Rational design and selection of bivalent peptide ligands of thrombin incorporating P4–P1 tetrapeptide sequences: from good substrates to potent inhibitors

Zhengding Su, Anna Vinogradova, Anatol Koutychenko, Dmitri Tolkatchev and Feng Ni1

Biomolecular NMR and Protein Research Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2

1 To whom correspondence should be addressed. E-mail: feng.ni{at}bri.nrc.ca


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tetrapeptide Phe-Asn-Pro-Arg is a structurally optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or some variants to a C-terminal fragment of hirudin, we were able to generate a series of new bivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P3 and P'3 sites of the active-site directed sequence, Phe(P4)-Xaa(P3)-Pro(P2)-Arg(P1)-Pro(P'1)-Gln(P'2)-Yaa(P'3). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P'3 sites for bivalent and optimized two-site binding. Very significantly, panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-clotting activities in the low nanomolar range and yet interfering only partially the catalytic active site of thrombin. Modes of action of the newly discovered bivalent inhibitors are rationalized in light of the allosteric properties of thrombin, especially the interplay between the proteolytic action and regulatory binding occurring at thrombin surfaces remote from the catalytic active site.

Keywords: bivalent inhibitors/peptide ligands/tetrapeptide sequences/thrombin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thrombin is the ultimate protease resulting from the coagulation cascades of protein–protein interaction and enzyme activation reactions (Furie and Furie, 1988Go; Mann, 1999Go). Upon generation, thrombin induces formation of the fibrin clot from the soluble fibrinogen, activates the fibrin cross-linking factor XIII, stimulates the aggregation of platelets and catalyzes the conversion of factors V, VIII and XI into Va, VIIIa and XIa to amplify its own production. Thrombin also binds to the cell-anchored thrombomodulin to form the thrombin–thrombomodulin complex, which in turn activates protein C and the thrombin-activatable fibrinolysis inhibitor (TAFI), initiating the natural anticoagulation and anti-fibrinolysis pathways (Nesheim et al., 1997Go). In addition, thrombin is a powerful stimulant of inflammatory responses and cell proliferation, processes that underline many pathological conditions (Libby, 2002Go; Esmon, 2003Go; Loynes and Zacharski, 2003Go). The critical role of thrombin in making blood clots and in thrombotic diseases has stimulated in-depth studies on the structure and function of thrombin (Berliner, 1992Go) and the design of thrombin inhibitors as novel anticoagulants (Fenton et al., 1993Go; Song and Ni, 1998Go; Weitz and Buller, 2002Go).

Efforts to design specific inhibitors of human thrombin (DiMaio et al., 1990Go; Maraganore et al., 1990Go; Fenton et al., 1993Go) have started to pay off as a bivalent peptide mimicking the action of hirudin has recently proved its clinical efficacy (Weitz and Buller, 2002Go; Hirsh, 2003Go; Salam, 2003Go; Wykrzykowska et al., 2003Go). Bivalent peptide inhibitors of thrombin contain two covalently linked motifs that bind to the catalytic active site and a protein recognition exosite of thrombin, respectively. One of these peptide molecules, P53, for example, has an amino acid sequence of acetyl-(d)F-P-R-P-Q-S-H-N-D-G-D-F-E-E-I-P-E-E-Y-L-Q (DiMaio et al., 1990Go), which inhibits human {alpha}-thrombin with a Ki of ~2.8 nM. The clinically tested peptide known as hirulog or bivalirudin has a sequence of (d)F-P-R-P-G-G-G-G-N-G-D-F-E-E-I-P-E-E-Y-L, which is also a strong inhibitor of human {alpha}-thrombin (Ki {approx} 2.3 nM) (Maraganore et al., 1990Go). Both of these two classes of peptide inhibitors of thrombin contain an amino acid residue in the d-configuration, i.e. (d)Phe, thereby requiring chemical synthesis. However, restriction to chemical synthesis hinders many other potential applications that may benefit from the high-affinities and exquisite specificity of this unique class of polypeptide-based inhibitors of thrombin.

Structurally, the tripeptide, (d)Phe-Pro-Arg, mimics the specific binding of human fibrinopeptide A to thrombin. The aromatic side chain of the (d)Phe residue occupies a binding subsite on thrombin for an (l)Phe at the P9 site, i.e. eight amino acids away from the N-terminal side of the Arg(P1)–Gly(P'1) peptide bond (Ni et al., 1989Go, 1995Go; Stubbs et al., 1992Go). Alternatively, the same binding subsites on thrombin can accommodate tetrapeptide sequences as long as there is an aliphatic, or more preferably an aromatic residue, at the P4 subsite. The P4 residue of the tetrapeptides fulfils the binding of the natural (l)Phe residue at the P9 position of FpA (Ni et al., 1992Go, 1995Go; Rose and Di Cera, 2002Go). Some bivalent peptides, although having weak binding properties, have been derived from the activation sequences of thrombin-activated receptors, also referred to as protease-activated receptors (PARs). These peptides carry the LDPR sequence and binding motifs targeting the fibrinogen-recognition exosite of thrombin (Liu et al., 1991Go; Mathews et al., 1994Go). More recent work using substrate libraries has shown that thrombin preferentially cleaves after tetrapeptides carrying the L/FXPR sequence motif (Backes et al., 2000Go; Edwards et al., 2000Go; Furlong et al., 2002Go). Indeed, there is an FNPR tetrapeptide sequence in human prothrombin, which is cleaved off efficiently by thrombin to generate the free N-terminus of the A-chain of human thrombin (Ni et al., 1995Go; Rose and Di Cera, 2002Go).

There has been renewed interest in peptide inhibitors of thrombin composed of genetically encodable natural amino acids, as these peptides can be expressed through recombinant DNA or used in gene therapy. For example, Shen and co-workers (Xue et al., 2001Go) replaced the (d)Phe moiety of hirulog with a 12-residue sequence derived from human PAR1 to generate a hirulog-like peptide (HLP). This peptide, containing the LDPR sequence, exhibited an improved safety/efficacy profile with reduced bleeding complications compared with hirulog, despite having a significantly decreased binding affinity to thrombin (Xue et al., 2001Go; Chen et al., 2003Go). The (d)Phe-Pro-Arg sequence of P53 or hirulog can also be replaced by part of the natural sequence of human FpA, e.g. Asp-Phe-Leu-Ala-Gln-Gly-Gly-Gly-Val-Arg as proposed (Fenton et al., 1993Go) and synthesized previously (Maraganore et al., 1992Go). Such a bivalent conjugate of FpA has been used along with an N-terminal extension to include a binding moiety targeting integrins on platelets (Mu et al., 2002Go). This targeting strategy was also attempted in earlier work using both hirudin and synthetic peptide inhibitors of thrombin (Church et al., 1991Go; Knapp et al., 1992Go; Leblond et al., 1999Go). A more recent study created a targetable hirudin molecule through linking to an antibody moiety recognizing fibrin (Peter et al., 2003Go). Therefore, it would be very desirable to design optimized tetrapeptide sequences for use as inhibitory elements of the thrombin active site within expressible bivalent polypeptides. The availability of a new generation of this clinically successful class of thrombin inhibitors opens the door for much wider applications, e.g. through the incorporation of targeting peptides for site-specific delivery to improve clinical effacacy/safety of anticoagulant therapy.

In this work, we first used the Phe-Asn-Pro-Arg sequence to substitute for the active site binding element of the P53 bivalent peptide inhibitor of thrombin (DiMaio et al., 1990Go). The resulting new bivalent peptide served as a template for studying the nature of the P3 and P'3 sites required for enhanced thrombin inhibition. Furthermore, we constructed a phage-displayed library of bivalent peptides in order to explore residue preferences at the P4, P3 and P'3 sites that confer strong bivalency for thrombin binding and at the same time resistance against thrombin cleavage. Synthetic peptides derived from the phage-selected sequences were found to have unique and rather unexpected antithrombin and anticoagulant activities.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

All the restriction enzymes were purchased from New England Biolabs (Beverly, MA). The T4 DNA ligase was obtained from Amersham Biosciences (Piscataway, NJ). Bovine and human {alpha}-thrombins were supplied by Haematologic Technologies Inc (Essex Junction, VT). The stock solution of human {alpha}-thrombin had a concentration of 12.6 mg/ml with a specific activity of 3300 NIH units/mg. The stock solution of bovine {alpha}-thrombin had a concentration of 14.6 mg/ml with a specific activity of 3290 NIH units/mg. The chromogenic substrate Tos-Gly-Pro-Arg-pNA, poly(ethylene glycol)-8000, clottable bovine fibrinogen and the C-terminal peptide of hirudin (Hirudin54–65) were purchased from Sigma (St Louis, MO).

Peptide preparation

The two leading peptides, referred to as FN22 and FD22 (Figure 1A), were prepared with a recombinant DNA approach essentially as described previously (Osborne et al., 2003Go). The carrier protein termed MFH (Figure 2A), a variant of the SFC120 fusion carrier (Osborne et al., 2003Go), was used to fuse with the targeted peptide. Essentially, MFH is a methionine-free mutant of SFC120 with a six-histidine tag. Other peptides were synthesized by the solid-phase method using FMOC chemistry either at the Sheldon Biotechnology Centre of McGill University or at the Peptides Facility of the Biotechnology Research Institute, except where indicated otherwise.



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Fig. 1. Amino acid sequences of bivalent peptide inhibitors of thrombin. (A) Sequences of the FN22 and FD22 peptides in comparison with two related peptides, P53 and hirulog, both of which contain a residue in the d-configuration, i.e. dF or (d)Phe. All peptides contain three distinctive segments, the active site binding moiety (ABM), the fibrinogen-recognition exosite binding moiety (EBM) and a linker sequence. The FN22 and FD22 peptides were prepared by recombinant DNA techniques as described in Materials and methods. (B) Depicted here are two primers used to synthesize the gene encoding the FN22 peptide.

 


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Fig. 2. Preparation and characterization of recombinant FN22 and FD22 peptides. (A) The expression vector (pMFH) for the preparation of FN22 and FD22, collectively referred to as FX22. (B) SDS–PAGE analysis of the expressed fusion proteins containing FN22 and FD22. Lane M: molecular weight markers (LMW, Amersham Bioscience). Lane 1: cell lysate without ß-D-isopropylthiogalactopyranoside (IPTG) induction. Lane 2: cell lysate with IPTG induction. The total protein of cells harboring pMFH-FN22 was prepared by dissolving the cell pellets in the SDS sample buffer. Lanes 3 and 4: cell lysate with IPTG induction. The total protein of cells harboring pMFH-FN22 was prepared by dissolving the cell pellets in buffer A under denaturing conditions. Lanes 5 and 6: cell lysate with IPTG induction. The total protein of cells harboring pMFH-FD22 was prepared by dissolving the cell pellets in buffer A under denaturing conditions. (C) HPLC purification of the FN22 peptide after CNBr treatment and cleavage of the fusion protein. Peak 1 with a retention time of 17.16 min was collected and confirmed to contain the intact FN22 peptide by mass spectrometry. (D) 1H–15N HSQC spectrum of 15N-labeled FD22 peptide. The HSQC cross-peaks of residues D45, Q49 and D53 are marked with asterisks and can only be seen at lower contour levels.

 
CNBr cleavage was used to release the target peptides from the fusion proteins. The fusion proteins were dissolved in 50% formic acid solution except that the FD22-containing fusion protein was dissolved in 0.1 M HCl and 6 M guanidine hydrochloride (at 10 mg protein/ml). Crystalline CNBr was added to a final molar ratio of 100:1 of the fusion protein. The solution was allowed to stand for 12–24 h. The samples were then purified with Ni-nitrilotriacetic acid (Ni-NTA) agarose beads to remove the MFH fusion carrier and undigested fusion protein if any. The flow-through was desalted and lyophilized.

Both the recombinant and synthetic peptides were finally purified using HPLC on a C18 reversed-phase column with a water–acetonitrile gradient with added 0.1% trifluoroacetic acid. The identities of all purified peptides were verified by electrospray mass spectrometry. Peptide concentrations were determined by comparing the OD280 values of peptide stock solutions in the clotting buffer (see below) with the predicted extinction coefficient for each peptide (Gill and von Hippel, 1989Go).

Clotting assays

The clotting assays were carried out by use of the protocols described previously (DiMaio et al., 1990Go; Witting et al., 1992Go) with a Spectramax plate reader. The assay employs bovine plasma fibrinogen dissolved at 0.1% in 50 mM Tris–HCl, 100 mM NaCl, 0.1% PEG-8000 at pH 7.6 (i.e. the clotting buffer). The concentration of a peptide needed to double the clotting time (DCT) was defined as IC50 (DiMaio et al., 1990Go).

Inhibition of the amidolydic activity of thrombin

Kinetics of thrombin-catalyzed hydrolysis of the chromogenic substrate Tos-Gly-Pro-Arg-p-nitroanilide were followed by measuring the absorbance at 405 nm on a Spectramax plate reader thermostated at either 25 or 37°C according to the method of Maraganore et al. (1990)Go. The inhibition assays were performed in the clotting buffer with a certain fixed concentration of {alpha}-thrombin (~0.3 nM) such that linear progress curves were achieved within at least 15 min in the absence of inhibition. The concentrations of Tos-Gly-Pro-Arg-p-nitroanilide ranged from 2 to 400 mM. Initial rates were calculated under conditions of <15% hydrolysis of the total substrate. The concentration of the peptides ranged from 0.5 nM to 10 mM. The Ki values of the inhibitors were determined using the equation Ki = [I]/[(SL0/SL1) – 1], where [I] is the inhibitor concentration, SL0 is the slope of the reaction in the absence of inhibitors and SL1 is the slope of the reaction in the presence of the inhibitor.

Following peptide cleavage by HPLC

Previously reported methods (DiMaio et al., 1990Go; Witting et al., 1992Go) were employed with some modifications to determine whether some bivalent peptides are cleaved by thrombin. Each peptide was dissolved in the clotting buffer at a final concentration of 2.0 µM and incubated at 25°C with or without 2 µM of human {alpha}-thrombin for up to 40 h. The samples were then boiled for 2 min to stop the reaction and centrifuged. The supernatant was acidified by addition of concentrated acetic acid before being loaded on a C18 analytical HPLC column and eluted with a linear gradient of 0–50% (v/v) acetonitrile.

NMR experiments

NMR spectra were acquired with a Bruker 500 or 800 MHz NMR spectrometer at 25°C using standard pulse sequences (Mori et al., 1995Go). The NMR samples were prepared by dissolving the peptides in an aqueous solution containing 50 mM Tris–HCl and 100 mM NaCl at pH 6.8. Heteronuclear NMR experiments including heteronuclear single quantum coherence (HSQC) (2D), HSQC–nuclear Overhauser effects spectroscopy (NOESY) (3D) and HSQC–total correlation spectroscopy (TOCSY) (3D) were carried out on the 800 MHz NMR spectrometer. Spectral processing, display and analysis were performed using the XwinNMR software package supplied with the spectrometer system. Sequence-specific assignment of the peptide HSQC spectrum was carried out with the NMRview 4.0 software program.

Construction of phage library

The phage vector fd-tetGIIID (MacKenzie and To, 1998Go) was a generous gift from Dr R.MacKenzie (Institute of Biological Sciences, Ottawa). The phage library was constructed essentially as described by Tanha et al. (Tanha et al., 2001Go). Double-stranded DNA fragments, encoding a bivalent peptide library with an N-terminal His6-tag and randomized at a number of residue locations (Figure 5), were generated and amplified by polymerase chain reaction (PCR) using a mixture of four synthetic and partly complementary synthetic DNA primers: (1) 5'-cat gac cac agt gca cag cac cac cac cat cac cat ggc tct ggc-3', (2) 5'-ttc ctc aaa atc acc gtc gtt atg lnn ttg agg gcg cgg lnn lnn aga gcc aga gcc atg gtg atg-3', (3) 5'-aac gac ggt gat ttt gag gaa att cct gaa gag tat tta caa ggt ggt-3' and (4) 5'-cga ttc tgc ggc cgc aga aga acc acc ttg taa ata ctc-3', where l and n stand for a/c and a/t/g/c, respectively. A total of 1.7 µg of the ligated product yielded a library of ~1.0 x 105 transformants, which allowed ~99% sampling of the library.



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Fig. 5. A structure model showing the contacts between the P4–P'3 residues of the FX22 peptides with human {alpha}-thrombin. The model was built using the crystal structure of heparin cofactor II in complex with thrombin, capturing subsite interactions from P4–P'3 (Baglin et al., 2002Go). The peptide segment is colored generally in yellow with the oxygen atoms shown in red and nitrogen atoms in blue. Thrombin is shown in surface representation with the green color marking hydrophobic areas (residues) and red color highlighting negatively charged residues. In making the model, Asp(P3), Arg(P1) and Arg(P'3) were substituted into the corresponding positions of the P4–P'3 sites of heparin cofactor II, i.e. residues Phe-Asn(P3)-Pro-Leu(P1)-Ser-Thr-Gln(P'3). Very similar positioning of the Arg(P'3) side chain was found in the crystal structure of an analog of human FpA in complex with thrombin (Martin et al., 1996Go).

 
Selection of proteolytically stable peptides from the phage library

Phage particles (109) were incubated with 0.55 pM of human thrombin at 37°C for 30 min in 26 µl of PBS buffer, pH 7.4. The proteolytic reaction was stopped with an excess of the inhibitor (d)Phe-Pro-Arg-chloromethyl ketone (PPACK) (Calbiochem) (to a final concentration of 0.4 µM). The reaction mixture was mixed with 700 µl of Ni-NTA agarose resin (Qiagen) in PBS (50% slurry) and phage particles were allowed to bind with gentle agitation for 2 h at 0°C. Cleaved phage particles were separated from the resin by washing with 9 ml of PBS buffer, pH 7.4. Bound phage particles were eluted from the resin by 0.7 ml of PBS adjusted to pH 4.4 and immediately neutralized by the addition of 30 µl of 1 M Tris–HCl, pH 8.0. Exponentially growing TG1 cultures (0.3 ml) were infected with the eluted phage at 37°C for 30 min. Serial dilutions were used to estimate phage recovery.

Panning the phage library against human thrombin

Individual wells of MaxiSorp plates were coated with 150 µl of 2.2 µM human {alpha}-thrombin in PBS buffer, pH 7.4, at 4°C under shaking for 2 h. Wells were rinsed three times with PBS, blocked with 400 µl PBS–2% (w/v) skim milk (2% MPBS) at 4°C for 2 h and rinsed as above. A 200 µl volume of the phage particles (~1012 plaque-forming units) in 2% MPBS were added to the thrombin-coated wells and incubated with shaking either for 2 h at 4°C or for 0.5–1 h at room temperature (25°C) without shaking. The wells were rinsed 15 (or 25) times with PBS–0.1% (v/w) Tween-20 and then 15 (or 25) times with PBS at the corresponding temperature. Bound phage was eluted by adding 200 µl of freshly prepared 100 mM triethylamine, neutralized with 100 µl of 1 M Tris–HCl, pH 7.4 and used to infect TG1 cells as described above. Alternatively, TG1 cells were infected directly by the addition of 300 µl of cell culture in the wells (Stoop and Craik, 2003Go).


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Incorporation of the tetrapeptide Phe-Asn-Pro-Arg into bivalent inhibitors of thrombin: recombinant expression and characterization

The Phe-Asn-Pro-Arg peptide fragment was conjugated to a peptide derived from the C-terminus of hirudin (or hirudin48–65) as done previously (DiMaio et al., 1990Go). The resulting 22-residue chimeric peptide was designated FN22 and contained one proline residue inserted between the two parts in order to decrease the rate of peptide cleavage by thrombin (Figure 1A). The FN22 peptide was then expressed as a fusion protein to a mutant, named MFH, of the SFC120 fusion carrier (Osborne et al., 2003Go). The mutant carrier had all the methionine residues of SFC120 replaced by leucines and had an attached His-tag in order to simplify peptide purification (Figure 2A). A single methionine residue was inserted between the carrier protein and the FN22 peptide sequence to facilitate release of the peptides by CNBr cleavage.

The recombinant FN22 peptide had enhanced anticlotting activity as it further increased the time of fibrinogen clotting catalyzed by {alpha}-thrombin (Figure 3B and E and Table I) compared with the hirudin peptide (Figure 3A and E and Table I). The FN22 peptide showed inhibitory activities in the concentration range from 50 nM to 6 mM (Figure 3A and E). The IC50 values for FN22 determined were 289 nM for bovine {alpha}-thrombin and 150 nM for human {alpha}-thrombin (Table I). In comparison, a hirudin tail peptide has an IC50 of 447 nM for human {alpha}-thrombin (Table I). Similar experiments showed that the FNPRP peptide alone was a much weaker inhibitor (Table I) with submillimolar IC50 values.



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Fig. 3. Inhibition of thrombin by different peptides including hirudin54–65, FN22, FD22 and hirulog. The clotting progress curves in (A), (B), (C) and (D) were recorded in the presence of the different peptides with increasing peptide concentrations. In each panel, the leftmost curve represents the clotting progress curve in the absence of the peptide and others appear from left to right with increasing peptide concentrations. (A) Hirudin54–65; the peptide concentrations were 0, 0.1, 0.2, 0.5, 0.7 and 1.0 mM. (B) FN22; the peptide concentrations were 0, 0.04, 0.08, 0.12, 0.2, 0.4, 0.8 and 1.0 mM. (C) FD22; the peptide concentrations were 0, 0.01, 0.02, 0.04, 0.08, 0.1, 0.3 and 0.5 mM. (D) Hirulog; the peptide concentrations were 0, 1.35, 2.7, 13.5 and 27 nM. (E) Transition clotting time as a function of the peptide concentration. Hirudin54–65 (solid square), FN22 (open square), FD22 (solid circle) and hirulog (open circle). In the absence of the peptides, the onset of the clotting time for all assays was ~75 s. The peptide concentration for doubling the clotting time (DCT) was defined as the IC50 value. The IC50 value of each peptide was determined by the peptide concentration when the clotting time was 150 s and is listed in Table I. (F) Dixon plot of the inhibition of human {alpha}-thrombin-catalyzed hydrolysis of Tos-Gly-Pro-Arg-pNA by FD22. Assays were performed (as described in Materials and methods) in the presence of 5 (open square), 10 (open circle), 20 (full square), 50 (full circle) and 80 mM (open circle) of the chromogenic substrate.

 

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Table I. Inhibitory activities of the FX22 peptides containing P4–P1 tetrapeptides

 
The FN22 peptide also inhibited {alpha}-thrombin-catalyzed hydrolysis of the chromogenic substrate, Tos-Gly-Pro-Arg-p-nitroanilide, at submicromolar concentrations (Table I). Under the same experimental conditions, micromolar concentrations of the hirudin tail peptide exhibited no measurable inhibition of thrombin-catalyzed substrate turnover (Table I). Likewise, the presence of the FNPRP pentapeptide at concentrations as high as 100 µM exhibited no significant inhibition. These observations indicated that at micromolar concentrations, the individual components of FN22, i.e. the active site- and the fibrinogen recognition exosite (FRE)-directed moieties, were unable by themselves to inhibit the thrombin hydrolysis of the tripeptide substrate. In contrast, the combination of these components in a single polypeptide as in FN22 resulted in potent inhibition of the thrombin active site.

The inhibition constant (Ki) of the FN22 peptide was then determined at substrate concentrations near the KM of the substrate for human {alpha}-thrombin (KM = 4.2 mM). Interestingly, the inhibition by FN22 was found to be competitive, which showed a Ki of ~100 nM (Table I). In the presence of the FN22 peptide at 80 nM, the KM for thrombin-catalyzed hydrolysis of the chromogenic substrate increased from 4.2 to 6.4 mM.

The FN22 peptide was incubated with thrombin at 25°C in order to investigate the stability of the peptide in complex with thrombin. The progress of peptide cleavage by thrombin was monitored and analyzed through the HPLC profiles of the FN22 peptide in the absence or presence of thrombin for different incubation times. It was found that around 30% of the peptide was cleaved by thrombin after 40 h. In the clotting assay, the FN22 peptide at a concentration of 400 nM was premixed with thrombin for different times of incubation before initiating the clotting reaction. The cleavage of the peptide by thrombin is indicated by a reduced clotting time with increasing incubation time. After 30 h of incubation, the clotting time was reduced by 41% compared with the control.

Asp residue at the P3 site confers enhanced bivalency for thrombin binding

We studied whether a charged residue at the P3 site would enhance the inhibition of a substituted FN22 peptide to human thrombin. We first considered the replacement by negatively charged residues (e.g. Asp) as an Asp residue at this site is unfavorable for peptide cleavage (Le Bonniec and Esmon, 1991Go). Even so, an Asp at the P3 site can still confer binding to the thrombin active site as shown for the LDPR sequence (Ni et al., 1992Go). The new bivalent peptide, referred to as FD22, was prepared by use of the same recombinant procedure as for the FN22 peptide (Figure 2A and B). Figure 2D show the 1H–15N HSQC spectrum of the 15N-labeled FD22 peptide. The narrow proton chemical shift dispersion (~1 p.p.m., Figure 2D) indicates that the FD22 peptide lacks a folded structure in solution, as shown previously for related peptides (DiMaio et al., 1992Go). Clotting assay showed that FD22 had a stronger inhibition of thrombin than FN22 (Figure 3C and Table I), with an IC50 of ~50 nM (at 37°C) and Ki values of ~55 nM at 25°C.

Compared with FN22, the FD22 peptide is much more stable against thrombin cleavage. After the peptide had been incubated with thrombin at 25°C for 40 h, ~90% of the intact FD22 peptide still remained, as shown by HPLC analysis. In the clotting assay, the FD22 peptide at 100 nM was premixed with thrombin for different times before initiation of clotting. The peptide thus prepared had the clotting time reduced by only 12% after 30 h of incubation with thrombin. Indeed, the 15N-labeled FD22 with a concentration of ~1 mM (Figure 2D) was digested completely at the Arg(P1)–Pro(P'1) bond by a 5 µM concentration of human {alpha}-thrombin only after more than 60 h of incubation.

Nature of the P3 and P'3 residues and effects on thrombin inhibition

We then explored the P3 and P'3 positions of the active-site binding sequence Phe(P4)-Asp(P3)-Pro(P2)-Arg(P1)-Pro(P'1)-Gln(P'2)-Ser(P'3) for their contributions to thrombin binding and inhibition by the bivalent peptide. For these experiments, both the FN22 and FD22 peptides and all other substituted ones (see below) were generated synthetically using solid-phase methods. Essentially, the synthetic FN22 and FD22 peptides appear to have similar activities to the recombinant equivalents (Table I). It is noted that these peptides were also slowly proteolyzed at the Arg(P1)–Pro(P'1) peptide bonds, as shown in previous sections and for the P53 and hirulog peptides in previous studies (DiMaio et al., 1990Go; Witting et al., 1992Go).

Activities of the synthetic bivalent peptides were best ranked by use of the IC50 (or DCT) values for the inhibition of fibrinogen clotting. Therefore, elongation of the negatively charged side chain through the Asp(P3) to Glu(P3) substitution decreased the anticlotting activity of the bivalent peptide from an IC50 of 45 nM (for FD22) to 80 nM (for FE22) (Table I). The positively charged and somewhat aromatic His residue at P3 is not well tolerated, as the FH22 peptide had a further decreased anticlotting activity (Table I). Interestingly, the P'3 site appears to be insensitive to the presence of a negatively charged residue, as the FD22-D peptide with a Ser(P'3) to Asp(P'3) substitution had a similar activity to the FD22 peptide. On the other hand, the FE22-D peptide had a significantly reduced activity (Table I), indicating that an elongated and negatively charged Glu(P3) is unfavorable when a negatively-charged Asp is present at the P3 position.

We examined further the residue requirements at the P'3 position by reversing the charge of the residue at this site. A His or a Lys residue is not as favorable as a Ser residue at P'3 since both FD22-H and FD22-K had somewhat reduced activities compared with the FD22 peptide (Table I). Very surprisingly, however, an Arg(P'3) residue appears to confer enhanced anticlotting activity as the FD22-R peptide had an IC50 of ~ 32 nM compared with ~45 nM for the FD22 peptide (Table I). Inhibition of the thrombin turnover of the chromogenic substrate Tos-Gly-Pro-Arg-pNA was then studied for all the further-substituted bivalent peptides. Changes of Ki values compared with that of FD22 reflect the impact of P3 and P'3 modifications on binding to the thrombin active site. Interestingly, most of the further-substituted peptides lost the capacity for inhibiting the active site of thrombin with the exception of only two peptides, FD22-D and FD22-R (Table I). The FD22-R peptide with Arg(P'3) had a similar Ki to FD22 with Ser (P'3) even though FD22-R appears to be slightly more active (IC50 {approx} 32 nM) in the anticlotting assay. The FD22-D peptide with Asp(P'3) had the largest Ki value (or the least active) among the three, indicating that the negatively charged Asp is not a favorable P'3 residue.

Selection of bivalent peptide inhibitors of thrombin by phage display

Residue preferences at the P4, P3 and P'3 sites were then searched through sequences displayed on phages and panning the displayed peptide library. The framework of the peptide library (Figure 4) includes two flexible linkers of glycine/serine residues to separate the displayed peptide ligand from the His-tag and the gIII protein. These Gly/Ser linkers were introduced to minimize possible interference with thrombin binding. Sequencing of 10 clones randomly chosen showed the lack of a noticeable sequence bias (Figure 4). Three slightly different protocols were used for phage selection. In the first approach, we incubated the phage particles at 37°C with active thrombin, followed by purification using Ni-NTA affinity resin of the phages containing uncleaved sequences. The amount of added thrombin was chosen empirically to retain 10–50% of the intact phages. The collected phage was amplified and panned against active thrombin immobilized on a solid support. Only two sequences were identified after these two steps of selection (Table II).



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Fig. 4. Construction of the phage-displayed bivalent peptide library. (A) Cloning sites are shown as DNA sequences. Underlined are the inserted flexible linkers of Gly/Ser residues, which were introduced to minimize interference with thrombin binding to the displayed peptide. Boxed are randomized amino acids, with putative subsite positions displayed on top. (B) The sequences of nine randomly selected clones from a small-scale production of the phage library.

 

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Table II. Sample sequences of retained phages after the first round of selectiona

 
Phage selection was then carried out with less stringent cleavage conditions, i.e. no thrombin cleavage was performed before the phage panning. We reasoned that upon binding to immobilized active thrombin, some cleavage would take place and therefore enrich propagated phages with the sequences possessing lower proteolytic susceptibility plus higher affinity. One round of panning was performed at room temperature (25°C), another at 4°C and the TG1 cells were infected with the retained phages directly in the panning well (see Materials and methods). For panning rounds carried out at 25 and 4°C, 60 and 4 x 103 plaques were recovered, respectively. Peptide sequences were determined for eight and 10 randomly-chosen phage particles from these two experiments (Table II). A clear tendency of the P'3 position was observed, with Ile/Leu/Met dominating the collected phages. In addition, P4 is occupied preferentially by aromatic, heterocyclic or long-chain aliphatic amino acids. Interestingly, the rationally designed tetrapeptide sequence Phe-Asn-Pro-Arg was found in one of the clones obtained from panning at 4°C (Table II).

The phage particles collected after the first round of panning at 4°C were amplified and used for a second round of panning, at two temperatures. A total of 2 x 107 plaques and 3 x 107 plaques were recovered at 25 and 4°C, respectively. The increase in phage recovery compared with the first round of panning is consistent with amplification of sequences with higher affinity towards thrombin, while less dependence of phage recovery on temperature suggested decreased proteolytic cleavage. In the second round of selection, the P'3 position retained its strong preference for Ile/Leu/Met amino acids (Table II). The P3 position showed a preference for Gln, while aromatic and heterocyclic amino acids at P4 were outnumbered by long-chain aliphatic residues. Apparently, trivalent peptides may have been selected by the process of panning, with the invariant C-terminal hirudin-derived tail targeting the fibrinogen-recognition exosite, optimized prime-site sequences with Ile/Leu/Met in the P'3 position and the P1–P4 tetrapeptide sequences binding to the active site of thrombin.

Antithrombin activities of representative peptides selected from the phage library

Five bivalent peptides were derived from two sequences obtained from the first round of panning (Table II) and three sequences from the second round of selection (Table III). Two peptides, FN22-I and FQ22-M, had a Phe residue at P4, Gln or Asn at P3 and Ile or Met at P'3, which resemble the FNPR sequence of the prototypic peptide FN22 (Figure 1 and Table I). Three peptides, GS-AV22-I, GS-IQ22-I and IQ22-I, from the second round had an Ala or Ile at P4, Val or Gln at P3 and Ile at P'3, all of which are frequently occurring in the panning hits (Table III). Two of the peptides, GS-AV22-I and GS-IQ22-I, included the two residues Gly-Ser at the N-termini, in accordance with the context of phage-displayed sequences (Figure 4).


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Table III. Sample sequences of retained phages after the second round of selectiona

 
All five peptides displayed IC50 values in the range 10–45 nM (Table I). However, they were, with the exception of IQ22-I, generally much less efficient at inhibiting the amidolytic activity of thrombin (Table I). The significantly increased Ki with respect to IC50 may reflect less efficient binding and/or the existence of additional binding modes of these peptides near the active site of thrombin. Nevertheless, these bivalent peptides derived from the phage library exhibit potent inhibition of fibrinogen clotting catalyzed by thrombin, in accordance with the phage selection process. The FQ22-M and the GS-IQ22-I peptides with sequences Phe(P4)-Gln(P3)-Pro-Arg-Pro-Gln-Met(P'3) and Gly-Ser-Ile(P4)-Gln(P3)-Pro-Arg-Pro-Gln-Ile(P'3), respectively, even had IC50 values down to 10 nM, which approaches the inhibitory potencies of other bivalent thrombin inhibitors such as hirulog or P53 (DiMaio et al., 1990Go; Maraganore et al., 1990Go).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The specificities of substrate recognition by thrombin are commonly analyzed at the P1, P2 and P3 subsites using a tripeptide sequence framework (Le Bonniec and Esmon, 1991Go; Le Bonniec et al., 1991Go; Liu et al., 1991Go; Vindigni et al., 1997Go). Optimal binding interactions with thrombin occur only if these tripeptide substrates contain an amino acid residue in the d-configuration, such as (d)Phe, (d)Leu or (d)Val at P3 (Blomback et al., 1969Go; Butenas et al., 1997Go), which mimics the natural P9 residue in FpA (Ni et al., 1989Go; Martin et al., 1992Go; Stubbs et al., 1992Go). However, these minimalistic peptide substrates probe only the active site apparatus of thrombin and related binding events, which were found to be mildly sensitive to interactions of thrombin with regulatory proteins (Liu et al., 1991Go). On the other hand, cleavages after tetrapeptide sequences by thrombin, e.g. VDPR in human protein C, LDPR in human PAR1 and ISPR in the thrombin-activatable fibrinolysis inhibitor, are dramatically enhanced by binding interactions at thrombin exosites remote from the active site (Le Bonniec and Esmon, 1991Go; Le Bonniec et al., 1991Go; Ishii et al., 1995Go; Boffa et al., 2000Go; Schneider et al., 2002Go). Our previous NMR and structural studies suggested that binding specificity at the active site of thrombin may be captured by minimally a four-residue consensus motif, Phe(P4)-Xxx(P3)-Pro(P2)-Arg(P1) or FXPR, where the P3 residue (i.e. Xxx) can be a charged or a neutral polar residue contacting specific structural features of the S1–S4 subsites (Ni et al., 1995Go). We speculated that the FXPR tetrapeptide sequences could be good candidates for active site binding in designing novel bivalent peptide inhibitors of thrombin (Song and Ni, 1998Go), that are composed of only (l)-amino acids.

Both the FNPR and FDPR sequence motifs turned out to confer bivalent and two-site bridge binding, as the peptides FN22 and FD22 had significantly enhanced anticlotting activities compared with the hirudin peptide that bind only to the fibrinogen recognition exosite of thrombin (Table I). The FN22 peptide was also found to be cleaved by thrombin at the Arg(P1)–Pro(P'1) peptide bond as with all substrate-type bivalent inhibitors such as P53 or hirulog (DiMaio et al., 1990Go; Maraganore et al., 1990Go). In addition, thrombin can cleave much more efficiently (>50-fold) recombinant fusion proteins containing the FNPR sequence in place of the commonly used LVPR sequence (unpublished observations). Screening combinatorial substrate libraries containing the P4–P1 tetrapeptides has confirmed thrombin's preference for a Pro(P2) residue and Phe or long-chain aliphatic residues at the P4 position (Backes et al., 2000Go; Edwards et al., 2000Go; Furlong et al., 2002Go). On the other hand, the P3 subsite was found to be rather promiscuous with both Asn and Asp among the least preferred residues for substrate cleavage. Even so, substitution of Asn(P3) for Asp(P3) led to an enhancement of the anticlotting activity of the FD22 peptide in addition to a significant decrease of the proteolytic sensitivity to thrombin. This latter observation is very much in line with the notion that a P3 aspartate in protein C partially inhibits the proteolytic cleavage at the VDPR site by thrombin (Le Bonniec and Esmon, 1991Go; Rezaie and Esmon, 1994Go; Ishii et al., 1995Go).

Using extended peptide substrates, it was shown previously that thrombin prefers a positively charged residue, e.g. a lysine, at the P'3 position over negatively charged residues (Le Bonniec et al., 1996Go). Interestingly, a Ser(P'3) to Asp(P'3) substitution resulted in little change in the inhibitory activities of the FD22-D peptide, indicating that the thrombin active site can also tolerate negatively charged aspartates at both the P3 and P'3 positions, at least for inhibitory binding. However, a further change of Asp(P3) to Glu(P3) impaired active site binding as the FE22-D peptide showed diminished anticlotting activities to a level approaching the hirudin peptide alone (Table I). On the other hand, the bivalent peptide with an aspartate at P3 indeed accommodates well a large and positively charged residue at the P'3 position, such as Lys or Arg (Table I). With an arginine at the P'3 position, the FD22-R peptide showed a somewhat enhanced anticlotting activity compared with the FD22 and FD22-D peptides (Table I) with Ser(P'3) and Asp(P'3), respectively. Furthermore, the Arg residue at the P'3 position may create a more favorable binding environment for the Asp(P3) residue as the FD22-R peptide showed the strongest inhibition (or lowest Ki) of the thrombin active site (Table I). These findings raise the possibility of intricate communications between the P3 and P'3 sites not only through charge–charge interactions, but also with defined spatial requirements as shown in studies of protein C activation by thrombin (Le Bonniec and Esmon, 1991Go; Le Bonniec et al., 1992Go; Rezaie and Esmon, 1994Go; Rezaie and Yang, 2003Go). The three-dimensional structures of an uncleavable analogue of human FpA containing P'1–P'3 residues (Martin et al., 1996Go) and of heparin cofactor II (Baglin et al., 2002Go) in complexes with thrombin showed that the P'3 residue can project its side chain in the direction of the P3 site, bringing both residues into side chain to side chain contacts (Figure 5). Such specific interactions would account for the enhanced anticlotting activity of the FD22-R peptide containing Asp(P3) and Arg(P'3) residues.

Complementary to rational design, panning of a phage library revealed additional bivalent sequences with unique P4, P3 and P'3 residues (Tables II and III). Using the current panning procedure, we were able to select peptide sequences that bind strongly to thrombin and are resistant to proteolysis in the presence of thrombin. The P4 site of the panned phage sequences had a preference for a hydrophobic or an aromatic residue, such as Ile/Leu, Phe or Tyr, in agreement with screening of substrate libraries (Backes et al., 2000Go; Edwards et al., 2000Go; Furlong et al., 2002Go). Surprisingly, many phage sequences contain an alanine at the P4 position (Table III), which is not a favorable residue for substrate turnover (Backes et al., 2000Go; Edwards et al., 2000Go; Furlong et al., 2002Go) and is not found in sequences of natural substrates of thrombin (Ni et al., 1995Go; Rose and Di Cera, 2002Go). The P3 site was a great deal more variable, having either hydrophobic or polar residues such as Asn and Gln. The high occurrence of a long-chain hydrophobic residue, i.e. Leu/Met/Ile, at the P'3 position (Tables II and III) was not noted in previous studies using extended peptide substrates of thrombin (Le Bonniec et al., 1996Go), or within protein sequences that are natural substrates of thrombin (Le Bonniec et al., 1996Go; Rose and Di Cera, 2002Go). The long-chain P'3 hydrophobic residues found through phage panning may also be a consequence of the hydrophobic nature around the S'2 site of thrombin (Figure 5), as the P'3 residue can sample an alternative conformation, projecting its side chain towards the P'2-binding pocket (Martin et al., 1996Go). In addition, the P'3 residue lies near the negatively charged residues Gln39 and Glu192 of thrombin, which are at the center of an allosteric relay system linking the active site with the fibrinogen-recognition exosite of thrombin (Rezaie and Yang, 2003Go, and references cited therein).

All representative peptides derived from panning hits showed strong anticlotting activities in the low nanomolar range (Table I). Two of the five phage-derived peptides, i.e. FQ22-M and GS-IQ22-I, had anticlotting IC50 values as low as 10 nM, an inhibitory potency that is close to that of hirulog (Table I). Very interestingly, however, the FQ22-M and GS-IQ22-I peptides had rather large Ki values for the inhibition of the amidolytic activity of thrombin (Table I). Even the less potent peptide FN22-I exhibited a Ki value (360 nM), which is eight times the IC50 for clotting inhibition (45 nM). These consistently increased Ki values over the anticlotting IC50 values indicate that these bivalent peptides may also bind to alternative sites on thrombin, through the P4–P1 and P'1–P'3 sequences, other than the catalytic active site. In fact, the very potent GS-IQ22-I peptide containing residues Gly-Ser before the putative Ile(P4) residue may not even bind to the active site of thrombin as the amidolytic activity of thrombin was little affected in the presence of GS-IQ22-I up to 400 nM concentration (Table I). Indeed, it was reported recently that a peptide derived from the N-terminus of the thrombin receptor PAR3 also confers strong inhibition of the fibrinogen-clotting activity while leaving open the catalytic active site of thrombin (Owen, 2003Go).

The frequent occurrence of Ala at P4 in phage sequences (Table III) is of further interest since thrombin is not known to favor this residue and yet the GSAVPR sequence (as in the GS-AV22-I peptide) confers enhanced bridge-binding inhibition of the thrombin active site (Table I). On the other hand, thrombin is a highly allosteric enzyme, featuring, in addition to the catalytic active site, the FRE, which the hirudin sequence of the bivalent peptides targets and binds. It was shown in a recent work that the active site of thrombin could assume a closed and inactive conformation, which in solution may be in equilibrium with active conformations of thrombin (Huntington and Esmon, 2003Go). This inactive conformation of thrombin has an occluded P4-binding subsite, leaving little room for the bulky side chain of an aromatic or aliphatic P4 residue. The binding properties of the thrombin active site may be biased toward those of the closed and inactive conformations by ligand binding at the fibrinogen-recognition exosite. As such, it is conceivable that binding of the GS-AV22-I peptide may target selectively the inactive and closed conformation of thrombin, achieving inhibition of catalysis indirectly instead of direct binding to catalytically active structures at the active site.


    Acknowledgments
 
We thank Andy Ng and Barbara Requesens for technical assistance. This work was supported by the International Collaboration Programs and a Genomics and Health Initiative of the National Research Council of Canada (NRCC Publication No. 46218), sponsored by the Government of Canada.


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Received August 30, 2004; accepted September 6, 2004.

Edited by Andre Menez





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