Proc. Nati. Acad. Sci. USA Vol. 88, pp. 6775-6779, August 1991 Biochemistry

Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin (protein C/serine proteases/anion-binding exosite)

QINGYU WU*tt, JOHN P. SHEEHANt*, MANUEL TSIANG*tt, STEVEN R. LENTZt*, JENS J. J. EVAN SADLER*tt§

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*Howard Hughes Medical Institute, Departments of tMedicine and of tBiochemistry and Molecular Biophysics, The Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, MO 63110

Communicated by Robert L. Hill, May 15, 1991

ABSTRACT Thrombin is a serine protease that acts as a procoagulant by clotting fibrinogen and activating platelets and as an anticoagulant by activating protein C in a thrombomodulin-dependent reaction. Fibrinogen and thrombomodulin bind competitively to an anion-binding exosite on thrombin. We prepared recombinant normal human thrombin and mutant thrombins with single amino acid substitutions in order to localize and distinguish the fibrinogen- and thrombomodulinbinding sites. Normal and mutant thrombins had similar amidolytic activity. Thrombin K52E had ==2.5-fold increased protein C-activating activity but only =17% of normal fibrinogen-clotting activity. Thrombin R70E had normal fibrinogenclotting activity but only -7% of normal protein C-activating activity. Thrombin R68E had markedly reduced activity in both assays. Decreased activation of protein C correlated with decreased binding affinity for thrombomodulin, and ability to activate platelets correlated directly with fibrinogen-clotting activity. These results demonstrate that thrombins with predominantly anticoagulant or procoagulant activity can be created by mutagenesis and that thrombomodulin- and fibrinogen-binding sites on thrombin may overlap but are not identical.

Thrombin is a multifunctional serine protease that has procoagulant and anticoagulant activities. As a procoagulant enzyme thrombin clots fibrinogen, activates clotting factors V, VIII, and XIII, and activates platelets (1, 2). Thrombin also binds to thrombomodulin, a glycoprotein expressed on the surface of vascular endothelial cells. In the thrombinthrombomodulin complex the procoagulant activities of thrombin are reduced and its capacity to activate protein C, a serine protease zymogen, is greatly enhanced (3, 4). Activated protein C degrades plasma factors Va and VIIIa and thereby inhibits blood coagulation (5, 6). Thus the formation of the thrombin-thrombomodulin complex converts thrombin from a procoagulant to an anticoagulant enzyme, and the normal balance between these opposing activities is critical to the regulation of hemostasis. The structures on thrombin required for binding to other macromolecules are not understood in detail. A variety of experiments suggest that a positively charged surface region located some distance from the active site, the so-called anion-binding exosite (7, 8), contributes to the remarkable specificity of thrombin interactions with many substrates, cofactors, and inhibitors. For example, the carboxyl-terminal domain of hirudin, a potent thrombin-specific inhibitor from leech saliva (9), makes many ionic and hydrophobic contacts within the thrombin anion-binding exosite (10, 11) (Fig. 1). Fibrinogen, thrombomodulin, and hirudin all appear to bind The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

competitively to this exosite (12, 13). Although these proteins may interact with some common residues on the thrombin surface, other contacts are likely to be unique for each thrombin-ligand pair. Such unique protein-protein interactions provide a means to study thrombin structure-function relationships through the construction of mutant thrombins that interact with only certain ligands. This approach has been exploited to prepare thrombins that show selective loss of either fibrinogenclotting or thrombomodulin-binding activities and thereby to dissociate the procoagulant and anticoagulant properties of thrombin.

MATERIALS AND METHODS Plasmid Constructs. Plasmid pK30 containing a full-length human prothrombin cDNA insert was provided by S. J. F. Degen (University of Cincinnati, OH). Expression vector pCMV was constructed to contain the cytomegalovirus (CMV) promoter-enhancer (14) and simian virus 40 late poly(A) signal from pJC119 (15) flanking the polylinker of plasmid Bluescript-KS (Stratagene). The prothrombin cDNA insert was ligated into the EcoRP site of pCMV to give plasmid pCMVPT. Mutant constructs were made using the polymerase chain reaction (16) to create single nucleotide substitutions in the parent construct pCMVPT. The DNA sequence was confirmed for all segments derived from polymerase chain reactions, including the entire segment encoding the thrombin B chain. Amino acid residues are numbered sequentially beginning with the first residue of the thrombin B chain. Another commonly used numbering scheme relates the sequence of the thrombin B chain to that of chymotrypsinogen (17). For amino acid residues mentioned in this study, the correspondence between the two systems is as follows, with the thrombin numbering preceding the chymotrypsinogen numbering in parentheses: H43(57), K52(60f), N53(60g), R62(67), R68(73), R70(75), D99(102), S205(195). Expression, Purification, and Activation of Recombinant Prothrombins. For transfections, plasmids were linearized and introduced into CV-1 cells with a calcium phosphate method (18, 19) and with pSV2neo (20) at a 10: 1 molar ratio. Cells were grown for 10-14 days in Dulbecco's modified Eagle's medium, 10% fetal calf serum, and antibiotic G418. Clones were screened by Western blotting and ELISA methods with a rabbit polyclonal antibody to human thrombin (21). For production of recombinant prothrombins, cell lines were grown in serum-free medium containing 10 Ag of vitamin K1 per ml for 48 hr. §To whom reprint requests should be addressed at: Howard Hughes Medical Institute, Washington University, 660 South Euclid Ave8045, St. Louis, MO 63110.

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Prothrombins were purified to homogeneity by barium sulfate absorption (22), DEAE-Sephacel ion-exchange chromatography (23), and HPLC gel-filtration chromatography (TSK-G3000SW, Toyo Soda, Shinnanyo-shi, Japan). Purified prothrombins were quantitated by ELISA using a pure human plasma prothrombin standard and by absorption at 280 nm using an extinction coefficient (1 mg/ml) of 1.38 (24). Prothrombins were activated to thrombin with Echis carinatus venom (25). Complete activation was confirmed by Western blotting of reduced and unreduced samples. Amidolytic Activity Assays. The hydrolysis by thrombin of S-2238 (KabiVitrum, Stockholm, Sweden) at room temperature was assayed by monitoring absorbance at 405 nm. Assays were initiated by adding samples of thrombin to reaction mixtures containing 20 mM Tris HCI (pH 8.3), 150 mM NaCl, and S-2238 at six concentrations from 5-40 p&M. Fibrinogen Clotting. Samples of thrombin were incubated in 200 Al of 10 mM imidazole-HC1 (pH 7.4), 150 mM NaCl, 10 mM CaC12, and 6.6 mg of PEG-6000 per ml in polystyrene cuvettes at 37°C. Reaction was initiated by addition of 50 j1 of human fibrinogen at 2 mg/ml (KabiVitrum). Time in seconds from addition of fibrinogen to clot formation was measured with a fibrometer. Protein C Activation. Cell lysates were prepared from CV-1(18A) cells expressing recombinant human -thrombomodulin or from CV-1 cells, which do not express thrombomodulin (13). Cells were washed with phosphate-buffered saline and with assay buffer (50 mM Tris HCI, pH 8.0/2 mM CaCl2/100 mM NaCl/0.1% bovine serum albumin) and lysed in assay buffer containing 0.6% Triton X-100. Lysate equivalent to 2.3 x 106 cells was used for each 110 ,ul of protein C activation assay. For CV-1(18A) cell lysate this corresponds to -80 ng of thrombomodulin, for a final concentration of -8 nM thrombomodulin. Activation of protein C was measured in a two-stage assay (13). In the first stage thrombin and recombinant protein C were added to final concentrations of 3.8 nM and 0.9 ,uM, respectively, in a reaction volume of 110 p1 with or without recombinant human thrombomodulin with either 2 mM CaCl2 or 1 mM Na2EDTA. Reaction mixtures were incubated for 30 min at 37°C and stopped by addition of antithrombin III and heparin. The activated protein C generated was assayed by hydrolysis of substrate S-2366 (KabiVitrum). Thrombomodulin Binding. Competition equilibrium binding was performed as described (13). Diisopropyl fluorophosphate-inactivated 125I-labeled thrombin at 0.3 nM was incubated with CV-1(18A) cells expressing recombinant human thrombomodulin or control CV-1 cells, at 4°C for 2 hr in the presence of unlabeled thrombins. Cells were washed and solubilized with 1 M NaOH, and bound diisopropyl fluorophosphate-inactivated 125I-labeled thrombin was measured by 'y spectroscopy. Average values for nonspecific binding (0o) and maximal specific binding (100%) were 195 cpm and 1300 cpm, respectively. Binding data were analyzed as described (13). Molecular Modeling. A molecular model of thrombin was constructed using a knowledge-based comparative modelbuilding approach. This procedure has been described previously as applied to several serine proteases, including thrombin (26) and factor IX (27). The thrombin model has been related to the reported crystal structures of thrombin complexes (10, 11, 17).

RESULTS Four single amino acid substitutions in the human thrombin B chain were constructed. Three of these (K52E, R68E, R70E) replaced basic amino acids (arginine or lysine) with glutamic acid in the proposed substrate-binding groove in the region of the anion-binding exosite (Fig. 1). Mutant K52E

Proc. NatL. Acad. Sci. USA 88 (1991)

FIG. 1. Model of the Ca structure of human a-thrombin. Only a selection of amino acid side chains is shown. The active site residues His-43, Asp-102, and Ser-205 are shown in blue. In the active center a model substrate, Gly-Gly-Pro-Arg-Ala, is shown in purple. The surface of residues reported to interact with the carboxyl-terminal domain of hirudin (10, 11) is indicated by red stippling. Amino acid residues modified by mutagenesis in this study are shown in yellow. Looking from the active center from the left toward the top right of the figure, these are Lys-52, Arg-68, and Arg-70.

alters a lysine residue in a surface P-hairpin loop that contains a unique insertion of nine amino acids, compared to chymotrypsinogen, which projects into the substrate-binding cleft. Residue K52 appears to form part of the S1' subsite on the carboxyl side of the scissile bond (17). Mutant R68E alters an arginine residue that may be part of the S2' subsite, and R70E alters an arginine residue in a long groove extending from the S2' subsite. These three mutations affect residues that appear to interact with the carboxyl-terminal domain of hirudin (10, 11). The fourth mutation, S205A, replaces the active site serine residue to produce a catalytically inactive thrombin. Mutant and normal recombinant human prothrombins were expressed in CV-1 monkey kidney cell lines and purified to apparent homogeneity from serum-free conditioned medium. The electrophoretic mobility of recombinant prothrombins was slightly greater than that of plasma-derived prothrombin (Fig. 2A), but digestion of plasma and recombinant prothrombins with N-glycanase resulted in sharp bands of indistinguishable mobility, suggesting that differences in glycosylation of prothrombin by CV-1 cells may account for the differences in electrophoretic mobility (un-

published observations). After activation all recombinant and plasma-derived thrombins showed the same electrophoretic mobility (Fig. 2B). Mutant thrombin K52E contained in addition a minor species of faster mobility. This appears to be the result of incomplete glycosylation. Amino acid sequencing demonstrated that the major and minor forms of thrombin K52E contained the thrombin B chain, and digestion of all thrombins with N-glycanase yielded single bands with the same electrophoretic mobility as the minor K52E species (unpublished observations). Human thrombin has one N-linked

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-28 FIG. 2. Polyacrylamide gel electrophoresis of plasma and recombinatit prothrombins and of thrombins derived from them. (A) Prothrombins. Prothrombins (200 ng) were reduced with 2% 2-mercaptoethanol and analyzed by SDS/polyacrylamide gel electrophoresis (28) and silver staining. The apparent mass (kDa) of protein standards is indicated. S, standard proteins; pIT, plasma prothrombin; rIl, recombinant normal prothrombin; K52E, recombinant mutant prothrombin K52E; R68E, recombinant mutant prothrombin R68E; R70E, recombinant mutant prothrombin R70E; S205A, recombinant mutant prothrombin S205A. (B) Thrombins. Prothrombins (200 ng) were activated to thrombin, reduced, and subjected to SDS/polyacrylamide gel electrophoresis, transferred by electroblotting to nitrocellulose, and visualized with rabbit polyclonal antihuman thrombin antibody. Abbreviations are as described for A but refer to thrombins derived from the corresponding prothrombins.

oligosaccharide at Asn-53 (1, 2), adjacent to the mutation in thrombin K52E. This mutation may decrease the efficiency with which Asn-53 is glycosylated. The apparently nonglycosylated form of thrombin K52E accounts for no more than -30% of the total. In control experiments, recombinant normal thrombin and mutant thrombins K52E, R68E, and R70E were shown to have similar amidolytic activity with the small peptide substrate S-2238, with values for Km and k,.t similar to those of plasma-derived thrombin (Table 1). The thrombin active site mutant S205A did not cleave this substrate. Each active recombinant thrombin was inhibited completely by excess hirudin; at low concentrations of hirudin the extent of inhibition appeared to be decreased for certain mutant thrombins, but this variation was not studied systematically (unpublished observations). These data suggest that the tertiary structure ofthrombin was not significantly disrupted by these single amino acid substitutions. In contrast, the mutant thrombins showed marked differences in fibrinogen-clotting activity (Table 1). The clotting activity of thrombins K52E and R68E were -17% and