidic domain of HCII to the enhanced activity of rHCII-CHis6 by sequentially deleting the first 52, 68, or 75 amino acids from either wt-rHCII or rHCII-CHis6. In 1991, van Deerlin and Tollefsen (22van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Abstract Full Text PDF PubMed Google Scholar) described similar amino-terminal deletion mutants of HCII. Our data for deletions of wt-rHCII are in agreement with their results. No function has been assigned to the first 52 amino acids. However, within residues 53 and 75 there are 13 acidic amino acids (Asp, Glu, and sulfated-Tyr). These residues are grouped in two distinct clusters called “acidic region 1” and “acidic region 2” (AR-1 and AR-2). When glycosaminoglycans bind the D-helix of HCII it is believed that AR-2 is displaced, which allows AR-1 to be more accessible to bind ABE-1 of thrombin (22van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Abstract Full Text PDF PubMed Google Scholar). The removal of amino acids 1–52 should be relatively benign based on this model of HCII. However, the removal of amino acids 1–68 or 1–75 should influence the interaction of HCII with thrombin, especially in the presence of glycosaminoglycans.In the absence of glycosaminoglycan, the deletion mutants of wt-rHCII had no major loss of protease inhibition activity. Based on the results, the amino-terminal region of wt-HCII is not significantly involved in the inhibition of chymotrypsin or thrombin. In contrast, all three deletions in rHCII-CHis6 lead to significant losses of both thrombin and chymotrypsin inhibition. The losses found in inhibition must be due to the presence of the hexahistidine tag in rHCII-CHis6. Most likely the tag in the deletion mutants causes a change in the reactive site loop region of HCII since inhibition is dependent on this structure. These results suggest that either the amino-terminal acidic domain shields the reactive site loop from the hexahistidine tag or it interacts with the hexahistidine tag to then keep the tag from perturbing the reactive site loop.In the presence of glycosaminoglycan, the thrombin inhibition rates are only slightly affected by deletion of the first 52 amino acids of wt-rHCII. In the presence of heparin or dermatan sulfate, the loss of residues 1–68 or 1–75 leads to decreased antithrombotic activity, in agreement with the accepted model of HCII. However, the rates of thrombin inhibition by the rHCII-CHis6 deletions are somewhat different. The enhanced heparin cofactor activity of rHCII-CHis6 is lost with the deletion of the first 52 amino acids. As expected, the 1–68 and 1–75 deletions caused a large loss of activity with both heparin and dermatan sulfate. The progressive loss of activity indicates that the protective effect the amino-terminal region of HCII imparts on the hexahistidine tag is partially mediated between residues 52 and 75. We believe these data provide evidence for the importance and specificity of an HCII carboxyl terminus (hexahistidine tag) and amino-terminal acidic domain interaction. Since there is no crystal structure of HCII, the data imply that the amino terminus may be in close proximity to the carboxyl terminus.We compared rHCII-CHis6, rHCII-CAla6, and rHCII-CLys6 to provide information about the character of the carboxyl-terminal hexapeptide tag. We hypothesized that if the enhanced activity of rHCII-CHis6 was a result either of the extra length or of partial positive charge on the tag, then a hexa-alanine or a hexalysine tag could be used to probe this phenomenon further. We found that only the hexahistidine or hexa-alanine tag increased the rate of thrombin inhibition in the absence of glycosaminoglycan. The inhibition of another serine protease, chymotrypsin, is not affected by the addition of each tag. These experiments provide evidence that the rate increases seen with the hexahistidine tag are not fully a result of charge on the tag, but changes in thrombin inhibition do further suggest that the acidic domain-D-helix interaction is perturbed.In the presence of heparin we see a large increase in the rate of thrombin inhibition by rHCII-CHis6 and a shift to a lower heparin requirement. In contrast, with rHCII-CAla6 and rHCII-CLys6, we see either a loss or no change in activity. In the presence of dermatan sulfate we do not observe increased rates of thrombin inhibition when comparing rHCII-CHis6 to wt-rHCII. The hexa-alanine and hexalysine tags actually are detrimental to the inhibition of thrombin in the presence of dermatan sulfate. The increase in both dermatan sulfate and heparin binding by rHCII-CLys6 implies that this protein may have less specific glycosaminoglycan binding abilities than the altered binding properties of rHCII-CHis6. These data indicate that neither the positive charge nor the addition of six amino acids to the carboxyl terminus of HCII is solely responsible for increased heparin binding. However, these results do suggest that the increase in rates of rHCII inhibition with heparin seems to be specific to the hexahistidine tag.Current antithrombotic therapies include heparin, low molecular weight heparin, and other heparinoids, oral anticoagulants such as warfarin, synthetic molecules such as Argatroban, and naturally occurring peptides isolated from hematophagous parasites, most notably hirudin (46Mueller R.L. Scheidt S. Circulation. 1993; 89: 432-449Crossref Google Scholar, 47Weitz J.I. N. Engl. J. Med. 1997; 337: 688-698Crossref PubMed Scopus (1447) Google Scholar, 48Leung L. Bennett J.S. 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