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Structure-based Design of a Potent Chimeric Thrombin Inhibitor

1997; Elsevier BV; Volume: 272; Issue: 32 Linguagem: Inglês

10.1074/jbc.272.32.19938

1083-351X

Robert Morenweiser, Ennes A. Auerswald, Andreas van de Locht, Hans Fritz, Jörg Stürzebecher, Milton T. Stubbs,

Neuropeptides and Animal Physiology

Using the three-dimensional structures of thrombin and the leech-derived tryptase inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the inhibitor reactive site loop to fit thrombin's narrow active site cleft resulted in inhibition constants (K i ) in the 10 nmconcentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin's tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (K i in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays. Using the three-dimensional structures of thrombin and the leech-derived tryptase inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the inhibitor reactive site loop to fit thrombin's narrow active site cleft resulted in inhibition constants (K i ) in the 10 nmconcentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin's tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (K i in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays. The action of thrombin is central to coagulation (1Stubbs M.T. Bode W. Trends Biochem. Sci. 1995; 20: 23-28Abstract Full Text PDF PubMed Scopus (177) Google Scholar). Physiologically, its activity is regulated by serpins such as antithrombin III, heparin cofactor II, and protease nexin I as well as the general proteinase scavenger α2-macroglobulin (2Olson S.T. Björk I. Berliner L.J. Thrombin: Structure and Function. Plenum Press, New York1992: 159-217Crossref Google Scholar). Inhibition by antithrombin III and heparin cofactor II is strongly accelerated by the acidic glycosaminoglycan heparin. Thrombin is inhibited only weakly by other typical serine proteinase inhibitors such as the Kunitz inhibitor BPTI 1The abbreviations used are: BPTI, basic pancreatic trypsin inhibitor; LDTI, leech-derived tryptase inhibitor; rLDTI, recombinant leech-derived tryptase inhibitor; HPLC, high performance liquid chromatography. (3Ascenzi P. Coletta M. Amiconi G. de Cristofaro R. Bolognesi M. Guarneri M. Menegatti E. Biochem. Biophys. Acta. 1988; 956: 156-161Crossref PubMed Scopus (27) Google Scholar). The structure of human α-thrombin (4Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (821) Google Scholar, 5Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar) reveals an unusually deep and narrow active site cleft, which is a major determinant of its restricted specificity. Although the number of endogenous thrombin inhibitors is small, various hematophagous parasites have developed potent antithrombotic agents, of which hirudin from the medicinal leech Hirudo medicinalis is presently the best known (6Walsmann P. Markwardt F. Pharmazie. 1981; 36: 653-660PubMed Google Scholar). The structure determination of the hirudin-thrombin complex (7Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton J.W., II Science. 1990; 249: 277-280Crossref PubMed Scopus (641) Google Scholar, 8Grütter M.G. Priestle J.P. Rahuel J. Grossenbacher H. Bode W. Hofsteenge J. Stone S.R. EMBO J. 1990; 9: 2361-2365Crossref PubMed Scopus (314) Google Scholar) revealed primarily a two-site interaction, namely limited penetration into the active site cleft and extensive electrostatic interaction between the acidic carboxyl-terminal “tail” of hirudin with the basic fibrinogen recognition exosite of thrombin. More recent structure elucidations of complexes of thrombin with rhodniin (9van de Locht A. Lamba D. Bauer M. Huber R. Friedrich T. Kröger B. Höffken H.W. Bode W. EMBO J. 1995; 14: 5149-5157Crossref PubMed Scopus (170) Google Scholar) (from the assassin bugRhodnius prolixus (10Friedrich T. Kröger B. Biolajan S. Lemaire H.G. Höffken H.W. Reuschenbach P. Otte M. Dodt J. J. Biol. Chem. 1993; 268: 16216-16222Abstract Full Text PDF PubMed Google Scholar)) and ornithodorin (11EMBO J. 15, 6011–6017van de Locht, A., Stubbs, M. T., Bode, W., Friedrich, T., Bollschweiler, C., Höffken, W., and Huber, R. EMBO J., 15, 6011–6017.Google Scholar) (from the soft tick Ornithodoros moubata) also show this two-site interaction. Despite its Kunitz-like fold, ornithodorin binds thrombin in a manner that is completely different from that of the well known BPTI-serine proteinase interaction (12Bode W. Huber R. Eur. J. Biochem. 1992; 204: 433-452Crossref PubMed Scopus (1006) Google Scholar); contacts are made between the amino terminus of ornithodorin and residues at the active site, thus resembling the interaction of hirudin with thrombin. Rhodniin is composed of two Kazal-type domains (9van de Locht A. Lamba D. Bauer M. Huber R. Friedrich T. Kröger B. Höffken H.W. Bode W. EMBO J. 1995; 14: 5149-5157Crossref PubMed Scopus (170) Google Scholar). The first domain binds in a canonical manner (12Bode W. Huber R. Eur. J. Biochem. 1992; 204: 433-452Crossref PubMed Scopus (1006) Google Scholar), with its reactive site loop occupying the active site of thrombin as would a substrate. The unconventional disulfide bridge arrangement of rhodniin, which it shares with the plasmin inhibitor bdellin B-3 (13Fink E. Rehm H. Gippner C. Bode W. Eulitz M. Machleidt W. Fritz H. Biol. Chem. Hoppe-Seyler. 1986; 367: 1235-1242Crossref PubMed Scopus (51) Google Scholar) and the tryptase inhibitor LDTI (leech-derived tryptase inhibitor) (14Sommerhoff C.P. Söllner C. Mentele R. Piechottka G.P. Auerswald E.A. Fritz H. Biol. Chem. Hoppe-Seyler. 1994; 375: 685-694Crossref PubMed Scopus (108) Google Scholar), allows a particularly narrow reactive site loop, which is able to fit into thrombin's restrictive active site canyon. The second acidic domain binds at the fibrinogen recognition exosite. Despite the close structural homology of LDTI to rhodniin (15Mühlhahn P. Czisch M. Morenweiser R. Habermann B. Engh R.A. Sommerhoff C.P. Auerswald E.A. Holak T.A. FEBS Lett. 1994; 355: 290-296Crossref PubMed Scopus (44) Google Scholar, 16Stubbs M.T. Morenweiser R. Bauer M. Bode W. Huber R. Piechottka G.P. Matschiner G. Sommerhoff C.P. Fritz H. Auerswald E.A. J. Biol. Chem. 1997; 272: 19931-19937Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), the leech-derived inhibitor does not inhibit thrombin. This prompted us to attempt modification of the LDTI molecule to convert it into a thrombin inhibitor. The synthetic gene for rLDTI, expressed in Saccharomyces cerevisiae (17Auerswald E.A. Morenweiser R. Sommerhoff C.P. Piechottka G.P. Eckerskorn C. Gürtler L.G. Fritz H. Biol. Chem. Hoppe-Seyler. 1994; 375: 695-703Crossref PubMed Scopus (27) Google Scholar), facilitates the construction of mutants to probe aspects of specificity and selectivity for serine proteinases. The volume of structural data available for thrombin (18Stubbs M.T. Bode W. Thromb. Res. 1993; 69: 1-58Abstract Full Text PDF PubMed Scopus (445) Google Scholar) allows a rational approach to the design of specific inhibitors, for which its restricted active site canyon poses particularly stringent conditions. In this paper, we show that trimming the reactive site loop of LDTI to reduce collisions with residues lining the active site cleft of thrombin can produce an inhibitor with an inhibition constant in the 10 nm range. An inhibitor with similar affinity is obtained upon introduction of a hirudin tail fragment to untrimmed rLDTI, showing that thrombin can accept unfavorable substituents in its active site cleft upon favorable binding at the fibrinogen exosite. A mutant combining both favorable properties inhibits thrombin with aK i value in the 10 pm range, indicating that the interactions at the active site and exosite are additive. With the exception of the modifications outlined below, all reagents and methods were used or carried out as described in the accompanying paper (16Stubbs M.T. Morenweiser R. Bauer M. Bode W. Huber R. Piechottka G.P. Matschiner G. Sommerhoff C.P. Fritz H. Auerswald E.A. J. Biol. Chem. 1997; 272: 19931-19937Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Human thrombin was kindly provided by M. Otte (LMU, München, Germany); thromboplastin was purchased from Dade (Unterschleißheim, Germany). The substrate Tos-Gly-Pro-Arg-pNA was purchased from Sigma, and the reagent for measuring prothrombin time was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). For design of the variants, coordinates of the thrombin-fibrinopeptide A-hirugen complex (19Stubbs M.T. Oschkinat H. Mayr I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (198) Google Scholar), the solution structure of rLDTI (15Mühlhahn P. Czisch M. Morenweiser R. Habermann B. Engh R.A. Sommerhoff C.P. Auerswald E.A. Holak T.A. FEBS Lett. 1994; 355: 290-296Crossref PubMed Scopus (44) Google Scholar), the rhodniin-thrombin complex (9van de Locht A. Lamba D. Bauer M. Huber R. Friedrich T. Kröger B. Höffken H.W. Bode W. EMBO J. 1995; 14: 5149-5157Crossref PubMed Scopus (170) Google Scholar), and the rLDTI-trypsin complex (16Stubbs M.T. Morenweiser R. Bauer M. Bode W. Huber R. Piechottka G.P. Matschiner G. Sommerhoff C.P. Fritz H. Auerswald E.A. J. Biol. Chem. 1997; 272: 19931-19937Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) were superimposed and displayed using the program O (20Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). Variants were obtained by cassette mutagenesis. Substitutions and insertions of the desired sequences were performed with the cloning vector pRM 5.1.5 harboring the synthetic rLDTI gene (17Auerswald E.A. Morenweiser R. Sommerhoff C.P. Piechottka G.P. Eckerskorn C. Gürtler L.G. Fritz H. Biol. Chem. Hoppe-Seyler. 1994; 375: 695-703Crossref PubMed Scopus (27) Google Scholar). After digestion of the vector with AgeI/HindIII,SphI/NsiI, or ClaI/HindIII, respectively, the fragments coding for the reactive area of rLDTI (Ala5–Ile26) and the fragment coding for the C terminus of rLDTI (Thr42–Asn46 and Ser32–Asn46) were deleted. The vector fragments were isolated by agarose gel electrophoresis and religated with the appropriate hybridized oligonucleotides. The oligonucleotide sequences for theAgeI/HindIII fragment of rLDTI-varθ1 and rLDTI-varθ2 were 5′-CCG GTG AAC CAG ACG AAG ACG AAG ACG TTT AAT A and 3′-A CTT GGT CTG CTT CTG CTT CTG CAA ATT ATT CGA or 5′-CCG GTG ACT TCG AAG AAA TTC CAG AAG AAT ACT TGC AAT AAT A and 3′-A CTG AAG CTT CTT TAA GGT CTT CTT ATG AAC GTT ATT ATT CGA; sequences for theSphI/NsiI fragment of rLDTI-varθ3 were 5′-C CCA AAG GCT TTG CAC AGA GTC TGT GGT TCT GAC GGT CGT ACA TAT GCT AAC CCA TGC and 3′GT ACG GGT TTC CGA AAC GTG TCT CAG ACA CCA AGA CTG CCA GCA TGT ATA CGA TTG GGT; and sequences for theClaI/HindIII fragment of rLDTI-varθ4 were 5′-CGA TCA AGT CTG AAG GTT CTT GTG GTG GTG GCA CCG GTG ACT TCG AAG AAA TTC CAG AAG AAT ACT TGC AAT AAT A and 3′-TAG TTC AGA CTT CCA AGA ACA CCA CCA CCG TGG CCA CTG AAG CTT CTT TAA GGT CTT CTT ATG AAC GTT ATT ATT CGA. The new cloning vectors were named pHB 6.1.1 (rLDTI-varθ1), pHB 1.1.1 (rLDTI-varθ2), pRM 13.1.1 (rLDTI-varθ3), and pHB 2.1.2 (rLDTI-varθ4). The gene for the double variant rLDTI-varθ5 was constructed by digestion of vector pHB 2.1.2 (rLDTI-varθ4) withSphI/NsiI and religation with theSphI/NsiI oligonucleotide cassette shown above. The resulting cloning vector was called pHB 3.1.1. The vectors were cloned in Escherichia coli TG1. For expression in S. cerevisiae, the modified rLDTI genes were isolated by XbaI/HindIII cleavage and ligated into yeast shuttle vector pVT102U/α (17Auerswald E.A. Morenweiser R. Sommerhoff C.P. Piechottka G.P. Eckerskorn C. Gürtler L.G. Fritz H. Biol. Chem. Hoppe-Seyler. 1994; 375: 695-703Crossref PubMed Scopus (27) Google Scholar). The resulting expression vectors pMH 2.1.1 (rLDTI-varθ1), pMH 1.1.3 (rLDTI-varθ2), pRM 14.1.2 (rLDTI-varθ3), pHB 4.1.1 (rLDTI-varθ4), and pHB 5.1.1 (rLDTI-varθ5) were used to transform S. cerevisiae S-78 (21Becker D.M. Guarente L. Methods Enzymol. 1991; 194: 182-187Crossref PubMed Scopus (673) Google Scholar). Standard yeast expression experiments were performed as described previously (17Auerswald E.A. Morenweiser R. Sommerhoff C.P. Piechottka G.P. Eckerskorn C. Gürtler L.G. Fritz H. Biol. Chem. Hoppe-Seyler. 1994; 375: 695-703Crossref PubMed Scopus (27) Google Scholar). Yeast culture broth was harvested after 168 h of fermentation (6000 × g for 20 min at 4 °C). The crude supernatant was additionally centrifuged at 9000 × gfor 10 min at 4 °C and concentrated using an ultrafiltration membrane with a 3-kDa cut-off value (YM3 membrane, Amicon). The buffer was exchanged by dialysis (1-kDa cut-off value, Spectra-Por 6 Membrane; Spectrum, Houston, TX) against 20 mmNaH2PO4, pH 7.8 (rLDTI-varθ3) or against 20 mm Tris/HCl, pH 7.2 (rLDTI-varθ1, -varθ2, -varθ4, and -varθ5). The variant rLDTI-varθ3 was purified by cation exchange chromatography (Fractogel® EMD SO3- 650(S) column 150–10; Merck) similar to wild-type rLDTI (17Auerswald E.A. Morenweiser R. Sommerhoff C.P. Piechottka G.P. Eckerskorn C. Gürtler L.G. Fritz H. Biol. Chem. Hoppe-Seyler. 1994; 375: 695-703Crossref PubMed Scopus (27) Google Scholar). Yellow pigments in the dialyzed supernatants of rLDTI-varθ1, -varθ2, -varθ4, and -varθ5 were separated using anion exchange chromatography (Fractogel®EMD TMAE 650(Q) column) with a flow rate of 1.5 ml/min. The flow-through fraction harboring the rLDTI variants was dialyzed against 20 mm sodium phosphate buffer, pH 4.0, and purified by cation exchange chromatography (Fractogel® EMD SO3–650(S) column 150–10, Merck) at a flow rate of 1.5 ml/min with a linear gradient from 0 to 500 mm NaCl. Equilibrium dissociation constants (K i ) for the complexes of rLDTI variants with human thrombin were determined as described in the accompanying paper (16Stubbs M.T. Morenweiser R. Bauer M. Bode W. Huber R. Piechottka G.P. Matschiner G. Sommerhoff C.P. Fritz H. Auerswald E.A. J. Biol. Chem. 1997; 272: 19931-19937Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In the case of rLDTI-varθ3 and rLDTI-varθ4, K i values were determined using conditions for “classical inhibitors” (22Morrison J.W. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (726) Google Scholar) and the “specific velocity plot” of Ref. 23Baici A. Eur. J. Biochem. 1981; 119: 9-14Crossref PubMed Scopus (66) Google Scholar. For determination of prothrombin time, 0.1 ml of thromboplastin and 0.1 ml of inhibitor (dissolved in 25 mm CaCl2, 5% ethanol) were incubated at 37 °C for 2 min. Coagulation was initiated by the addition of 0.1 ml of citrated human plasma. For determination of activated partial thromboplastin time, citrated human plasma was incubated at 37 °C with 0.1 ml of prothrombin time reagent. After 3 min, 0.1 ml of inhibitor (dissolved in 25 mm CaCl2, 5% ethanol) was added. For determination of thrombin time, 0.1 ml of citrated human plasma was mixed with 0.05 ml of inhibitor dissolved in 0.154 m NaCl, 5% ethanol, and coagulation was started by the addition of 0.05 ml of thrombin (10 units/ml). Clotting times were determined in duplicate using the coagulometer Thrombotrack 8 (Immuno GmbH, Heidelberg, Germany). Inhibitor concentrations required to double the respective clotting times (IC50) were read from semilogarithmic graphs of clotting times versus inhibitor concentrations. The rLDTI variants, displayed schematically in Fig.1, were constructed by cassette mutagenesis using the cloning vectors pRM 5.1.5 and pHB 2.1.2, harboring the synthetic rLDTI and rLDTI-varθ4 genes, respectively. The initial mutants were designed on the basis of the solution structure of rLDTI (15Mühlhahn P. Czisch M. Morenweiser R. Habermann B. Engh R.A. Sommerhoff C.P. Auerswald E.A. Holak T.A. FEBS Lett. 1994; 355: 290-296Crossref PubMed Scopus (44) Google Scholar) and the sequence of rhodniin (10Friedrich T. Kröger B. Biolajan S. Lemaire H.G. Höffken H.W. Reuschenbach P. Otte M. Dodt J. J. Biol. Chem. 1993; 268: 16216-16222Abstract Full Text PDF PubMed Google Scholar) only,i.e. prior to the crystal structure elucidations of the complexes of rhodniin-thrombin (9van de Locht A. Lamba D. Bauer M. Huber R. Friedrich T. Kröger B. Höffken H.W. Bode W. EMBO J. 1995; 14: 5149-5157Crossref PubMed Scopus (170) Google Scholar) and rLDTI-trypsin (16Stubbs M.T. Morenweiser R. Bauer M. Bode W. Huber R. Piechottka G.P. Matschiner G. Sommerhoff C.P. Fritz H. Auerswald E.A. J. Biol. Chem. 1997; 272: 19931-19937Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The rhodniin-derived peptide EPDEDEDV, presumed to bind to the fibrinogen recognition exosite, was added to the flexible native C terminus of LDTI (rLDTI-varθ1), as was the hirudin tail sequence DFEEIPEEYLQ (rLDTI-varθ2). Three residues of the reactive site loop (Ile9, Lys11, and Ser24) were identified as yielding potential clashes with the characteristic thrombin 60-loop (Fig.2) and were therefore replaced with the corresponding residues of rhodniin (Ala, His, and Pro, respectively). Furthermore, Pro12 was replaced by its rhodniin counterpart Arg to avoid any possible adverse main chain conformational rigidity. This resulted in mutant rLDTI-varθ3. Subsequent elucidation of the rLDTI-trypsin complex crystal structure (16Stubbs M.T. Morenweiser R. Bauer M. Bode W. Huber R. Piechottka G.P. Matschiner G. Sommerhoff C.P. Fritz H. Auerswald E.A. J. Biol. Chem. 1997; 272: 19931-19937Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and superposition with the ternary complex thrombin-fibrinopeptide A-hirugen (19Stubbs M.T. Oschkinat H. Mayr I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (198) Google Scholar) revealed the need for a polyglycine spacer between the rLDTI C terminus and the hirudin tail peptide, leading to variants rLDTI-varθ4 and rLDTI-varθ5 (Fig.3). Cloning vectors pHB 6.1.1 (rLDTI-varθ1), pHB 1.1.1 (rLDTI-varθ2), pRM 13.1.1 (rLDTI-varθ3), and pHB 2.1.2 (rLDTI-varθ4) were used to transform E. coli TG1, and the corresponding DNA sequences were confirmed. For expression in yeast, the singleXbaI-HindIII gene cassettes were subcloned in the yeast shuttle vector pVT102U/α (24Chen X.-M. Qian Y.-W. Chi C.-W. Gan K.-D. Zhangh M.-F. Chen C.-Q. J. Biochem. ( Tokyo ). 1992; 112: 45-51Crossref PubMed Scopus (27) Google Scholar), and S. cerevisiae strain S-78 was transformed with the resulting expression vectors pMH 2.1.1 (rLDTI-varθ1), pMH 1.1.3 (rLDTI-varθ2), pRM14.1.2 (rLDTI-varθ3), pHB 4.1.1 (rLDTI-varθ4), and pHB 5.1.1 (rLDTI-varθ5). Transformed yeast cells cultivated under standard conditions produced recombinant material. For each variant, trypsin-inhibitory activity was detectable in the culture broth as well as a distinct protein band migrating at a M rcorresponding to the theoretical mass as analyzed by SDS-polyacrylamide gel electrophoresis (data not shown). Yields of the variants were >5.3 mg/liter. The isolated material of each variant was homogeneous and >95% pure as judged by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, and HPLC analysis (data not shown). Automated N-terminal sequencing verified the correct processing of the α mating type leader fusion protein. With the exception of rLDTI-varθ5, mass spectroscopy of the variants yielded molecular masses in agreement with those calculated (given in parentheses): 5326.8 (5325.9) Da for rLDTI-varθ1, 5791.3 (5790.5) Da for rLDTI-varθ2, 4773.5 (4779.5) Da for rLDTI-varθ3 5863.6 (5864.6) Da for rLDTI-varθ4, and 5772.9 (5900.6) Da for rLDTI-varθ5. The lower mass of 127.7 Da for rLDTI-varθ5 is probably due to truncation of the C-terminal Gln56 by endogenous yeast proteinases. The trypsin-specific inhibitory activity of the isolated variants was found to be >40% of the theoretical value, which is comparable with recombinant wild-type LDTI (17Auerswald E.A. Morenweiser R. Sommerhoff C.P. Piechottka G.P. Eckerskorn C. Gürtler L.G. Fritz H. Biol. Chem. Hoppe-Seyler. 1994; 375: 695-703Crossref PubMed Scopus (27) Google Scholar). Equilibrium dissociation constants (K i ) were determined for the complexes of recombinant LDTI (17Auerswald E.A. Morenweiser R. Sommerhoff C.P. Piechottka G.P. Eckerskorn C. Gürtler L.G. Fritz H. Biol. Chem. Hoppe-Seyler. 1994; 375: 695-703Crossref PubMed Scopus (27) Google Scholar) and its variants with human thrombin (TableI). The acidic C-terminal extension in rLDTI-varθ1 and rLDTI-varθ2 failed to produce the anticipated increase in affinity for human α-thrombin, while theK i value for bovine trypsin was identical to that of wild-type rLDTI.Table IEquilibrium dissociation constants (Ki) for the inhibition of thrombin by various LDTI variants and concentrations required for doubling thrombin time, activated partial thromboplastin time, and prothrombin time in plasmaK i for thrombin inhibitionConcentration required for doubling ofThrombin timeActivated partial thromboplastin timeProthrombin timeμ mrLDTI>300 nm>10ND1-aND, not determined.NDrLDTI-varθ1>300 nm>10ND 1-aND, not determined.NDrLDTI-varθ2>300 nm>10ND 1-aND, not determined.NDrLDTI-varθ3 9.4 nm0.243.7NDrLDTI-varθ414.9 nm0.0320.430.76rLDTI-varθ516.0 pm0.0130.090.20Hirudin27.0 fm0.0110.110.231-a ND, not determined. Open table in a new tab The amino acid substitutions at the reactive site of rLDTI (rLDTI-varθ3) and the additional insertion of a glycine spacer in rLDTI-varθ2 to give rLDTI-varθ4 resulted in a remarkable improvement in affinity for human α-thrombin, leaving that for bovine trypsin unchanged but strongly reducing that for bovine chymotrypsin in both cases. The highest affinity toward α-thrombin was achieved by combining the mutations of rLDTI-varθ3 and rLDTI-varθ4 to produce rLDTI-varθ5, which resulted in an over 18,000-fold increase in affinity for thrombin compared with the wild-type form. This increase in affinity was paralleled by an increased selectivity for thrombin versusthe other serine proteinases tested. The degree of anticoagulatory activity displayed by the variants in clotting assays (Table I) correlates with the K i values determined for thrombin inhibition. Despite the 1000-fold lowerK i value measured for the complex of thrombin with rLDTI-varθ5 compared with that with hirudin, very little difference is seen between their anticoagulatory activities. The results presented here show that serine proteinase inhibitors can be suitably modified for a specified target enzyme using a structure-based approach. The failure of rLDTI variants varθ1 and varθ2 to yield the anticipated inhibition of thrombin, however, highlights the difficulties of such an approach when insufficient structural information is available at the outset. The design of rLDTI-varθ3 involved identifying LDTI residues colliding with thrombin residues of the 60-insertion loop and replacing them with the corresponding residues of rhodniin. Closer inspection of the proposed interaction suggests that the major obstruction for binding to thrombin comes from the side chain of Ile9, which would clash with the side chain of Lys60F (Fig. 2; thrombin residue numbering according to Ref. 4Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (821) Google Scholar). Indeed, antithrombin Denver, which has Leu in this position instead of Ser, is incapable of inhibiting thrombin (25Stephens A.W. Thalley B.S. Hirs C.H.W. J. Biol. Chem. 1987; 262: 1044-1048Abstract Full Text PDF PubMed Google Scholar), a fact confirmed by site-directed mutagenesis experiments (26Stephens A.W. Siddiqui A. Hirs C.H.W. J. Biol. Chem. 1988; 263: 15849-15852Abstract Full Text PDF PubMed Google Scholar, 27Theunissen H.J.M. Dijkema R. Grootenhuis P.D.J. Swinkels J.C. de Poorter T.L. Carati P. Visser A. J. Biol. Chem. 1993; 268: 9035-9040Abstract Full Text PDF PubMed Google Scholar); a similar mutation in factor VIII results in mild hemophilia A (28Johnson D.J. Pemberton S. Acquila M. Mori P.G. Tuddenham E.G. O'Brien D.P. Thromb. Haemostasis. 1994; 71: 428-433Crossref PubMed Scopus (14) Google Scholar). The contributions of the remaining three substitutions require further assessment. Clearly, rLDTI-varθ3 has the potential for further development as a potent thrombin inhibitor. In particular, it is likely that the substitution Lys8 → Arg, which would better match thrombin's primary specificity, should lead to a more potent inhibitor. Preliminary phage display experiments support this assumption. 2A. S. Tanaka, C. A. Sampaio, M. T. Stubbs, H. Fritz, and E. A. Auerswald, unpublished results. Occupancy of the fibrinogen recognition exosite, a feature of all hematophage-derived thrombin inhibitors whose structures have been solved so far (7Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton J.W., II Science. 1990; 249: 277-280Crossref PubMed Scopus (641) Google Scholar, 8Grütter M.G. Priestle J.P. Rahuel J. Grossenbacher H. Bode W. Hofsteenge J. Stone S.R. EMBO J. 1990; 9: 2361-2365Crossref PubMed Scopus (314) Google Scholar, 9van de Locht A. Lamba D. Bauer M. Huber R. Friedrich T. Kröger B. Höffken H.W. Bode W. EMBO J. 1995; 14: 5149-5157Crossref PubMed Scopus (170) Google Scholar, 11EMBO J. 15, 6011–6017van de Locht, A., Stubbs, M. T., Bode, W., Friedrich, T., Bollschweiler, C., Höffken, W., and Huber, R. EMBO J., 15, 6011–6017.Google Scholar), facilitates targeting of the thrombin molecule. For all of these inhibitors, binding to the active site cleft is achieved with no noticeable conformational change in thrombin. This does not appear to be the case for rLDTI-varθ4. Binding of this variant would require adjustment of the Lys60F side chain, such as that observed in a recent thrombin-inhibitor complex (29Matthews J.H. Krishnan R. Costanzo M.J. Maryanoff B.E. Tulinsky A. Biophys. J. 1996; 71: 2830-2839Abstract Full Text PDF PubMed Scopus (56) Google Scholar). The binding energy for hirudin's tail at the fibrinogen recognition exosite is about 36 kJ/mol (30Schmitz T. Rothe M. Dodt J. Eur. J. Biochem. 1991; 195: 251-256Crossref PubMed Scopus (37) Google Scholar). Using this value, theK i value for rLDTI-varθ4, and the formula ΔG = RTlnK i, we can make make a rough estimate of ∼10−5m for theK i value of rLDTI for thrombin. Trimming of the active site loop (rLDTI-varθ3) therefore represents more than a 106 increase in affinity; the additional binding at the exosite (rLDTI-varθ5) corresponds to a greater than 109increase in affinity. Thus, occupation of the fibrinogen recognition exosite can facilitate binding at the active site, reminiscent of “allosteric linkage” (31de Cristofaro R. Rocca B. Bizzi B. Landolfi R. Biochem. J. 1993; 289: 475-480Crossref PubMed Scopus (32) Google Scholar). The action of rLDTI-varθ4 resembles that of the serpin heparin cofactor II. Unusual among inhibitors of serine proteinases with trypsin-like specificity, this serpin possesses a Leu residue at P1 rather than the preferred Arg (32Griffith M.J. Noyes C.M. Tyndall J.A. Church F.C. Biochemistry. 1985; 24: 6777-6782Crossref PubMed Scopus (39) Google Scholar, 33Ragg H. Nucleic Acids Res. 1986; 14: 1073-1088Crossref PubMed Scopus (49) Google Scholar). Accommodation of this unfavorable residue requires the addition of heparin, whose action is 2-fold; 1) it links the heparin binding sites of thrombin and heparin cofactor II, and 2) it simultaneously exposes the acidic N-terminal peptide of heparin cofactor II, making it available for binding to the fibrinogen recognition exosite (34Van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Abstract Full Text PDF PubMed Google Scholar, 35Ragg H. Ulshofer T. Gerewitz J. J. Biol. 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Biol. Chem. 1994; 269: 18395-18400Abstract Full Text PDF PubMed Google Scholar). We have recently solved the structure of thrombin E192Q in complex with BPTI (39van de Locht A. Bode W. Huber R. Le Bonniec B.F. Stone S.R. Esmon C.T. Stubbs M.T. EMBO J. 1997; 16: 2977-2984Crossref PubMed Scopus (89) Google Scholar), which shows dramatic rearrangements of the surface loops surrounding the active site including a remodeling of the fibrinogen recognition exosite. The results presented here corroborate our conjecture that access to thrombin's active site cleft can be increased upon energetically favorable exosite binding, which might be necessary for thrombin to perform some of its diverse functions. It is conceivable that progressively tighter binding at the exosite(s) could widen the active site cleft to varying degrees, although there exists as yet no direct structural evidence for this. Interactions at the active site and at the fibrinogen recognition exosite are additive, as shown by the inhibition data obtained for rLDTI-varθ5. The best inhibitor designed by our approach, variant rLDTI-varθ5, could be useful as a potential novel anticoagulant. The favorable combination of both active site and fibrinogen recognition exosite binding yields a recombinant equivalent of the synthetic divalent inhibitors epitomized by hirulog (40Maraganore J.M. Bourdon P. Jablonski J. Ramachandran K.L. Fenton J.W., II Biochemistry. 1990; 29: 7095-7101Crossref PubMed Scopus (443) Google Scholar) and hirutonin (41DiMaio J. Gibbs B. Munn D. Lefebvre J. Ni F. Konishi Y. J. Biol. Chem. 1990; 265: 21698-21703Abstract Full Text PDF PubMed Google Scholar). Despite the 1000-fold lower affinity for thrombin compared with hirudin, the anticoagulatory activity of rLDTI-varθ5 matches that of hirudin in plasma-based clotting assays. The suitability of this variant for possible therapeutic or in vivo applications will depend on several aspects, however: its potential antigenicity, its bioavailability, and its effectiveness in preventing bleeding. These items must be addressed thoroughly before a therapeutic application can be envisaged. In conclusion, we have been able to construct three potent inhibitors of thrombin. They represent suitable models for the design of tighter, more specific or more selective coagulation inhibitors. We are grateful to Professors Robert Huber and Wolfram Bode for continuous support and encouragement.