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J. Biol. Chem., Vol. 277, Issue 22, 19823-19830, May 31, 2002

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

Jennifer W. Mitchell and Frank C. Church^§¶

From the Departments of  Pathology and Laboratory Medicine, ^§ Pharmacology, and ^¶ Medicine, Center for Thrombosis and Hemostasis, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, North Carolina 27599-7035

Received for publication, January 22, 2002, and in revised form, February 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We used site-directed mutagenesis to investigate the role of Glu⁶⁹, Asp⁷⁰, Asp⁷¹, Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵ in the second acidic region (AR2) of the serpin heparin cofactor II (HCII) during formation of the thrombin·HCII complex with and without glycosaminoglycans. E69Q/D70N/D71N recombinant (r)HCII, D72N/Y73F/D75N rHCII, and E69Q/D70N/D71N/D72N/Y73F/D75N rHCII were prepared to localize acidic residues important for thrombin inhibition. Interestingly, D72N/Y73F/D75N rHCII had significantly enhanced thrombin inhibition without glycosaminoglycan (4-fold greater) and with heparin (6-fold greater), showing maximal activity at 2 µg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) with maximal activity at 20 µg/ml heparin. The other rHCII mutants had lesser-enhanced activities, but they all eluted from heparin-Sepharose at significantly higher ionic strengths compared with wt-rHCII. Neutralizing and reversing the charge of Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵ were done to characterize their individual contribution to HCII activity. Only Y73K rHCII and D75K rHCII have significantly increased heparin cofactor activity compared with wt-rHCII; however, all of the individual rHCII mutants required substantially less glycosaminoglycan at maximal inhibition than did wt-rHCII. Inhibition of either -thrombin/hirugen or _T-thrombin (both with an altered anion-binding exosite-1) by the AR2 rHCII mutants was similar to wt-rHCII. D72N/Y73F/D75N rHCII and D75K rHCII were significantly more active than wt-rHCII in a plasma-based thrombin inhibition assay with glycosaminoglycans. These results indicate that improved thrombin inhibition in the AR2 HCII mutants is mediated by enhanced interactions between the acidic domain and anion-binding exosite-1 of thrombin and that AR2 may be a "molecular rheostat" to promote thrombin inhibition in the presence of glycosaminoglycans.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The serine protease thrombin is a critical component in coagulation, inflammation, and wound healing (1-9). Thrombin activity must be carefully regulated to maintain an appropriate balance within the vasculature. One mechanism of thrombin regulation is by serine protease inhibitors (serpins)¹ such as antithrombin (ATIII) and heparin cofactor II (HCII) (10-14). Heparin cofactor II and ATIII belong to a sub-class of serpins whose activity is greatly enhanced upon binding to glycosaminoglycans like heparin and heparan sulfate (HCII and ATIII) and dermatan sulfate (HCII) (15-18). These glycosaminoglycans are found in vivo on cell surfaces and in extracellular matrix to support these inhibition reactions (19-23).

Heparin cofactor II has several features for thrombin inhibition and specificity that render it novel among heparin-binding serpins (24-26). Heparin cofactor II has an atypical Leu⁴⁴⁴ reactive center residue, whereas the more typical Arg is found in thrombin inhibitors like ATIII or protein C inhibitor (10, 12, 13). The inhibition of thrombin by HCII is enhanced by both heparin and dermatan sulfate, and the glycosaminoglycan binding site is primarily contained within the D-helix region of HCII (15, 27-34). Although HCII is ~30% identical in sequence to antithrombin and other serpins, it has a unique N-terminal extension of ~80 residues that contains a tandem repeat of negatively charged acidic residues (9 Asp, 5 Glu, and 2 Tyr-sulfate) (25, 26). Interestingly, the acidic domain of HCII is homologous to the C terminus of the leech anticoagulant protein hirudin, which is a potent thrombin inhibitor. Within the acidic domain of HCII, the two regions are designated acidic region 1 (AR1) from residues 49-62 (⁴⁹DWIPEGEEDDDYLD⁶²) and acidic region 2 (AR2) from residues 63-75 (⁶³LEKIFSEDDDYID⁷⁵). There is substantial evidence that the acidic domain of HCII is involved in facilitating thrombin inhibition, especially in the presence of glycosaminoglycans (27, 29, 30). Ragg et al. (29, 30) initially examined the role of the acidic domain in HCII by either deleting or neutralizing various acidic amino acids and found that AR1 and AR2 have separate functions for thrombin inhibition. Van Deerlin and Tollefsen (27) demonstrated that, by deleting portions of the acidic domain (AR1 and AR2), HCII was a less effective inhibitor of thrombin; interestingly, these mutants also had substantially enhanced binding to heparin compared with wild-type (wt) recombinant (r)-HCII. These studies suggested that the acidic domain of HCII both is involved in and is imperative for effective thrombin inhibition. The two current models to describe the mechanism for glycosaminoglycan-accelerated inhibition of thrombin by HCII are the "double-bridge" and the "allosteric" mechanisms (31, 35). In both models it is assumed that the acidic domain position is altered following glycosaminoglycan binding to the D-helix region of HCII, thereby allowing the N-terminal acidic domain to bind thrombin anion-binding exosite-1 (exosite-1) (27, 28, 31, 33, 34, 35, 37).

This paper investigates the role of six AR2 acidic residues in HCII during thrombin inhibition in the absence and presence of glycosaminoglycans and with thrombin derivatives with and without a functional exosite-1. The results indicate that Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵ promote intramolecular interactions of the acidic domain with HCII in the absence of glycosaminoglycans, and altering the charge of these acidic residues substantially changes glycosaminoglycan-dependent activities of HCII.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Human plasma -thrombin and HCII were prepared and purified in our laboratory as previously described (38, 39). We prepared human _T-thrombin by limited proteolysis of -thrombin with trypsin as previously described (28). Monoclonal antibody 2-4-34 to purified human plasma HCII was made in our laboratory using standard techniques. Human antithrombin-deficient plasma (catalog no. 203) and normal hemostasis reference plasma (catalog no. 258N) were purchased from America Diagnostica (Greenwich, CT). Heparin was from Diosynth (Oss, The Netherlands). Dermatan sulfate was from Calbiochem (La Jolla, CA) and was nitrous acid-treated to remove contaminating heparin/heparan sulfate (40). Hi-Trap Heparin-Sepharose was from Amersham Biosciences, Inc. (Piscataway, NJ), and Q-Sepharose was from Sigma Chemical Co. Tosyl-Gly-Pro-Arg-4-nitroanilide acetate (tosGly-Pro-Arg-NA, Chromozym TH) was from Roche Molecular Biochemicals (Indianapolis, IN), and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (sucAla-Ala-Pro-Phe-NA) was from Sigma. Hirugen and the reverse peptide to HCII's acidic domain were synthesized and purified by the University of North Carolina at Chapel Hill Peptide Synthesis and Protein Core Facility.

Mutagenesis of Recombinant Proteins-- The QuikChange site-di- rected mutagenesis kit from Stratagene was used to introduce the following mutations E69Q/D70N/D71N, D72N/Y73F/D75N, E69Q/D70N/D71N/D72N/Y73F/D75N (QNNNFN), D72N, D72K, Y73F, Y73K, D75N, and D75K in the double-stranded plasmid HCII-pVL1392, following the protocol as described by the supplier using these primers: 5'-GGACCTGGAGAAGATATTCAGTCAAAACAACGACTACATCGACATCGTCGAC-3' (forward, E69Q/D70N/D71N), 5'-TCGACGATGTCGATGTAGTCGTTGTTTTGACTGAATATCTTCTCCAGGTCC-3' (reverse, E69Q/D70N/D71N), 5'-GATATTCAGTGAAGACGACAACTTCATCAACATCGTCGACAGTCTGTCAGTTTCC-3' (forward, D72N/Y73F/D75N), 5'-GGAAACTGACAGACTGTCGACGATGTTGATGAAGTTGTCGTCTTCATCGAATATC-3' (reverse, D72N/Y73F/D75N), 5'-GAAGATATTCAGTCAAAACAACAACTTCATCAACATCGTCGACAGTCTGTCAGTTTCC-3' (forward, QNNNFN), 5'-GGAAACTGACAGACTGTCGACGATGTTGATGAAGTTGTTGTTTTGACTGAATATCTTC-3' (reverse, QNNNFN), 5'-CCTGGAGAAGATATTCAGTGAAGACGACAACTACATCGACATCGTCGACAGTCG-3' (forward, D72N), 5'-CAGACTGTCGACGATGTCGATGTAGTTGTCGTCTTCACTGAATATCTTCTCCAGG-3' (reverse, D72N), 5'-CCTGGAGAAGATATTCAGTGAAGACGACAAATACATCGACATCGTCGACAGTCTG-3' (forward, D72K), 5'-CAGACTGTCGACGATGTCGATGTATTTGTCGTCTTCACTGAATATCTTCTCCAGG-3' (reverse, D72K), 5'-CCTGGAGAAGATATTCAGTGAAGACGACGACTTCATCGACATCGTCGACAGTCTG-3' (forward, Y73F), 5'-CAGACTGTCGACGATGTCGATGAAGTCGTCGTCTTCACTGAATATCTTCTCCAGG-3' (reverse, Y73F), 5'-CCTGGAGAAGATATTCAGTGAAGACGACGACAAAATCGACATCGTCGACAGTCTG-3' (forward, Y73K), 5'-CAGACTGTCGACGATGTCGATTTTGTCGTCGTCTTCACTGAATATCTTCTCCAGG-3' (reverse, Y73K), 5'-CCTGGAGAAGATATTCAGTGAAGACGACGACTACATCAACATCGTCGACAGTCTG-3' (forward, D75N), 5'-CAGACTGTCGACGATGTTGATGTAGTCGTCGTCTTCACTGAATATCTTCTCCAGG-3' (reverse, D75N), 5'-CCTGGAGAAGATATTCAGTGAAGACGACGACTACATCAAAATCGTCGACAGTCTG-3' (forward, D75K), 5'-CAGACTGTCGACGATTTTGATGTAGTCGTCGTCTTCACTGAATATCTTCTCCAGG-3' (reverse, D75K). PCR was performed to generate double-stranded HCII cDNA, including the mutations in the expression vector. After amplification, parental DNA was removed by digestion with DpnI endonuclease and the nicked vector DNA containing the desired mutation was transformed into Epicurian Coli XL1-Blue supercompetent cells (Stratagene). Full-length sequencing of each clone by Sequenase kit (version 2.0, Amersham Biosciences, Inc., Cleveland, OH) confirmed the incorporation of each of these mutations into the HCII-pVL1392 construct.

Protein Expression and Purification-- Each mutant rHCII and wt rHCII was cotransfected with linearized BaculoGold (BD PharMingen) Autographa californica nuclear polyhedrosis virus (AcNPV) into Spodoptera frugiperda (Sf9, Invitrogen) insect cells using established protocols in our laboratory (41). We expressed rHCII in HighFive insect cells (Invitrogen, Carlsbad, CA) maintained at 25 °C in Excel 405 medium (JRH Biosciences, Lenexa, KS) as previously detailed. Three days post-baculovirus transfection, the media was collected and centrifuged to remove cell debris. The protein was purified essentially as described previously (41). Protein was then dialyzed against 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% PEG8000 and 0.02% NaN₃. Protein was aliquoted and stored at 80 °C. The production of rHCII was verified by immunoblot analysis of whole cell lysates using a monoclonal antibody to HCII (mAb 2-4-34). The concentration of rHCII was determined by enzyme-linked immunosorbent assay using purified plasma HCII as the standard as described previously (32). We obtained ~70 µg of recombinant protein of each mutant from 100 ml of HighFive cells in a 200-ml shaker flask infected with the various recombinant viral stocks. Immunoblot and SDS-PAGE confirmed that each mutant rHCII had the same electrophoretic mobility as wt rHCII (data not shown).

Protease Inhibition-- Protease inhibition rates were determined as described previously (32, 42, 43). All assays were performed at room temperature in 96-well microtiter plates previously coated with 2 mg/ml BSA. In the absence of glycosaminoglycan, 40-100 nM rHCII (wt and rHCII mutants) was incubated with 1 nM -thrombin, 1 nM _T-thrombin, or 4 nM chymotrypsin, in the presence of 1 mg/ml Polybrene and 2 mg/ml BSA in HNPN, pH 7.4 (20 mM Hepes, pH 7.4, 150 mM NaCl, 0.2% PEG, and 0.02% NaN₃). In the presence of glycosaminoglycans, 10 nM rHCII was incubated with 0-2 mg/ml heparin or 0-2 mg/ml dermatan sulfate with 1 nM thrombin in the presence of 2 mg/ml BSA in HNPN, pH 7.4, in the absence of Polybrene.

In the presence of 10 µg/ml glycosaminoglycan, either hirugen or a control peptide corresponding to the reverse sequence of the HCII acidic domain (residues 47-61) at 20 µM and rHCII (10 nM) was incubated with 0.5 nM thrombin in the presence of 1 mg/ml Polybrene and 2 mg/ml BSA in HNPN, pH 7.4. In the presence of either heparin (5 or 20 µg/ml) or dermatan sulfate (20 or 200 µg/ml), 0.5 nM _T-thrombin was incubated with 10 nM rHCII in the presence of 2 mg/ml BSA in HNPN, pH 7.4.

Association time courses ranged from 1 to 240 min for -thrombin, _T-thrombin, and chymotrypsin depending upon the assay conditions. Residual thrombin activity was measured with 150 µM tosGly-Pro-Arg-NA and 1 mg/ml Polybrene in the absence of glycosaminoglycan or 2 mg/ml Polybrene in the presence of heparin or dermatan sulfate. Residual chymotrypsin activity was measured with 150 µM sucAla-Ala-Pro-Phe-NA. Substrate cleavage was measured by color development at 405 nm on a V_max Kinetic Microplate Reader (Molecular Devices). Assays were performed at least in triplicate on two or more recombinant protein preparations. Second order inhibition rate constants were calculated as described (32, 42, 43).

Heparin-Sepharose Affinity Chromatography-- We determined the relative heparin affinities by using 1-3 µg of rHCII protein diluted in 20 mM Hepes, 50 mM NaCl, 0.1% PEG, 0.05% NaN₃, pH 7.4. We ran each protein on a 1-ml Hi-Trap heparin-Sepharose column equilibrated in 20 mM HEPES, 0.1% PEG, 50 mM NaCl, 0.05% NaN₃, pH 7.4, using an fast-protein liquid chromatography system (Amersham Biosciences, Inc.). After the sample was loaded onto the column, protein was eluted with a 40-ml gradient of 20 mM Hepes, pH 7.4, from 50 to 1500 mM NaCl, and 0.5-ml fractions were collected. For each fraction, 100 µl was aliquoted onto a 96-well microtiter plate and an enzyme-linked immunosorbent assay was performed. The peak elution ionic strength was determined by plotting 405-nm color development and NaCl concentration against the fraction number. All samples were run at least in triplicate using two or more protein preparations.

Human Plasma Assays-- The assay used to evaluate rHCII in plasma was designed based on previously published methods (32, 44, 45). We performed this assay using normal hemostasis reference plasma (REF), human antithrombin-deficient plasma (DEF), or a 50:50 mixture of both types of plasma (REF/DEF) (American Diagnostica) at room temperature in 96-well microtiter plates previously coated with 2 mg/ml BSA (32). We incubated 10 nM wt rHCII or mutant rHCII with either 1 µg/ml heparin or 5 µg/ml dermatan sulfate, in the presence of each type of plasma (diluted 1:100). Thrombin inhibition was started when 1 nM thrombin was added to each well. After 15 s, 150 µM tosGly-Pro-Arg-NA was added, and the residual thrombin activity was measured by color development at 405 nm on a kinetic microplate reader.

Statistical Analysis-- The statistical significance of the data was evaluated using Tukey's Student t test; p values  0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibition of -Thrombin, _T-Thrombin, and Chymotrypsin by the AR2 rHCII Derivatives in the Absence of Glycosaminoglycans-- Wild-type rHCII inhibits -thrombin at a rate of ~1 × 10⁴ M¹ min¹ in the absence of glycosaminoglycans, whereas E69Q/D70N/D71N rHCII, D72N/Y73F/D75N rHCII, and QNNNFN rHCII inhibit -thrombin 1.2-, 4.2-, and 3.2-fold faster than wt-rHCII, respectively (Table I). The results with these "shotgun" HCII mutants suggest that neutralizing Asp⁷², Asp⁷⁵, and Tyr-sulfate⁷³ had the biggest influence on thrombin inhibition. Comparing the effect of these individual residues in HCII individually for thrombin inhibition (using neutral and reverse-charge mutations), we found only D75K had a significantly increased rate of -thrombin inhibition (2.6-fold) when compared with wt-rHCII (Table I). _T-Thrombin (-thrombin with an altered exosite-1) inhibition by wt-rHCII and all of the AR2 rHCII derivatives was ~2-3 × 10³ M¹min¹ (Table I), which further demonstrates the importance of exosite-1 in thrombin during HCII inhibition. Chymotrypsin inhibition by HCII requires neither the acidic domain nor glycosaminoglycan binding, and we found no significant difference for any of the AR2 rHCII derivatives compared with wt-rHCII (Table I). These results indicate that altering the charge of AR2 in HCII creates a better -thrombin inhibitor in the absence of glycosaminoglycans, and that Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵ contribute the most to this effect.

View this table: [in this window] [in a new window]   Table I HCII Inhibition of -thrombin, T-thrombin, and chymotrypsin in the absence of glycosaminoglycans The data summarized below are the rate constants for -thrombin (1 nM), T-thrombin (1 nM), or chymotrypsin (4 nM) inhibition by wild-type and mutant rHCIIs (100 nM rHCII for -thrombin and T-thrombin; 40 nM rHCII for chymotrypsin). The values represent the mean ± S.D. of three to six separate determinations with at least three different protein preparations. All assays were performed as described under "Experimental Procedures."

Glycosaminoglycan-accelerated -Thrombin Inhibition and Heparin-Sepharose Binding by the AR2 rHCII Derivatives-- With heparin, each of the shotgun rHCII derivatives inhibit -thrombin 3- to 6-fold faster than wt-rHCII (Fig. 1, top panel).² Interestingly, less heparin was required for each rHCII mutant to reach maximal thrombin inhibition (1-2 µg/ml) compared with wt-rHCII (~20 µg/ml) (Table II). In the presence of dermatan sulfate, wt-rHCII inhibits thrombin at a rate of 3.3 × 10⁸ M¹min¹ (Fig. 1, bottom panel). D72N/Y73F/D75N rHCII inhibits thrombin at a rate 3.3 times faster than that of wt-rHCII whereas E69Q/D70N/D71N rHCII and QNNNFN rHCII inhibit thrombin similar to wt-rHCII (Fig. 1, lower panel and Table II). However, the amount of dermatan sulfate needed for maximal thrombin inhibitory activity is also significantly decreased for the shotgun AR2 rHCIIs (5-20 µg/ml) as compared with wt-rHCII (100-200 µg/ml) (Table II). The individual AR2 rHCII derivatives of Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵ all required less heparin and dermatan sulfate to reach maximal thrombin inhibition compared with wt-rHCII (Fig. 2); however, only Y73K rHCII and D75K rHCII have significantly increased activity in the presence of heparin whereas none have significantly increased activity in the presence of dermatan sulfate (Table II and Fig. 2). Because the individual Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵ rHCII mutants do not have increased glycosaminoglycan-accelerated thrombin inhibitory activity to the same extent, as does D72N/Y73F/D75N rHCII, this implies an additive influence of these AR2 residues to accelerate the HCII-glycosaminoglycan thrombin inhibition reaction.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Inhibition of -thrombin by wild-type and shotgun AR2 rHCIIs in the presence of heparin and dermatan sulfate. -Thrombin was incubated with wild-type rHCII (), E69Q/D70N/D71N rHCII (), D72N/Y73F/D75N rHCII (), or QNNNFN rHCII () in the presence of increasing amounts of heparin (top panel) or dermatan sulfate (bottom panel). The data represent an average of at least three separate protein preparations performed in triplicate.

View this table: [in this window] [in a new window]   Table II HCII inhibition of -thrombin in the presence of heparin and dermatan sulfate The data summarized below are the rate constants for -thrombin (0.5 nM) inhibition by wild-type and mutant rHCIIs (5 nM) in the presence of heparin or dermatan sulfate (DSO4). The maximal inhibition of each curve was used in the calculation of the average inhibition rate. The values represent the mean ± S.D. of three to six separate determinations with at least three different protein preparations. The final column indicates the peak NaCl concentration at which each variant eluted from Hi-Trap heparin-Sepharose. All assays were performed as described under "Experimental Procedures."

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Inhibition of -thrombin by wild-type and individual AR2 rHCIIs in the presence of heparin and dermatan sulfate. -Thrombin was incubated with rHCII in the presence of increasing amounts of heparin (top panels) or dermatan sulfate (bottom panels). For all graphs, wild-type rHCII is indicated by , in top and bottom left panels, the neutral AR2 residues are D72N (), Y73F (), and D75N (); in the top and bottom right panels, the basic AR2 residues are D72K (), Y73K (), and D75K (). The data represent an average of at least three separate protein preparations performed in triplicate.

Because the AR2 rHCII derivatives required substantially less glycosaminoglycan to reach maximal thrombin inhibition, we next addressed whether this implied a more accessible glycosaminoglycan-binding site. We used NaCl gradient elution from heparin-Sepharose to determine whether the AR2 rHCII mutations have altered apparent affinity for immobilized heparin (Table II). We found that the AR2 rHCII derivatives (except D75N) required more NaCl to elute from heparin-Sepharose compared with wt-rHCII, ranging from 525-550 mM NaCl for wt-rHCII and D75N rHCII to 650-925 mM NaCl for the remainder of the AR2 rHCII derivatives. Collectively, the activity data and the heparin-Sepharose elution results suggest that altering the charge of AR2 allows heparin to bind with higher affinity to the D-helix region of HCII.

-Thrombin/Hirugen and _T-Thrombin Inhibition by the AR2 rHCII Derivatives-- We further studied the role of thrombin exosite-1 during AR2 rHCII inhibition (with heparin or dermatan sulfate). Consistent with past studies, hirugen drastically reduced the rate of -thrombin inhibition with heparin or dermatan sulfate by both wt-rHCII and all of the AR2 rHCII derivatives (Fig. 3, shown for dermatan sulfate in the top panel). Although the inhibition rates for _T-thrombin are much reduced compared with -thrombin, E69Q/D70N/D71N rHCII, D72N/Y73F/D75N rHCII, and D75K rHCII have slightly increased rates of _T-thrombin inhibition as compared with wt-rHCII with heparin or dermatan sulfate (Fig. 3, shown for dermatan sulfate in the bottom panel). Overall, these results imply that the process of thrombin recognition by all of the rHCII's is similar whether using hirugen to sterically hinder access to thrombin exosite-1 or _T-thrombin where the -loop of exosite-1 is absent.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Inhibition of -thrombin/hirugen and of T-thrombin by wild-type and AR2 rHCIIs in the presence dermatan sulfate. Top panel, inhibition of -thrombin by AR2 rHCII mutants in the presence of 10 µg/ml dermatan sulfate with hirugen (black bars), buffer (gray bars), or a control acidic peptide (white bars). The data represent an average of at least two separate protein preparations performed in triplicate. Bottom panel, T-thrombin inhibition by the AR2 rHCII mutants in the presence of 20 µg/ml dermatan sulfate (black bars) or 200 µg/ml dermatan sulfate (white bars). The data represent an average of at least two separate protein preparations performed in triplicate.

Physiological-based Thrombin Inhibition Studies by the ARII rHCII Derivatives-- Our results presented so far show that some AR2 rHCII mutants are more active than wt-rHCII. We next assessed the potential of three AR2 rHCII derivatives (E69Q/D70N/D71N rHCII, D72N/Y73F/D75N rHCII, and D75K rHCII) to inhibit -thrombin with a more physiologically relevant assay setting (Table III). With heparin or dermatan sulfate, E69Q/D70N/D71N rHCII and D72N/Y73F/D75N rHCII both exhibited a 2- to 3-fold greater thrombin inhibitory rate than wt-rHCII in the presence of all three types of plasma (normal reference, antithrombin-deficient, and a 50:50 mix to mimic a heterozygous antithrombin deficiency), whereas the inhibitory activity of D75K rHCII was slightly less than the other rHCII mutants (Table III). These results show that the AR2 rHCII mutants are potent thrombin inhibitors in the presence of glycosaminoglycans and plasma and imply that they may have potential as therapeutic anticoagulants.

View this table: [in this window] [in a new window]   Table III HCII inhibition of -thrombin in the presence of glycosaminoglycans and plasma The data summarized below are the rate constants for -thrombin (1 nM) inhibition by wild-type and mutant rHCIIs (10 nM) added to the various plasmas. The values represent the mean ± S.D. of three to six separate determinations with at least three different protein preparations. All assays were performed as described under "Experimental Procedures."

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although HCII is ~30% identical to ATIII and other serpins, it contains an N-terminal extension of ~80 residues that is found in no other serpin (10, 14, 25, 26). The N terminus contains a tandem repeat of two acidic stretches that have the following sequences: Asp⁴⁹-Trp⁵⁰-Ile-Pro-Glu-Gly-Glu⁵⁵-Glu-Asp-Asp-Asp-Tyr⁶⁰-Leu-Asp⁶² and Leu⁶³-Glu-Lys⁶⁵-Ile-Phe-Ser-Glu-Asp⁷⁰-Asp-Asp-Tyr-Ile-Asp⁷⁵. Metabolic labeling of HepG2 cells demonstrated that the Tyr residues in each acidic repeat are sulfated (46). These acidic repeat elements are homologous to a thrombin-binding region of hirudin, the extremely potent thrombin inhibitor from leech salivary glands. HCII deletion mutants lacking either the first (67-rHCII) or both (74-rHCII) acidic domains were found to have greatly increased affinity for heparin as assessed by salt elution from heparin-Sepharose (27). This observation has led to the hypothesis that the D-helix of HCII is part of an "internal binding site" for the acidic domain. Because the nature of heparin-HCII interaction is mostly anionic, the increased heparin affinity could also be due to the removal of negative charges concentrated in the acidic domain. The goal of this study was to study the structure-activity relationships of the second acidic region (AR2) in HCII using site-directed mutagenesis. Our working hypothesis was that, by altering the charge of AR2 acidic residues in HCII, we would increase both progressive antithrombin activity (inhibition in the absence of added glycosaminoglycans) and heparin- and dermatan sulfate-cofactor activities (thrombin inhibition in the presence of added glycosaminoglycans).

-Thrombin inhibition rates of two of the three shotgun AR2 rHCII mutants in the absence of glycosaminoglycans were significantly increased suggesting that the largest change in activity was influenced by the final three acidic residues (Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵). There was an additive effect of these acidic residues, as only D75K (when residues were individually mutated) had a substantially increased rate compared with wt-rHCII yet this inhibition rate was still lower when compared with D72N/Y73F/D75N rHCII. The increased inhibition activity appears to be caused by an enhanced interaction between the HCII acidic domain and exosite-1 of thrombin, because inhibition rates of both _T-thrombin and -thrombin in the presence of hirugen were all reduced to rates similar to wt-rHCII. Finally, the comparable inhibition rates of wt-rHCII and the AR2 rHCII mutants with chymotrypsin (which requires neither the HCII acidic domain nor glycosaminoglycans for inhibition) indicate that the reactive site loop has not been altered to an "activated" conformation. The results further implicate a role for the acidic domain due to altered HCII intramolecular interactions that increased activity to -thrombin.

We initially hypothesized that the AR2 rHCII mutants would have significant changes related both to glycosaminoglycan binding by HCII and to glycosaminoglycan-mediated inhibitory activity of HCII with thrombin. This hypothesis is due to the unique role postulated for AR2 to be an intramolecular mimic of a glycosaminoglycan (27, 29, 30). The majority of the AR2 rHCII mutations did affect both heparin- and dermatan sulfate-accelerated thrombin inhibition. Interestingly, the glycosaminoglycan-dependent inhibition curves for most of the AR2 rHCII mutants were "left-shifted" when compared with wt-rHCII. The optimal glycosaminoglycan concentration, which is the concentration corresponding to the maximum inhibition rate, is expected to be a function of the affinity of HCII for glycosaminoglycan because -thrombin was used for all experiments and all other variables were constant. We found that AR2 mutants, like D72N/Y73F/D75N rHCII and Y73K rHCII, had maximum heparin and dermatan sulfate optima clearly reduced from wt-rHCII, in some cases as much as 10- to 20-fold less glycosaminoglycan was required for rates that were increased 2-to 6-fold compared with that for wt-rHCII. Additionally, the inhibition values were still enhanced for the AR2 rHCII mutants compared with wt-rHCII when tested with a mixture of plasma and buffer with added glycosaminoglycans. Even with _T-thrombin or -thrombin/hirugen in the presence of glycosaminoglycans, some AR2 mutants were more active than wt-rHCII (although all of these rates were drastically reduced compared with -thrombin), most likely because the acidic domain binding site on exosite-1 is neither totally removed in _T-thrombin nor completely blocked by hirugen in -thrombin (for instance, with hirugen (20 µM) and heparin (10 µg/ml) the inhibition rates (k₂, M¹ min¹) for D72N/Y73F/D75N rHCII and D75K rHCII were reduced to 2.0 ± 0.5 × 10⁸ and 1.0 ± 0.3 × 10⁸, respectively, as compared with 0.4 ± 0.1 × 10⁸ for wt-rHCII). We have shown previously that dermatan sulfate accelerates _T-thrombin inhibition by HCII by 30-fold, indicating that the acidic domain still interacts with the remaining portions of exosite-1 in a productive manner. These results imply that for wild-type HCII, Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵ help maintain critical intramolecular interactions that attenuate thrombin inhibition activity until glycosaminoglycans bind, which expels the acidic domain to then contact exosite-1 of thrombin.

To complement the above results, the optimum glycosaminoglycan concentration was inversely related to apparent heparin affinity for each AR2 rHCII mutant as found using heparin-Sepharose elution profiles. Because the intermolecular interactions between glycosaminoglycans and HCII are thought to be primarily ionic, the concentration of NaCl required to elute the proteins from heparin-Sepharose is a measure of their relative affinity. We found that some of the AR2 rHCII mutants required from 1.4- to 1.7-fold higher NaCl concentrations to elute from heparin-Sepharose when compared with wt-rHCII. What is remarkable about these AR2 rHCII mutants is that the majority of the negatively charged acidic domain is present while still requiring increased NaCl concentrations to elute from heparin-Sepharose. These elution differences are somewhat analogous to N-terminal deletions when the negative charges were removed from the intact HCII molecule, where 1-67 rHCII and 1-74 rHCII eluted from heparin-Sepharose at 1.4- and 2.1-fold higher NaCl concentrations, respectively, compared with wt-rHCII (27, 32). We found previously that a synthetic peptide corresponding to the glycosaminoglycan binding site in HCII (from residues 183 to 200) bound with 2.6-fold greater affinity to heparin-Sepharose when compared with native HCII, indicating that the glycosaminoglycan-binding site was either masked or in an altered conformation compared with the peptide (47). It is intriguing to speculate that both Y73K and D75K (which had the highest increase in NaCl elution from heparin-Sepharose) might be directly contributing to glycosaminoglycan binding and be juxtaposed to the D-helix glycosaminoglycan region of HCII. Compared with wt-rHCII, the AR2 rHCII mutants are generally more permissive to glycosaminoglycan binding. These results indicate that for wild-type HCII, Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵ support crucial intramolecular interactions that hinder glycosaminoglycan binding.

The mechanism of glycosaminoglycan-accelerated thrombin inhibition by HCII is unique among thrombin-inhibiting serpins (15, 16, 47). HCII appears to employ an allosteric mechanism whereby binding of glycosaminoglycans to the D-helix region is thought to alter the acidic domain, which then serves as a "tethered-ligand" for exosite-1 of thrombin (27, 28, 31-37). The acidic domain/exosite-1 interaction is a critical component of the inhibition mechanism in the presence of glycosaminoglycans, whereas ternary complex formation bridging HCII, glycosaminoglycan, and thrombin appears to play a more minor role in the inhibition mechanism. The proposed interaction between the acidic domain and exosite-1 is well supported by a variety of data. A synthetic peptide corresponding to residues 54-75 of HCII inhibited thrombin cleavage of fibrinogen but not thrombin chromogenic substrate activity (48). Because proteolysis of fibrinogen requires binding between exosite-1 and fibrinogen, this suggested that the HCII acidic peptide interacts with exosite-1. Deletion of HCII residues 1-52 by recombinant DNA technology did not affect thrombin inhibition in either the absence or presence of glycosaminoglycans (27). By contrast, deletion of residues 1-67 (first acidic region) greatly decreased HCII inhibition of thrombin in the presence of glycosaminoglycans but only slightly reduced it in the absence of glycosaminoglycan, suggesting that the first acidic domain interacted with thrombin (27). Interestingly, deletion of the entire acidic domain (residues 1-74) caused no further decreases in activity, suggesting that only the first acidic domain interacts directly with thrombin. Our laboratory (28, 32, 34) and others (27, 33) showed that glycosaminoglycan-accelerated HCII inhibition of _T-thrombin (in which exosite-1 has been removed by limited proteolysis) was reduced by greater than 1000-fold as compared with -thrombin, whereas inhibition by antithrombin was relatively unaffected by the absence of exosite-1. Site-directed mutants of the basic residues in exosite-1 showed specificity similar to where hirugen binds exosite-1, and large decreases in activity with HCII-glycosaminoglycans were seen for some of the mutants (31, 35, 37).

The report by Baglin et al. (49) of the determination of the structures of native HCII and S195A thrombin-complexed HCII revealed some of the expected features based on the above discussion; however, the location of the acidic domain in HCII was unequivocal. The resolved native structure of HCII was a dimer formed by anti-parallel -sheet interaction between strands 1C of each monomer with the acidic domain and the N terminus undefined. Recently, Liaw et al. (33) employed the exact opposite strategy to our study, where they replaced the basic residues in the D-helix region of HCII with neutral residues. They found an increase in progressive antithrombin activity of more than 100-fold compared with wt-rHCII (33). As expected, thrombin exosite-1 was essential for inhibition by these rHCII mutants (33). More recently, Brinkmeyer et al. (36) characterized an rHCII mutant with a re-formable disulfide bond between P52C and F195C. The oxidized form of this mutant was unable to form stable HCII·thrombin complexes; however, after reduction with -mercaptoethanol the reduced HCII mutant was an active thrombin inhibitor in the presence of glycosaminoglycans.

Collectively, these data allow us to provide an updated proposal of the mechanism of thrombin inhibition by HCII (Fig. 4). The historical depiction of HCII is given Fig. 4A, and this shows the acidic domain docked to the D-helix region (27, 29, 30, 32, 36). The mutants and data described by Liaw et al. (33) suggest that the acidic domain is no longer bound to the D-helix, and its location is altered and may resemble that shown in Fig. 4B. Following glycosaminoglycan binding, the acidic domain is fully extended and interaction with exosite-1 in thrombin is favored (Fig. 4C). The structural data from Baglin et al. (49) show that the HCII·S195A thrombin complex resembles that predicted with the acidic domain and exosite-1 in direct contact with the expected interaction between the reactive center of HCII and active site of thrombin (Fig. 4D). The AR2 rHCII mutants described here would appear to be in equilibrium between the HCII structures depicted in Fig. 4, A and B (comparing differences in activity found here to those reported by Liaw et al. (33)). The enhanced sensitivity of the AR2 rHCII mutants (especially Asp⁷², Tyr-sulfate⁷³, and Asp⁷⁵) to glycosaminoglycan interactions is connected to increased thrombin inhibition activity, which is consistent with a more permissive glycosaminoglycan binding site due to altered intramolecular interactions. Thus, AR2 may act as a "molecular rheostat" to help maintain HCII in a relatively inactive form in the absence of glycosaminoglycans, but after glycosaminoglycan binding it allows the acidic domain to tether and recruit exosite-1 of thrombin that leads to favorable thrombin inhibition.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Schematic model for thrombin inhibition by HCII. Shown here are the following possible structures for HCII (Leu444 reactive center residue in green). A, as historically depicted with acidic domain (AR1 and AR2 shown in yellow) ionically bound to the glycosaminoglycan-binding site (D-helix shown in blue); B, interpreting the data from Liaw et al. (33) implies that the acidic domain is not associated with the D-helix after neutralization of the D-helix by mutagenesis; C, following interaction with glycosaminoglycan, the acidic domain is liberated and is able to effectively bind to exosite-1 of thrombin; and (D) complexed to thrombin (active site Ser of thrombin (in gray) and exosite-1 (in red) and exosite-2 (in light blue)) with the acidic domain bound to exosite-1 (as shown historically by biochemical/molecular biological approaches and recently confirmed by crystallization of the HCII·S195A thrombin non-covalent complex (Baglin et al. (49)). The mechanism of HCII·thrombin interaction predicts Asp72, Tyr-sulfate73, and Asp75 of AR2 promote intramolecular interactions during glycosaminoglycan binding to the D-helix to support a conformational change that allows for acidic domain recognition of thrombin exosite-1. Collectively, the data can be used to interpret an equilibrium between conformations A, B, and C above, which, when in the presence of glycosaminoglycan, the encounter between the active site of thrombin and the reactive center of HCII is enhanced and stabilized by acidic domain and exosite-1 orientation and interaction (drawing is based on a schematic by D. M. Tollefsen).

	ACKNOWLEDGEMENTS

We thank Dr. Susannah Bauman for advice and support in the early part of this work. We also thank Dr. Dougald Monroe and Dr. Herbert Whinna for scientific assistance and advice throughout this study.

	FOOTNOTES

^* This work was supported by Research Grant HL-32656 from the National Institutes of Health (to F. C. C).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Campus Box 7035, Division of Hematology-Oncology/Medicine, 932 Mary Ellen Jones Bldg., University of North Carolina, Chapel Hill, NC 27599-7035. Tel.: 919-966-3311; Fax: 919-966-7639; E-mail: fchurch@email.unc.edu.

Published, JBC Papers in Press, February 20, 2002, DOI 10.1074/jbc.M200630200

² We determined the stoichiometry of inhibition values (SI; the number of serpin molecules consumed before an inactivated serpin·protease complex is formed) for the shotgun rHCII mutants compared to wtrHCII in the presence of heparin and dermatan sulfate. SI values for wt-rHCII, E69Q/D70N/D71N rHCII, D72N/Y73F/D75N rHCII, and QNNNFN rHCII ranged from 1.9 to 2.1 (similar to values reported previously (42)). This suggests that the kinetics of inhibition for these rHCII mutants were very similar to wt-rHCII.

	ABBREVIATIONS

The abbreviations used are: serpin, serine protease inhibitor; HCII, heparin cofactor II; ATIII, antithrombin III; wt, wild-type; r, recombinant; E69Q/D70N/D71N/D72N/Y73F/D75N, QNNNFN; exosite-1, anion-binding exosite-1; BSA, bovine serum albumin; PEG, polyethylene glycol 8000; Gly-Pro-Arg-NA, tosyl-Gly-Pro-Arg-p-nitroanilide; Ala-Ala-Pro-Phe-NA, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; REF, normal hemostasis reference plasma; DEF, human antithrombin III-deficient plasma; DEF/REF, 1:1 mix of DEF and REF.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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