©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Mapping of the Surface Residues of Human Thrombin (*)

Manuel Tsiang (§) , Anant K. Jain , Kyla E. Dunn , Maria E. Rojas , Lawrence L. K. Leung (¶) , Craig S. Gibbs (§)

From the (1)Gilead Sciences Inc., Foster City, California 94404

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Utilizing site-directed mutagenesis, 77 charged and polar residues that are highly exposed on the surface of human thrombin were systematically substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleavage of the procoagulant substrate fibrinogen (Lys, Trp, Lys, Asn+Thr, Lys, His, Arg, Tyr, Arg, Lys, Lys+Lys, Asp+Lys, Glu, Glu, Arg, Asp) and the anticoagulant substrate protein C (Lys, Trp, Lys, His, Arg, Tyr, Arg, Lys, Lys+Lys, Glu, Arg), interactions with the cofactor thrombomodulin (Gln, Arg) and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor (Lys, His, Arg, Tyr, Arg). Although there is considerable overlap between the functional epitopes, distinct and specific residues with unique functions were identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. No functional residues were located on the opposite face of thrombin. Residues with procoagulant or anticoagulant functions were not spatially separated but interdigitated with residues of opposite or shared function. Thus thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the critical contact residues may vary.


INTRODUCTION

Thrombin is a multifunctional serine protease that plays a prominent role in the maintenance and regulation of hemostasis by blood coagulation(1) . Upon generation by factor Xa activation of prothrombin (2), thrombin recognizes and hydrolyzes multiple macromolecular substrates that have either procoagulant or anticoagulant functions and alters their activity (reviewed in Refs. 3 and 4). Thrombin cleaves two peptide bonds in fibrinogen, producing fibrin monomers that polymerize to form an insoluble clot. The fibrin polymers are subsequently stabilized through cross-linking by factor XIIIa which also results from thrombin-catalyzed activation(5) . Platelet activation and aggregation is mediated by the thrombin-catalyzed hydrolysis of the platelet thrombin receptor(6) , and the procoagulant stimulus is maintained by thrombin-mediated feedback activation of the serine protease factor XI (7) and the cofactors V and VIII(8) . Binding of thrombin to the cofactor thrombomodulin on the surface of endothelial cells alters the specificity of thrombin such that it no longer recognizes the procoagulant substrates described above but instead recognizes and activates the anticoagulant substrate, protein C. Activated protein C attenuates the coagulant stimulus by the cleavage and inactivation of activated cofactors Va and VIIIa and may function to localize blood coagulation at the site of vascular injury(9) .

The crystal structure of human -thrombin bound to the peptidyl inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK)()(10, 11) revealed that prominent structural features of the thrombin molecule are the location of the catalytic triad (His, Asp, Ser) within a deep canyon-like active site cleft and the presence of two extensive surfaces (referred to as exosite 1 (Arg, Lys, Arg, Arg, Arg, Lys, Lys, Lys, Lys) and exosite 2 (Arg, Arg, Arg, Arg, Arg, Lys, Arg, Arg, Lys, Lys, Lys)) that are mainly comprised of positively charged residues. Considerable insight into the interaction of the thrombin active site with substrate residues immediately proximal to the cleavage site has been provided by the structure of the PPACKthrombin complex and the complex of human -thrombin with fibrinopeptide A(12) . However, the exquisite specificity of thrombin for the macromolecular substrates described above is thought to involve interactions with secondary sites on the surface of thrombin (13). The crystal structure of thrombin bound to hirudin, an inhibitory protein from the medicinal leech, revealed that while the amino-terminal domain of hirudin occupies the active site of thrombin, the carboxyl terminus makes extensive contacts with exosite 1(14, 15) . Although some insight into the interactions of fibrinogen, thrombomodulin, and the platelet thrombin receptor with thrombin has been derived by analogy with the COOH terminus of hirudin (reviewed in Ref. 16), the interactions of these macromolecular substrates with secondary sites on thrombin are not well defined.

The current thrombin inhibitors can be divided into four groups according to the location of their putative binding sites on the surface of thrombin: active site only (e.g. PPACK(10, 11) ), active site and exosite 1 (e.g. hirudin(14, 15) , heparin/heparin cofactor II(17) ), active site and exosite 2 (e.g. heparin/antithrombin III(18, 19) ), exosite 1 only (e.g. hirugen(20) , exosite 1 specific antisera(21) ). An ideal thrombin inhibitor would inhibit the procoagulant activities of thrombin while preserving its anticoagulant function. However, all the current inhibitors inhibit the activity of thrombin toward both the procoagulant substrates and the anticoagulant substrate, protein C. Thus, preliminary mutagenesis studies that indicated that the binding sites for fibrinogen and the platelet thrombin receptor can be dissociated from those of thrombomodulin and protein C (22) provoked speculation that there may be an epitope on the surface of thrombin, specific for procoagulant function, that could be selectively targeted by an inhibitor.

In this study we undertook an extensive mutagenesis study in order to map the secondary binding sites on the surface of thrombin required for fibrinogen clotting, thrombomodulin-dependent activation of protein C, and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor identified by a combinatorial selection strategy(23) , that does not interact with the active site and has been proposed to interact with both exosites 1 and 2(24, 25, 26) . The approach used involved the systematic substitution of all the charged and polar amino acids that are highly exposed on the surface of thrombin with the small neutral amino acid alanine. Functionally important residues were found only on a single hemisphere of the thrombin surface lining and flanking the active site cleft. Although residues were identified that are specifically required for unique functions, these residues were generally not spatially separated from residues with other activities.


EXPERIMENTAL PROCEDURES

Construction of the Vector for Expression of Human Prothrombin

The human prothrombin coding sequence from cDNA clone BS(KS)-hFII provided by Ross MacGillivray, University of British Columbia (27) was inserted into eukaryotic expression vector pRc/CMV (Invitrogen Corp.) to generate the construct, pRc/CMV-hPT which can be used to express prothrombin in mammalian cells and to generate a single-stranded template for mutagenesis and sequencing in Escherichia coli in the presence of a helper phage, M13K07 (28).

Oligonucleotide-directed Mutagenesis

The method utilized has been described previously in detail(29) . Oligonucleotide-directed mutagenesis was performed on a uracil-containing single-stranded template of pRc/CMV-hPT produced in dutungE. coli strain CJ236 (30) to allow selection against the parental strand in dutungE. coli strain, XL1-Blue. Single-stranded DNA from individual transformants was sequenced using dideoxy chain termination and Sequenase 2.0 (United States Biochemical) to confirm the identity of each mutation. 500 ml cultures of each pRc/CMV-hPT mutant in XL1-Blue were used to isolate plasmid DNA using the QIAGEN Maxi plasmid preparation kit for transfection of cultured COS-7 cells.

Expression and Activation of Recombinant Prothrombins

Plasmid DNA encoding pRc/CMV-hPT mutants (10 µg) was introduced into 1 10 COS-7 cells, grown in a 35-mm well, by the DEAE-dextran method of transfection(31) . Two days post-transfection, the cell monolayer was washed twice with PBS and incubated with 1 ml of serum-free Dulbecco's modified Eagle's medium at 37 °C for 24 h. The conditioned medium was concentrated 20-fold with a Centricon-30 ultrafiltration apparatus (Amicon). Prothrombin in 50 µl of this concentrated medium was activated with 1.5 µg of Echis carinatus venom (Sigma) at 37 °C for 45 min. Twenty-five µl of concentrated conditioned medium before and after venom activation were analyzed by Western blotting of reducing SDS-PAGE gels to ensure that processing was complete.

Quantitation of Recombinant Prothrombins in Conditioned Cell Culture Medium

Thrombin protein concentration was determined by quantitative Western slot-blotting using a Schleicher and Schuell Minifold II vacuum slot-blot apparatus. Prothrombin in 20-fold concentrated conditioned medium and purified prothrombin standards (American Diagnostica) were activated as described above, complete processing was demonstrated by Western blotting of reducing SDS-PAGE gels (Fig. 1A). Samples and standards were diluted with PBS and adjusted to the same concentration of conditioned medium from mock transfected cells. Duplicate 100-µl aliquots containing approximately 50 ng of the activated prothrombin unknown or duplicate aliquots of purified, activated prothrombin standards (1-200 ng) were aspirated through a 0.45-µm nitrocellulose filter in the slot-blot apparatus. Each slot was washed twice with 200-µl aliquots of PBS. The blot was washed twice with PBS and blocked with 5% non-fat skim milk (Carnation) in PBS. The blot was incubated with 11 µg/ml rabbit polyclonal immunoglobulins against human prothrombin that cross-react with thrombin (Dako) in 5% non-fat skim milk in PBS. The blot was washed with PBS containing 0.05% Tween-20 and incubated with 1 µCi/ml S-labeled donkey F(ab`) directed against rabbit immunoglobulins (Amersham Corp.). The blot was washed with PBS containing 0.05% Tween-20, and the radioactivity at each position was determined using an Ambis 4000 radioanalytic imaging detector. The thrombin concentration in each sample was determined from the standard curve which was linear over the range of 1-200 ng of prothrombin (Fig. 1B).


Figure 1: Processing and quantitation of prothrombin mutants. A, alignment of two Western blots (positions 1-3 and 4-8) of SDS-PAGE gels probed with the polyclonal antibody directed against prothrombin (Dako) used for quantitation of prothrombin mutants in the quantitative Western slot-blot. Lane 1, purified prothrombin (1 µg), fragments corresponding to prothrombin (72 kDa) and partially processed intermediates are visible. Lane 2, purified prothrombin (1 µg) processed with E. carinatus venom, fragments corresponding to the thrombin B chain (34 kDa), and the pro region fragments F1 (21 kDa) and F2 (12 kDa) are visible. Lane 3, purified thrombin (1 µg), a fragment corresponding to the thrombin B chain (34 kDa) is visible along with a possible degradation product. Lanes 4-8, (0.5 µg) recombinant prothrombin mutants (wild-type, mutants 3a, 8b, 10a, 36b) in concentrated cell culture medium following processing with E. carinatus venom, fragments corresponding to the thrombin B chain (34 kDa), and the pro-region fragment F1 (21 kDa) are visible. B, standard curve for quantitation of prothrombin mutants by Western slot-blot. Binding of the rabbit polyclonal antibody directed against prothrombin (Dako) to duplicate samples of purified prothrombin was quantitated following binding of S-labeled donkey antibodies (Amersham) directed against rabbit immunoglobulins. Linear correlation coefficient, r = 0.998.



Amidolytic Assay

The hydrolysis by thrombin of the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA) was performed as described previously(22) . Purified wild-type thrombin (1 µg) gave a rate of hydrolysis of 1012 mOD/min in 300 µl of 100 µM S-2238.

Fibrinogen Clotting

Fibrinogen clotting activity was determined using a standard amount of thrombin as measured by amidolytic activity. An aliquot of venom-activated conditioned medium containing 335 mOD/min of S-2238 amidolytic activity was added to each reaction. The reaction mixture contained 20 µl of conditioned medium and 180 µl of selection buffer (20 mM Tris acetate, pH 7.5, 140 mM NaCl, 5 mM KCl, 1 mM MgCl, 1 mM CaCl). The reaction was initiated by addition of 50 µl of human fibrinogen at 2 mg/ml, freshly diluted in selection buffer from a stock of 10 mg/ml made in calcium-free PBS. The time in seconds from addition of fibrinogen to clot formation was measured with a fibrometer. A plasma thrombin standard clotting curve was used to convert the clotting times into microgram/milliliter equivalent of plasma thrombin.

Inhibition of Clotting by the Thrombin Aptamer

The thrombin aptamer, an oligonucleotide thrombin inhibitor (GGTTGGTGTGGTTGG), was synthesized on an Applied Biosystems solid-phase synthesizer. For screening the susceptibility of thrombin mutants to inhibition by the thrombin aptamer, the fibrinogen clotting assay described above was used except that the thrombin aptamer was added in 180 µl of selection buffer to give a final concentration of 250 nM in 250 µl. For the determination of IC for selected mutants, a standard amount of venom-activated conditioned medium (S-2238 amidolytic activity that gives a clotting time of 30 s for wild-type thrombin) was assayed in the presence of increasing concentrations of thrombin aptamer (0-20 µM).

Protein C Activation

Cell lysates were prepared from TMnc cells expressing recombinant human thrombomodulin at the level of 504 ± 34 fmol/1 10 cells (32) as described previously (33). About 8 10 cells were lysed in 800 µl, giving a thrombomodulin concentration of 5 nM in the lysate. The commercially available human plasma protein C used contains detectable levels of contaminating prothrombin (0.005-0.02 pmol for each pmol of protein C). To circumvent this problem, 444 pmol of protein C were first treated with 10 µg of E. carinatus venom for 30 min at 37 °C to convert the contaminating prothrombin into thrombin, which was then inactivated by titration with PPACK. This venom-processed and PPACK-titrated protein C preparation was then used in a protein C activation assay(33) . The assay mixture contained venom-activated conditioned medium corresponding to a standard amount of S-2238 amidolytic activity (8.5 mOD/min), 20 µl of TMnc cell lysate and 887 nM protein C in a total volume of 50 µl. This mixture was incubated at 37 °C for 1 h and the reaction terminated by the addition of antithrombin III and heparin. For determination of thrombomodulin-independent protein C activation, the TMnc lysate was omitted and 2 mM CaCl was replaced with 5 mM NaEDTA. The activated protein C generated was assayed by hydrolysis of chromogenic substrate S-2366 (PyrGlu-Pro-Arg-pNA).


RESULTS

Mutagenesis Strategy to Identify Functional Residues on the Surface of Thrombin

A mutagenesis strategy was designed to systematically scan the surface of human -thrombin to identify residues important for fibrinogen clotting, thrombomodulin-dependent protein C activation and inhibition of clotting by the thrombin aptamer. The strategy was designed to maximize the chances of identifying functional residues while minimizing the possibility of nonspecific disruption of protein conformation. Only the charged (Arg, Lys, Asp, Glu, His) and polar (Ser, Thr, Gln, Asn, Tyr, Trp) amino acids were considered for mutation, as these residues are capable of participating in hydrogen bonds and electrostatic interactions that are likely to be important for the binding of charged ligands. Secondly, only the charged and polar residues on the surface of -thrombin that are highly exposed to solvent were selected for mutation. These residues are available for interactions with ligands and are more tolerant of sequence variation (34). The fractional accessibility (35, 36) to a solvent probe of radius 1.4 Å was determined for each residue in the 1.9-Å crystal structure of human -thrombin complexed with PPACK(10) . The 70 charged and polar residues with a fractional accessibility of >35% were selected for mutation. Only a single residue, Arg, was excluded from this list. Arg is located at the junction between the A and B chains of -thrombin and is required for the processing of prethrombin-2 to mature, two-chain -thrombin. However, several residues were added to the list (Arg, His, Lys, Arg, Lys, Arg, Trp) because functional studies (24, 25) and crystallography studies (26) indicated that residues in these locations may be involved in interactions with the thrombin aptamer. In addition, the catalytic serine residue (Ser) that participates in the formation of the acyl-enyzme intermediate was mutated to be used as a negative control in activity assays. A total of 77 residues were replaced with alanine by oligonucleotide-directed mutagenesis. Alanine was used for all substitutions because alanine is compatible with both and secondary structures(37) , tolerated in both buried and exposed locations in proteins(35, 37) , and the nonpolarity and small size of its side chain ensures that substitution with alanine is less likely to disrupt protein conformation. Multiple substitutions were made simultaneously when 2 or 3 targeted residues were clustered together. If such multiple mutants displayed a functional phenotype, then each residue was substituted individually. The complete list of alanine replacement mutants is included in .

Expression, Quantitation, and Amidolytic Activity of Prothrombin Mutants

Human prothrombin and prothrombin mutants were transiently expressed in COS-7 cells. Recombinant wild-type human thrombin produced in COS-7 cells was demonstrated to be functionally comparable to wild-type thrombin purified from human plasma (Haematologic Technologies Inc.) in the assays used in this study. In the fibrinogen clotting assay, recombinant thrombin displayed 88% of the clotting activity displayed by plasma-derived thrombin. The specific activity for generation of activated protein C by recombinant wild-type thrombin was 91.2% of plasma-derived thrombin.

Expression levels varied between 0.12-2.0 µg/1 10 cells. Mutants that expressed poorly at 37 °C showed improved expression at 27 °C. Only two mutants (K23aA,R26aA,E27aA) and W249A could not be expressed even at 27 °C (). When the 3 residues Lys, Arg, and Glu were replaced separately, all three mutants could be expressed at levels sufficient for analysis. The inability to express mutant W249A suggests that Trp plays a critical role in maintaining the structural integrity of prothrombin. All mutants except (N53A,T55A) displayed the same molecular weight as wild-type prothrombin as assessed by Western blots of SDS-PAGE gels, indicating that expression was stable with no evidence of proteolytic degradation and that there was no heterogeneity in glycosylation and proteolytic processing. The mutant (N53A,T55A) involves the 2 residues that define the single N-linked glycosylation site in mature -thrombin. Thus, this mutant is not glycosylated and exhibits a corresponding loss of molecular weight.

Concentrated cell culture medium containing prothrombin mutants was analyzed by Western blotting of reducing SDS-PAGE gels before and after processing to thrombin with E. carinatus venom (Fig. 1A). Processing was demonstrated to be complete for all mutants by the disappearance of the band (72 kDa) corresponding to prothrombin and the appearance of a band (34 kDa) corresponding to the B chain of mature -thrombin. Following processing there were no bands of higher molecular weight than the thrombin B chain, and there was no heterogeneity in the products except for the occasional appearance of trace amounts (<5%) of material corresponding to the thrombin autodegradation products -thrombin and -thrombin. The concentration of processed prothrombin was determined by quantitative Western slot-blot using a polyclonal antibody raised against prothrombin that recognizes prothrombin, thrombin A chain, thrombin B chain, and fragments (F1 and F2) derived from the pro region of prothrombin (Fig. 1B). A constant ratio of band intensities in Western blots of SDS-PAGE (Fig. 1A) indicated that there was no differential recognition of the B chain and pro-region fragments for different mutants, suggesting that mutations in the thrombin B chain did not affect recognition by the polyclonal antibody.

The specific amidolytic activity of each thrombin mutant toward the chromogenic peptidyl substrate S-2238 was assessed as a measure of the structural integrity of each mutant (). The mean specific activity of recombinant wild-type thrombin in cell culture medium from 16 separate transfections was 837 ± 168 mOD/min/µg compared to 1012 ± 61 mOD/min/µg for purified wild-type thrombin validating the slot-blot method for determination of prothrombin concentration and thrombin specific activity. The concentration of purified wild-type thrombin was determined by direct protein assay using the BCA (bicinchoninic acid) assay kit (Pierce). Medium from mock-transfected cells and from cells transfected with the active site mutant S205A displayed no detectable activity. Two additional mutants (E229A,R233A,D234A) and (R245A, K248A,Q251A) had undetectable amidolytic activity. Amidolytic activity was recovered when the residues that were simultaneously replaced in these triple mutants were substituted separately. All remaining mutants retained greater than 40% of the specific amidolytic activity of wild-type thrombin indicating that the overall tertiary conformation of each thrombin mutant was not perturbed.

Fibrinogen Clotting Activity of Thrombin Mutants

The procoagulant activity of each thrombin mutant was tested by determining their ability to clot fibrinogen (Fig. 2). Eighteen mutants had less than 50% of wild-type activity. Clotting-deficient mutants with multiple substitutions were reanalyzed with the residues replaced individually. When double mutant (W50A,D51A) was split, the mutation W50A was found to be solely responsible for impairing fibrinogen clotting. In double mutant (N74A,K77A) both mutations N74A and K77A decreased the clotting activity, with K77A having the greater effect. Both single mutants from mutant (K106A,K107A) were minimally affected in clotting activity (>50% of wild-type activity) but their effects were additive in the double mutant. Six mutants involving single amino acid substitutions retained less than 5% of the fibrinogen clotting activity of wild-type thrombin. Because this assay was normalized with respect to S-2238 amidolytic activity the effects of these mutations on clotting are not due to generalized disruption of the active site. Therefore the residues substituted in these mutants (Trp, Lys, His, Tyr, Glu, Arg) are likely to be critical for the recognition of fibrinogen.


Figure 2: Fibrinogen-clotting by thrombin mutants. The clotting times were converted into equivalent concentrations of plasma thrombin using a plasma thrombin standard clotting curve. Clotting activity of the mutant thrombins was then expressed as percent of wild-type activity. Wild-type thrombin gave a clotting time of 38 ± 6 s. Error bars represent the standard deviations of at least two independent experiments. The filled circle indicates that a mutant thrombin retained <50% of wild-type clotting activity, and the open circle indicates that a mutant thrombin retained <50% of wild-type protein C activation activity.



Protein C Activation by Thrombin Mutants

The anticoagulant properties of the mutant thrombins were initially screened for their ability to activate protein C in the presence of thrombomodulin (Fig. 3). Sixteen mutants had less than 50% of wild-type activity (). When the triple mutant (S22A,Q24A,E25A) was split into single mutations, mutation Q24A was found to be mainly responsible for the decrease in protein C activation. Similarly, mutation W50A in mutant (W50A,D51A) and mutation K77A in mutant (N74A,K77A) had the major effect on protein C activation. The result of splitting the double mutant (K106A,K107A) showed that neither mutation alone had much effect on protein C activation. Eight mutants involving single amino acid residues (Lys, Gln, Lys, His, Arg, Tyr, Lys, Glu) displayed less than 15% of the thrombomodulin-dependent protein C activating activity of wild-type thrombin. These residues are candidates for those that are important for the recognition of thrombomodulin or protein C. Again the effects of these mutations are not due to generalized disruption of catalytic activity because the assay was normalized with respect to S-2238 amidolytic activity.


Figure 3: Protein C activation by thrombin mutants. Protein C activation activity of the mutant thrombins is expressed as percent of wild-type activity. Wild-type thrombin activated protein C at a rate of 800 ± 122 pmol aPC/µg/h. The error bars represent the standard deviations of at least two independent experiments (see also Table II). The open circle indicates that a mutant thrombin retained <50% of wild-type protein C activation activity, and the filled circle indicates that a mutant thrombin retained <50% of wild-type clotting activity.



The mutants with less than 50% of wild-type activity in the thrombomodulin-dependent protein C assay were subsequently assayed for protein C activation in the absence of thrombomodulin and calcium ions (). Eleven of these also showed decreased activity in the thrombomodulin-independent protein C assay suggesting that the thrombin-protein C interaction was affected in these mutants. In contrast, mutants (S22A,Q24A,E25A), Q24A, and to a lesser extent R70A showed relatively unaltered or even enhanced activity, suggesting that the residues involved (Gln and Arg) participate in the thrombin-thrombomodulin interaction.

Inhibition of Clotting Activity of Thrombin Mutants by the Thrombin Aptamer

The ability of the thrombin aptamer to inhibit the mutant thrombins at 250 nM was assessed in a fibrinogen clotting assay (Fig. 4). Mutant E229A had virtually no clotting activity and was not tested in this assay. Five mutants were inhibited by less than 30% in this assay and were further assayed for their dose response to inhibition of clotting by the thrombin aptamer (Fig. 5). Mutant R70A had a 2800-fold increase in IC and was most refractory to inhibition. It was followed by K65A, R73A, H66A, and Y71A which increased the IC over wild-type by 220-, 94-, 28- and 7-fold, respectively. These residues are likely to be critical for interactions with the thrombin aptamer.


Figure 4: Inhibition of thrombin mutants by the thrombin aptamer. The inhibitory activity of 250 nM thrombin aptamer toward each thrombin mutant was assayed in a clotting assay. The inhibitory activity was expressed as percent inhibition of clotting activity relative to uninhibited control. The error bars represent the standard deviations of at least two independent experiments.




Figure 5: Dose dependence of inhibition by the thrombin aptamer for thrombin mutants. Inhibitory activity was determined in a clotting assay where the concentration of the thrombin aptamer was varied from 0 to 20 µM. The error bars represent the standard deviations of at least two independent experiments. The activities were normalized to that of uninhibited control. Filled circle, wild-type; open circle, Y71A; filled diamond, H66A; open diamond, R73A; filled triangle, K65A; open triangle, R70A; filled square, R70A with the following scrambled sequence of the thrombin aptamer: GGTGGTGGTTGTGGT. Values for IC were determined by curve fitting to the data using the SigmaPlot curve fitter (Jandel Scientific). Wild-type 0.058 ± 0.002 µM; K65A 21.9 ± 3.4 µM; H66A 1.5 ± 0.2 µM; R70A 161 ± 88 µM; Y71A 0.428 ± 0.038 µM; R73A 5.4 ± 0.5 µM).




DISCUSSION

Using a site-directed mutagenesis strategy to probe the highly exposed residues on the surface of thrombin, we have identified residues that are important for the recognition and cleavage of fibrinogen and protein C, interactions with thrombomodulin, and inhibition by the thrombin aptamer. Our approach was designed to minimize nonspecific structural disruption, and amidolytic activity toward the peptidyl substrate S-2238 was generally preserved among all the mutants () indicating that conformational integrity was unperturbed.

Nineteen residues were identified as being important for fibrinogen clotting activity (Lys, Trp, Lys, Asn+Thr, Lys, His, Arg, Tyr, Arg, Lys, Lys+Lys, Asp+Lys, Glu, Glu, Arg, Asp) (Fig. 2). Only 8 of these residues are absolutely conserved among 11 vertebrate thrombins (Lys, Trp, Lys, Asn+Thr, Arg, Glu, Glu)(38) . When the residues above were mapped onto the surface of thrombin (Fig. 6A) they all mapped to a single face of thrombin surrounding the active site cleft. Highly conserved residues, Trp, Lys, Glu, and Glu project into the active site cleft where they are well placed to interact with substrates. Trp occludes entry to the active site cleft from above and forms part of the apolar binding pocket and is in contact with the valine residue at P2 in the crystal structure of fibrinopeptide A bound to thrombin(12, 39) . Deletion of Trp along with Pro and Pro was previously shown to decrease k for fibrinopeptide A release(40) . In the complex with fibrinopeptide A, Glu is in contact with P5 glycine. Glu occludes entry to the active site cleft from below and could potentially contact residues at P3 or P3`(12) . Previous substitution of Glu with glutamine caused no change in fibrinopeptide A release but enhanced fibrinopeptide B release(41) . Modeling the interaction of fibrinogen residues on the carboxyl-terminal side of the cleavage site suggested that Lys could form part of the S2` subsite(12) . Previously Lys was nonconservatively substituted with glutamate and demonstrated to be important for fibrinogen clotting(22) .


Figure 6: Localization of functional epitopes on the surface of thrombin. Models generated are based on the coordinates of the PPACK-thrombin complex (10, 11) with PPACK removed, using MidasPlus (University of California, San Francisco). A, space filling model of human thrombin looking directly into the active site cleft. Exosite 1 is on the right side of the cleft. Catalytic residues (His and Ser) are colored red. Residues implicated only in recognition of fibrinogen are colored blue, residues implicated only in thrombomodulin-dependent protein C activation are colored yellow, and residues implicated in both functions are colored green. B, identical model as in A but rotated 180° about the vertical axis, illustrating that no functional residues were located on the opposite face of the molecule. C, same view as in A but highlighting residues implicated in binding the thrombin aptamer (Lys = blue, His = green, Arg = yellow, Tyr = aqua, Arg = magenta), clustered in the lower right corner of exosite 1.



Numerous studies suggested that a cluster of acidic amino acids located 20-24 residues COOH-terminal to the cleavage site of fibrinogen interacts with the highly basic exosite 1 on thrombin(21, 33, 42, 43) , including mutagenesis studies which demonstrated that the thrombin mutant R68E had no clotting activity(22) . The crystal structure of thrombin with the acidic COOH-terminal fragment of hirudin (hirugen) bound to exosite 1 has been used as a model for the interaction of fibrinogen with exosite 1(12, 20, 39) . In our mutagenesis study the loss of clotting activity for mutants involving residues Lys, His, Arg, Tyr, Arg, Lys, Lys, and Lys (Fig. 2) suggests that exosite 1, located to the right of the active site cleft (Fig. 6A), is extensively involved in the recognition of fibrinogen.

Additional residues implicated in fibrinogen recognition include residues Asp, Lys, Arg, and Asp. These residues are located below and to the left of the active site cleft (Fig. 6A). Residues Asn and Thr define the only site of N-linked glycosylation in -thrombin. Although the enzymatic removal of the sugar moiety was previously reported to have no effect on clotting activity(44) , the mutation of these residues caused a mild defect in fibrinogen clotting (43% of wild-type) that may be due to the loss of the oligosaccharide.

The crystal structure of fibrinopeptide A bound to thrombin predicted that residue Arg could form an ion pair with the glutamate residue at P6 in fibrinopeptide A (12) This interaction is conserved in the thrombin-hirudin complex. In our study Arg was substituted along with 2 other residues in mutant (R178A,R180A,D183A), and this mutant had fibrinogen clotting activity comparable to wild-type thrombin. Arg is non-conserved among vertebrate thrombins(38) , and the glutamate at P6 in fibrinopeptide A can be varied without effect(45) . Collectively, the data suggest that Arg is not as important for fibrinogen recognition by thrombin as the crystal structure of the fibrinopeptide A complex would suggest.

Fourteen residues were identified that are important for thrombomodulin-dependent protein C activation (Lys, Gln, Trp, Lys, His, Arg, Arg, Tyr, Arg, Lys, Lys+Lys, Glu, Arg) () Of these, only 5 (Lys, Gln, Trp, Arg, Glu) are absolutely conserved among 11 vertebrate thrombins(38) . Many of these residues are the same as those required for fibrinogen clotting, and they all map to the same hemisphere on the thrombin structure surrounding the active site cleft (Fig. 6A). No residues affecting protein C activation or fibrinogen clotting were found on the opposite face of thrombin (Fig. 6B). Residues Trp, Glu, and Arg line the active site cleft on the proximal side of the cleavage site. Deletion of Trp along with Pro and Pro was previously shown to decrease protein C activation(40) .

Unlike fibrinogen, protein C does not have an acidic domain on the COOH-terminal side of the cleavage site analogous to the COOH terminus of hirudin. However, an analogous domain can be found in thrombomodulin, and numerous studies have suggested that thrombomodulin binds to exosite 1 (21, 22, 32, 33, 42, 46-48). In our mutagenesis study, the replacement of 11 residues in exosite 1 (Lys, Gln, Lys, His, Arg, Arg, Tyr, Arg, Lys, Lys, Lys) (Fig. 6A) resulted in a decrease in thrombomodulin-dependent activation of protein C suggesting that protein C or thrombomodulin are involved in extensive interactions with exosite 1. Of these residues, substitution of Gln and to a lesser degree Arg did not affect thrombomodulin-independent activation of protein C () suggesting that these 2 residues may be exclusively involved in interactions with thrombomodulin. Previously, Arg and Arg were nonconservatively replaced with glutamate (22) with the same results as reported here.

A thrombin-based synthetic peptide corresponding to thrombin residues 147-158 was reported to bind thrombomodulin and block thrombin binding to thrombomodulin(46) . Analysis of modified peptides suggested residues Asn, Lys, and Gln were essential for thrombomodulin binding(47) . In our study, residues Thr, Trp, Thr, Asn, Lys, and Ser were all substituted with alanine in mutants 27-30 without any effect on thrombomodulin-dependent activation of protein C, suggesting that this region is not required for thrombomodulin binding. Human thrombomodulin can be isolated from transfected 293 cells in two forms with or without a covalently attached chrondroitin sulfate moiety that has been reported to enhance the affinity of thrombomodulin for thrombin 10-fold by interacting with exosite 2(48, 49) . In our study, mutation of residues in exosite 2 had no effect on thrombomodulin-dependent activation of protein C consistent with the observation that less than 10% of the thrombomodulin derived from the CV-1 cell line used in our study contains chrondroitin sulfate(32) .

Five residues were identified as being important for inhibition by the thrombin aptamer (Lys, His, Arg, Tyr, Arg) (Fig. 5). All of these residues are tightly clustered in the lower right corner of exosite I (Fig. 6C). Chemical modification studies demonstrated that Lys and Lys were protected by the thrombin aptamer (25) and nonconservative substitution of Arg with glutamate decreased affinity for the thrombin aptamer(24) . Crystallography studies of the thrombin aptamer complex with thrombin revealed that the thrombin aptamer was sandwiched between two symmetry related thrombin molecules, contacting exosite I on one thrombin molecule and exosite 2 on the other(26) . All the residues in exosite 1 predicted to make electrostatic interactions with the thrombin aptamer (His, Arg, Arg) were identified in our analysis. The residues in exosite 2 predicted to form hydrogen bonds or ion pairs with the thrombin aptamer (Arg, Arg, Arg, Lys, Trp, Lys) were substituted without affecting inhibition (Fig. 4) suggesting that either the thrombin aptamer does not bind to exosite 2 or that exosite 2 binding is noninhibitory. Binding of the thrombin aptamer to the exosite 1 mutant R70A was not detectable in solid-phase binding assays (data not shown) indicating that interactions with exosite 2 may be unique to the crystal complex.

The functional epitope for a procoagulant activity of thrombin, fibrinogen clotting is distinct from but overlaps with the residues required for thrombomodulin-dependent protein C activation, an anticoagulant function. Although unique residues involved in the contrasting functions of thrombin can be identified, they are not spatially separated and are located on a single face of thrombin that surrounds the active site cleft and includes exosite 1 (Fig. 6A). Thus, thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the specific contact residues may vary. The lack of spatial separation of these two epitopes is not conducive to the generation of thrombin inhibitors that can distinguish the procoagulant and anticoagulant functions of thrombin.

  
Table: Thrombin mutants: residues substituted and specific amidolytic activity


  
Table: Thrombin mutants with thrombomodulin-dependent protein C activation activity <50% of wild-type



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Gilead Sciences Inc., 353 Lakeside Dr., Foster City, CA 94404. Tel.: 415-574-3000; Fax: 415-573-4890.

Present address: Div. of Hematology, Stanford University School of Medicine, Rm. S-161, Stanford, CA 94305-5112.

The abbreviations used are: PPACK, D-Phe-Pro-Arg chloromethylketone; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We are grateful to Regan Shea, Terry Terhorst, Kim Sweetnam, Cathy Sueoka, Lisa Crow, and Melissa Klute for the synthesis of the oligonucleotides used in this study and Lisa Paborsky for critical comments on this manuscript.


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