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Arginine 200 of Heparin Cofactor II Promotes Intramolecular Interactions of the Acidic Domain IMPLICATION for THROMBIN INHIBITION*

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Arginine 200 of Heparin Cofactor II Promotes Intramolecular Interactions of the Acidic Domain IMPLICATION for THROMBIN INHIBITION* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 22, Issue of May 30, pp. 14074–14079, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Arginine 200 of Heparin Cofactor II Promotes Intramolecular Interactions of the Acidic Domain IMPLICATION FOR THROMBIN INHIBITION* (Received for publication, February 19, 1997) Angelina V. Ciaccia‡, Dougald M. Monroe§¶, and Frank C. Church§¶i** From the Departments of ‡Pharmacology, iPathology and Laboratory Medicine, and §Medicine, ¶Center for Thrombosis and Hemostasis, The University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 Heparin cofactor II (HCII) is presumed to be a physi- activity of serine proteinases involved in such processes as ological inhibitor of the serine proteinase thrombin. The coagulation, fibrinolysis, complement activation, inflamma- reaction between HCII and thrombin is quite unique, tion, and tumor metastasis (Refs. 1 and 2 and reviewed in Ref. because it involves an unusual HCII-reactive site loop 3). Heparin cofactor II (HCII) belongs to a subfamily of serpins 444 445 sequence of Leu -Ser , requires the presence of gly- whose activity is greatly accelerated upon binding to glycos- cosaminoglycans for optimal activity and involves a pro- aminoglycans, such as heparin, heparan sulfate, and derma- tein-protein interaction besides the reactive site loop- tan sulfate (4, 5). In vivo, glycosaminoglycan-containing pro- active site interaction characteristic of serine teoglycans found on cell surfaces and in extracellular matrix proteinase inhibitor-serine proteinase pairs. Two muta- serve to accelerate this reaction (6–8). The physiological target tions at a unique HCII residue, Arg200 3 Ala or Glu, were of HCII is presumed to be thrombin, a pluripotent coagulation generated by site-directed mutagenesis. The mutations proteinase that participates in inflammation and wound heal- did not alter either HCII binding to heparin-Sepharose ing processes based on its chemotactic, mitogenic, and cyto- or HCII inhibition of thrombin in the presence of hepa- rin or dermatan sulfate, suggesting that Arg200 is not kine-like action on vascular smooth muscle cells, monocytes, part of the glycosaminoglycan binding site of HCII. In and fibroblasts (9–12). Thrombin activity generated during the absence of glycosaminoglycan, there was a signifi- blood coagulation is regulated primarily by antithrombin, a cant increase in a-thrombin inhibition by the Arg200 heparin-binding serpin that inhibits most coagulation protein- mutants as compared with wild type recombinant HCII ases. However, extravascular thrombin activity associated (wt-rHCII), whereas inhibition rates with chymotrypsin with inflammation and wound healing processes is thought to were identical. Inhibition of gT-thrombin, which lacks be regulated by HCII, which exhibits remarkable specificity for anion-binding exosite 1 ((ABE-1), the region of a-throm- thrombin (13, 14). bin that interacts with the acidic domain of HCII), was HCII possesses several characteristics for thrombin specific- significantly reduced compared with a-thrombin, but ity that render it unique among heparin-binding serpins. HCII 200 the reduction was more dramatic for the Arg -rHCII is the only heparin-binding serpin that binds dermatan sulfate mutants. Hirugen, which binds to ABE-1 of a-thrombin, to accelerate thrombin inhibition (15). The heparin and derma- 200 also diminished inhibition of a-thrombin by the Arg - tan sulfate binding sites, which are distinct but overlapping, 200 rHCII mutants to nearly wt-rHCII levels. Both Arg - are localized primarily in the D-helix region (16–19). HCII is rHCII mutants had significantly increased k values as a also unique because it has Leu444 at the P1 position, whereas compared with wt-rHCII, whereas the k rates were un- d most thrombin-inhibiting serpins (like antithrombin and pro- changed. Collectively, these results suggest that the im- tein C inhibitor) and typical thrombin substrates contain an proved inhibitory activity of the Arg200-rHCII mutants is mediated by enhanced interactions between the acidic Arg at the P1 site (20, 21). The P1 residue, which is located on domain and ABE-1, resulting in an increased HCII- an exposed loop that interacts with the active site of the pro- thrombin association rate. teinase, determines in large part the proteinase specificity of the serpin (for a review, see Ref. 3). The presence of a P1 Leu in HCII enables it to inhibit chymotrypsin, a nonphysiological Serine proteinase inhibitors (serpins)1 are a superfamily of target, more rapidly than thrombin in the absence of glycos- proteins whose primary function is to regulate the proteolytic aminoglycans (22). HCII with an Arg substituted for the P1 Leu no longer inhibits chymotrypsin (23). Interestingly, this mutant has an increased thrombin inhibition rate in the ab- * This work was supported in part by Research Grants HL-32656 (to sence of glycosaminoglycans, but is also proteolytically inacti- F. C. C.) and 5T32-GM-07040-19 (stipend support for A. V. C.) from the vated by thrombin in the presence of heparin (23, 24). National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must Although HCII is ;30% identical in primary structure to therefore be hereby marked “advertisement” in accordance with 18 antithrombin and other serpins (25), it has an unusual amino- U.S.C. Section 1734 solely to indicate this fact. terminal extension of approximately 80 residues (26, 27). The ** To whom correspondence and reprint requests should be ad- amino terminus contains a tandem repeat of two acidic dressed: CB# 7035, Division of Hematology-Oncology/Medicine, 932 Mary Ellen Jones Bldg., University of North Carolina, Chapel Hill, NC stretches that are somewhat homologous to the carboxyl ter- 27599-7035. Fax: 919-966-7639; E-mail: [email protected]. minus of the leech thrombin inhibitor, hirudin (28). The acidic 1 The abbreviations used are: serpin, serine proteinase inhibitor; domains of both HCII and hirudin bind to anion-binding ex- HCII, heparin cofactor II; ABE-1, anion-binding exosite 1; wt-rHCII, osite-1 (ABE-1) of thrombin (29). The acidic domain of HCII is wild type recombinant heparin cofactor II; R200A-rHCII and R200E- also thought to bind intramolecularly to the D-helix, the gly- rHCII are the recombinant heparin cofactor II mutants with substitu- tions of Ala of Glu, respectively, for Arg200; Sf9, S. frugiperda insect cosaminoglycan binding site on HCII. Binding of glycosamin- cells. oglycans to the D-helix is thought to displace the acidic domain 14074 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Role of Arg200 in Heparin Cofactor II 14075 and promote its interaction with ABE-1 of thrombin (30, 31). with a linear 1.0 ml/min salt gradient of 50 mM to 0.5 M NaCl. 1.0 mgof The acidic domain interaction with ABE-1 appears to be the recombinant HCII was loaded, and 20 3 1-ml fractions were collected. driving force for the rapid inhibition of thrombin by HCII in the The fractions were analyzed by thrombin inhibition assays in the pres- ence of 10 mg/ml heparin, as described below. presence of glycosaminoglycans and compensates for the unfa- Proteinase Inhibition Assays—Human a-thrombin was isolated and vorable P1 Leu residue (19, 30, 32). prepared as described previously (36), and bovine chymotrypsin was We are studying HCII to better understand the role of spe- purchased from Sigma. gT-Thrombin was prepared by limited proteol- cific amino acid residues in this unique thrombin inhibition ysis of plasma-derived a-thrombin with L-1-tosylamido-2-phenylethyl reaction. A comparison of serpin sequences shows that HCII is chloromethyl ketone-treated trypsin (Cooper Biomedical) [(37) as mod- ified in (32)]. The activity of g -thrombin was then verified by chromo- the only heparin-binding serpin with a basic residue at Arg200 T 200 genic substrate cleavage and fibrin clotting assays. Chromogenic sub- (2). Arg of HCII is in strand 2 of b sheet A, adjacent to the strates were tosyl-Gly-Pro-Arg-r-nitroanilide (150 mM; Boehringer dermatan sulfate-binding region of the D-helix; thus it may be Mannheim) for thrombin and N-succinyl-Ala-Ala-Pro-Phe-r-nitroani- poised to play a unique role in regulating HCII activity. In this lide (500 mM; Sigma) for chymotrypsin. Hirugen (residues 53–64) was study, we have found that Arg200 promotes the interaction of from Multiple Peptide Systems. A control peptide corresponding to the the acidic domain with HCII in the absence of glycosaminogly- reverse sequence of the HCII acidic domain (residues 47–61) was syn- cans, but is not involved in glycosaminoglycan binding. We thesized on a Synergy™ peptide synthesizer (Applied Biosystems). Pro- 200 teinase inhibition assays for wt-rHCII and the R200-rHCII mutants propose that the function of Arg is to keep HCII essentially were performed in 96-well enzyme-linked immunosorbent assay plates “inactive” when it is circulating in the blood stream unbound to (previously coated with 2 mg/ml bovine serum albumin) at room tem- glycosaminoglycans. perature in HNPN, pH 7.4, buffer containing 2 mg/ml bovine serum albumin. In the absence of glycosaminoglycan, 100 nM HCII and 1 nM EXPERIMENTAL PROCEDURES thrombin were incubated together for 30–180 min in the presence of 50 Generation and Expression of R200A-rHCII and R200E-rHCII—Hu- mg/ml polybrene. The thrombin-HCII association time was 90 min for man wild type recombinant HCII (wt-rHCII) (cDNA kindly provided by assays performed in the presence of hirugen. For the heparin (Diosynth, Dr. Douglas M. Tollefsen, Washington University School of Medicine, Oss, The Netherlands) and dermatan sulfate (Calbiochem; nitrous acid- St. Louis, MO) was previously expressed in the baculovirus expression treated to remove contaminating heparin and heparan sulfate) tem- system and characterized (33).
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