INTRODUCTION

Top Abstract Introduction Procedures Results & Discussion References

Protein S (PS)¹ is a vitamin K-dependent plasma protein that functions as an anticoagulant cofactor to activated protein C (APC) in the degradation of the activated forms of coagulation factor V and factor VIII (1, 2). The biological importance of PS as an anticoagulant is clearly demonstrated by the massive thrombotic complications that affect infants with homozygous PS deficiency and by an increased risk of venous thrombosis in individuals with heterozygous PS deficiency (3-6).

The clear association between thrombosis and PS deficiency stands in sharp contrast to the weak APC cofactor function of PS in vitro. Thus, PS was reported to yield only a 2-fold increase in the rate of APC-mediated degradation of factor Va in a purified system (7). However, more recent results suggest that PS expresses rather specific APC cofactor function, because in the degradation of factor Va by APC, PS was found to stimulates only the APC-mediated cleavage at Arg³⁰⁶ and not the cleavage at Arg⁵⁰⁶ (8). That the protein C system indeed is more complicated than previously known became even more evident when factor V was found to function in synergy with PS as cofactor to APC (9, 10). Degradation of factors Va/VIIIa thus depends on the assembly of multimolecular complexes on the phospholipid membrane via molecular mechanisms that are still incompletely understood.

PS has a high affinity for negatively charged phospholipid (11), a property that is important for its APC cofactor activity because PS increases the affinity of APC for negatively charged phospholipid vesicles (12), endothelial cells (13), platelets (14, 15), and platelet microparticles (16). Recently, the interaction between APC and PS on the phospholipids was found to relocate the active site of APC about 10 Å closer to the membrane surface (17). The PS-induced conformational changes may be important for selectivity of the APC function. PS is also reported to have APC-independent anticoagulant function, which depends on direct interactions of PS with phospholipid membranes as well as with factor Va and factor Xa, and which leads to inhibition of factor X and prothrombin activation (18-20). However, the biological significance of the APC-independent functions of PS is still uncertain.

In human plasma, approximately 60% of PS is noncovalently complexed to C4b-binding protein (C4BP), a regulator of the classical complement pathway (21). Upon complex formation, PS loses its anticoagulant function (22) but retains its ability to bind negatively charged phospholipids (23).

The primary structures of human, monkey, bovine, rabbit, porcine, mouse, and rat PS have been elucidated (24-29). Mature PS is a mosaic protein composed of multiple modules. Starting from the NH₂ terminus, it contains a -carboxyglutamic acid (Gla)-rich module, a thrombin-sensitive region (TSR), four EGF-like modules, and a sex hormone-binding globulin-like module. The Gla module binds calcium and negatively charged phospholipids. The integrity of the TSR has been shown to be essential for the APC cofactor activity (30). Probing the structure-function relationships with well characterized monoclonal antibodies has suggested the TSR and EGF1 modules to be important for expression of full APC cofactor activity (31). Fab' fragments of monoclonal antibodies directed against these two modules completely inhibited the APC cofactor activity, whereas antibodies against other modules had no or very little effect. Additional support for the involvement of EGF1 in the PS-APC interaction has recently been gained from two studies. Thus, a thrombotic patient with type II protein S deficiency (functional defect) was found to carry an intron mutation in the PS gene resulting in exon 5 skipping and as a consequence a truncated PS molecule lacking EGF1 (32). The truncated PS was present at normal concentration in the patient plasma suggesting that the EGF1 deletion does not influence the synthesis of the protein. In the second report, a recombinant PS fragment comprising EGF1-4 was found to inhibit the APC cofactor activity of protein S, whereas an EGF2-4 construct had no such inhibitory activity (33).

The anticoagulant activity of bovine APC is reported to be species-restricted, which has been found to be due to the interaction between APC and PS (22, 34). Thus, bovine APC is inefficient as anticoagulant in human plasma unless bovine PS is also present. The interpretation of this is that bovine APC and human PS are unable to form an anticoagulant complex on the phospholipid surface. Human APC on the other hand is found to function both in human and bovine plasma, suggesting human APC to interact with both human and bovine PS (22). The structural determinants in human PS responsible for the species-restriction have been demonstrated to be located in both the TSR and EGF1 modules (35). Thus, human/bovine recombinant PS chimeras containing both these modules of bovine origin were found to function like bovine PS, whereas those chimeras having only one of the two modules of bovine origin did not. With respect to specificity of the APC cofactor function, porcine PS functions like bovine PS, whereas monkey PS is similar to its human counterpart (25). Rabbit PS is not an efficient cofactor to bovine APC but is stimulating the anticoagulant properties of human APC (27). By sequence comparison, a number of amino acid residues have been suggested to play a role for the species restriction. The candidate residues are Arg⁴⁹, Gln⁵², Thr⁵³, Gln⁶¹, Pro⁷⁷, Ser⁸¹, Ser⁹², Lys⁹⁷, Ser⁹⁹, Thr¹⁰³, and Pro¹⁰⁶ (25, 35).

Elucidation of the molecular basis of the specificity of the APC-PS interaction may provide valuable information as to which residues are functionally important in the interaction between APC and PS. The aim of this work was to identify amino acid residues in the TSR and EGF1 regions that are involved in determining the specificity of the APC cofactor function. This was achieved by expressing human recombinant PS mutants in which individual amino acids in the TSR and EGF1 were replaced with amino acid residues present at corresponding positions in bovine PS. The mutations were mapped on a preliminary model structure for the Gla-TSR-EGF1 region of human PS in order to further analyze our results.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results & Discussion References

Materials-- Kits for DNA sequencing and the T7-Gen in vitro mutagenesis were from U. S. Biochemical Corp.; Q-Sepharose Fast Flow was obtained from Amersham Pharmacia Biotech, and hygromycin B was from Calbiochem. Saline of phosphate buffer without calcium and carbonate, Lipofectin, Opti-MEM medium, and Dulbecco's modified Eagle's medium were from Life Technologies, Inc. Polystyrene latex beads were from Sigma. The chromogenic substrate pCa was from American Diagnostica. 1-Palmitoyl-sn-glycero-3-phosphatidylserine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine were from Avanti Polar Lipids, Inc. 1-Palmitoyl-2-[1-¹⁴C-oleoyl]PC was from NEN Life Science Products. The human PS deficiency plasma was prepared by incubating normal human plasma with immobilized polyclonal anti-human PS at 4 °C overnight as described (22). Bovine PS-deficient plasma was prepared in a similar way but using immobilized polyclonal anti-bovine PS.

Proteins-- PS and activated protein C were prepared from human and bovine plasma using methods previously described (31). The monoclonal antibodies that reacted with human PS have been previously characterized (31).

cDNA Clones-- Full-length human and bovine cDNA clones were previously isolated and characterized (26, 36). The original bovine PS cDNA clone was missing one nucleotide, an A in the second EGF-like module. To express wild-type bovine PS, site-directed mutagenesis was used to introduce the missing codon. BamHI sites were introduced at both 5'- and 3'-end of the coding regions of full-length human and bovine cDNA clones as described previously (35). The numbering of the nucleotides of human and bovine PS cDNA used in this paper is based on sequences which are available in the GenBank/EBI Data Bank having accession numbers M15036 and M13044, respectively.

In Vitro Mutagenesis-- Three human/bovine PS chimeras were prepared in which the bovine TSR, EGF1, or both modules were introduced into human PS, i.e. the bovine modules replaced corresponding human modules. Mutant 1 containing the bovine EGF1 module was prepared from the previously described chimeras V (35). This chimeras contained the Gla, TSR, and EGF1 modules of bovine origin with the rest being of human origin. A cleavage site for HincII (position 454) located between TSR and EGF1 in both human and bovine PS cDNA was used to create the mutant 1. BamHI-XbaI (XbaI cleaves at 1481) fragments of both human PS cDNA and chimeras V were cleaved with HincII. The isolated larger HincII-XbaI fragment from chimera V was ligated with the smaller BamHI-HincII fragment from human PS cDNA and the 3' XbaI-BamHI fragment to create full-length cDNA in BamHI-cleaved pUC18. This full-length construct, which contained EGF1 of bovine origin in a human PS background, was transferred to the expression vector pGT-h as described below. Mutant 6, which contained both TSR and EGF1 of bovine origin, was constructed in a similar way utilizing an NciI cleavage site which was present in both human (position 372) and bovine (position 261) PS cDNA between the Gla and TSR modules. In short, chimeras V was cut with NciI and BamHI, and the large fragment was isolated and ligated to the small BamHI-NciI fragment obtained from the human cDNA clone. Mutant 5, which contained only the TSR of bovine origin, was made from mutant 6 and human PS cDNA. BamHI-XbaI fragments of both these cDNAs were cut with HincII and the BamHI-HincII fragment from mutant 6 was ligated to the HincII-XbaI fragment from human PS together with the 3'-part XbaI-BamHI fragment to create full-length molecules. As a result, the mutant contained the TSR of bovine origin, and the rest was human PS.

Cell Culture and Expression-- The adenovirus-transfected human kidney cell line 293 was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and streptomycin, 10 µg/ml vitamin K1. Transfection was performed using the Lipofectin method (42). DNA (2-4 µg) was diluted to 0.1 ml with sterile water. Lipofectin was added (1 µg/µl), and samples were left at room temperature for 10-15 min. Cell monolayers (40-50% confluent in a 5-cm Petri dish) were washed twice in serum-free Opti-MEM medium (Life Technologies, Inc.). The DNA/lipid mixture was diluted to 1 ml in Opti-MEM medium, added to the cells, and incubated overnight (16-20 h). The cells were fed with 2 ml of complete medium containing 10% calf serum and left to recover for another 48-72 h. They were then trypsinized and seeded into 10-cm dishes with selection medium (Dulbecco's modified Eagle's medium containing 10% calf serum and 200 µg/ml hygromycin B) at 1:5 (43). Hygromycin-resistant colonies were obtained after 3-5 weeks of selection, pooled (30 colonies on average), grown to confluency, and the media screened for PS expression with an enzyme-linked immunosorbent assay (ELISA). Conditioned media were collected in the presence of 10 µg/ml vitamin K1.

Purification of Recombinant Wild-type and Mutant PS-- Tissue culture medium (approximately 2 liters) was filtered through glass wool and diluted with an equal volume of 10 mM Tris-HCl, pH 7.5, containing 10 mM benzamidine HCl and incubated with 50 ml of Q-Sepharose Fast Flow (Amersham Pharmacia Biotech) during gentle stirring at 4 °C overnight. The gel was allowed to settle for 1 h and the supernatant discarded. The gel was packed in a column (3 × 50 cm) and washed with 10 mM Tris-HCl, pH 7.5, containing 10 mM benzamidine HCl, and then with the same buffer also containing 50 mM NaCl. Elution was performed with 1.0 M NaCl in the same buffer. The column was run at 4 °C at a flow rate of 80 ml/h; 15-ml fractions were collected. Fractions containing PS were identified by electroimmunoassay or ELISA, pooled, and dialyzed against 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5, containing 5 mM benzamidine HCl. After the addition of Ca²⁺ to a final concentration of 2 mM and incubation for 2 h, the material was applied to an affinity column containing the Ca²⁺-dependent antibody HPS-21 (31, 44). The column was equilibrated with 50 mM Tris-HCl, 0.15 NaCl, pH 7.5, containing 5 mM benzamidine HCl and 2 mM Ca²⁺. After application, the column was washed with the same buffer containing 1.0 M NaCl, and PS was then eluted with 50 mM Tris-HCl, 1.0 M NaCl, 10 mM EDTA, pH 7.5, containing 5 mM benzamidine HCl. Fractions containing PS were pooled and dialyzed against 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5, overnight. After concentration by ultrafiltration using YM 10 filters (Amicon), the proteins were stored at 70 °C.

Polyacrylamide Gel Electrophoresis-- Polyacrylamide (10-15%) slab gel electrophoresis (PAGE) was run in the presence of 0.1% sodium dodecyl sulfate (SDS) under reducing and nonreducing conditions using methods described (31). Proteins were silver-stained on SDS-PAGE (45).

Enzyme-linked Immunosorbent Assay (ELISA)-- Quantification of recombinant wild-type and mutant PS was performed by ELISA using rabbit anti-PS IgG (human or bovine) as catching antibodies and biotinylated monoclonal antibody HPS-21 as detecting antibody. Polyclonal antibodies were used at appropriate dilution in 50 mM Na₂CO₃, pH 8.5, to coat microtiter plates (Costar, Cambridge, MA) overnight at 4 °C. Plates were washed three times with 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, and incubated with 200 µl of 1% BSA in the same buffer for 30 min. This was followed by three washes using 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, containing 0.1% Tween 20. Samples containing PS (diluted in 150 mM NaCl, 20 mM Tris-HCl, 0.1% BSA, pH 7.5, containing 2 mM CaCl₂) were incubated in the wells for 2 h at room temperature. After three washes, 100 µl of biotinylated HPS-21 (final concentration 1 µg/ml) was added and incubated for 1 h at room temperature. After three washes, a mixture of streptavidin and biotinylated horseradish peroxidase (50 µl) was added and incubated for 30 min. After three washes, 50 µl of 0.04% 1,2-phenylenediamine dihydrochloride and 0.015% hydrogen peroxide in 0.1 M sodium phosphate was added. Hydrolysis of p-nitrophenylphosphate was stopped by the addition of 50 µl of 2 N sulfuric acid, and the absorbance was measured at 492 nm using a V_max plate reader (Victor, Molecular Devices Corporation, Menlo Park, CA).

Amino Acid Analysis and Protein Sequencing-- After SDS-PAGE, proteins were transferred to Immobilon membranes, stained with Coomassie Brilliant Blue, and sequenced in a gas phase sequencer as described by Matsudaira (46). The amino acid composition was determined after acid hydrolysis (6 M HCl, 24 h, 110 °C in vacuum) using a Beckman 6300 amino acid analyzer. Gla was measured following alkaline hydrolysis as described (47). All measurements were performed at least in duplicate.

Measurement of APC Cofactor Activity of Different PS Mutants-- The APC cofactor activities of recombinant wild-type and mutant PS were characterized in a plasma-based activated partial thromboplastin time (APTT) system. Human PS-deficient plasma (100 µl) was incubated with 100 µl of APTT reagent (Chromogenix) for 5 min at 37 °C, after which 100 µl of 25 mM calcium chloride containing human or bovine APC (final concentration in assay of 0.3 µg/ml) and wild-type or mutant PS samples (final concentration in assay of 0-10 µg/ml) were added, and the clotting time was measured in an Amelung-Coagulometer KC4A. All dilutions were made in Michaelis buffer (0.036 M sodium acetate, 0.036 M sodium barbitone, 0.14 M NaCl, pH 7.4). Each data point was the mean of three determinations; mutants 7-18 were measured on two occasions each including three individual measurements. Control experiments with bovine PS-deficient plasma were also performed.

Interaction of PS Mutants with Human C4BP-- Binding of PS to C4BP was measured in a microtiter plate assay using immobilized human C4BP and detection of bound PS with biotinylated HPS-21. Human C4BP (10 µg/ml in 50 mM Na₂CO₃, pH 9.6) was incubated overnight at 4 °C in polyvinyl microtiter plates. After three washes with 50 mM Tris-HCl, 150 mM NaCl, 0.2% Tween 20, pH 7.5, containing 2 mM CaCl₂, wells were blocked with 200 µl of 1% BSA in the same buffer for 30 min at room temperature. After washing three times using the same buffer, 50 µl of different amounts of wild-type or mutant PS samples (0-10 µg/ml) in 50 mM Tris-HCl, 150 mM NaCl, 0.2% Tween 20, pH 7.5, containing 2 mM CaCl₂ were added and incubated overnight at 4 °C. Bound PS was detected with HPS-21 as described for the ELISA.

Adsorption of Phospholipid Vesicles to Polystyrene Latex Beads-- Phospholipid vesicles composed of 20% phosphatidylserine and 80% phosphatidylcholine (plus a tracer of [¹⁴C]phosphatidylcholine) were prepared using the method of Barenholz et al. (48) as modified by Smirnov and Esmon (49). In brief, the lipids were suspended (1 mg/ml) by vortexing in 0.15 M NaCl, 0.02 M Tris-HCl, pH 7.4 (TBS), containing 0.02% sodium azide and sonicated for 20 min in an ice-water bath under argon flow. After sonication, the suspension was centrifuged at 8000 × g for 15 min and passed through a 100-nm nucleopore membrane in an extruder (Avestin) to obtain homogenous vesicles. The vesicles were used immediately or stored at 80 °C. The vesicles were adsorbed to latex beads as described by Smirnov et al. (50). In brief, polystyrene latex beads (100 µl) were pelleted in an Eppendorf tube by centrifugation (8000 × g for 1 min), washed three times with TBS, and resuspended in 100 µl of TBS containing 5 mM CaCl₂. Liposomes (100 µl) were added and incubated for 2 h at 37 °C with shaking. After washing twice with TBS, the beads were suspended in TBS containing 1 mg/ml gelatin, 1 mg/ml ovalbumin, and 10 mg/ml BSA, pH 7.4. The suspension was continuously mixed at room temperature with shaking for 2 h. After washing twice with TBS, the beads were suspended in 1 ml of TBS. The concentration of phospholipid was determined by counting the [¹⁴C]phosphatidylcholine tracer which was included in the phospholipid mixture (Beckman model LS 6000 SE scintillation counter). The beads were used immediately or stored at +4 °C for up to 3 weeks without loss of absorbed phospholipid.

Fluorescent Labeling of Bovine APC-- Active site fluorescein labeling of bovine APC was prepared according to the method described by Bock (51) as modified by Smirnov et al. (50). In brief, 600 µl of bovine APC (1 mg/ml) was mixed with 80 µl of 1 M HEPES, pH 7.4, 3.5 µl of 0.2 M EDTA, and 20 µl of 4 mM N-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH₂Cl and incubated at room temperature for 10-15 min to form the ATA·FPR·APC complex. The inhibition of APC activity was examined using the chromogenic substrate pCa. After extensive dialysis, 90 µl of 1 M hydroxylamine (in 1 M HEPES, pH 7.4) and 100 µl of 1 mg/ml 5-(iodoacetamido)-fluorescein (Molecular Probes) (in 1 M HEPES, pH 7.4) were added to the ATA/FPR/APC mixture and incubated at room temperature for 1 h or at +4 °C overnight. The mixture was then subjected to gel filtration on a PD-10 column (Amersham Pharmacia Biotech), and the labeled APC peak was pooled and dialyzed overnight at +4 °C in TBS, 0.02% sodium azide, pH 7.4. With this approach, each molecule of labeled APC contains a single dye at the active site.

Facscan Analysis of Fluorescein-labeled APC Binding to Liposomes-- A procedure recently described by Smirnov et al. (50) was followed. In this method, latex beads carrying 0.5 µg of phospholipid/ml were suspended in TBS, 2.5 mM CaCl₂ containing 1 mg/ml gelatin, 1 mg/ml ovalbumin, and 10 mg/ml BSA, pH 7.4. Protein S (100 nM final concentration) and fluorescein-labeled bovine APC (between 0 and 2000 nM) were added. The protein/liposome mixtures were incubated at 25 °C for 20 min in the dark with gentle mixing. Binding of labeled APC to the phospholipid was measured using a FACScan flow cytometer (Becton Dickinson). Calcium-independent binding of the fluorescent-labeled APC to phospholipid was determined after addition of EDTA (10 mM final concentration). Each experiment was performed twice, and mean values were used for data analysis.

Data Analysis-- Binding data were analyzed by fitting the calcium-dependent binding parameter to the equation for single site binding model. The K_d was calculated using the ENZFITTER program (Elsevier Biosoft, Cambridge, UK). The free concentration of fluorescent labeled APC was calculated as described by Nelsestuen et al. (11).

	RESULTS AND DISCUSSION

Top Abstract Introduction Procedures Results & Discussion References

Comparison of TSR and EGF1 Sequences in Different Species and Site-directed Mutagenesis Strategy-- Bovine PS supports the activity of both human and bovine APC, whereas human PS is restricted in its activity and only expresses activity to human APC. Sequence differences in the TSR and EGF1 regions between human and bovine PS explain this difference in specificity (22, 34, 35). Species restriction of the APC-PS interaction is not only expressed by the human-bovine pair but also by other species combinations. Thus, monkey PS (mPS) is functionally similar to hPS (neither of them stimulate bovine APC), whereas porcine PS (pPS) is similar to bPS (stimulates both human and bovine APC) (25). Comparison of PS amino acid sequences from these four species (human, monkey, bovine, and porcine) reveals a limited number of amino acid differences in the TSR and EGF1 which possibly are involved in the species restriction of the PS function (Fig. 1). In the TSR, there are three amino acid residues that are identical in hPS and mPS and different from the bovine/porcine PS sequences. Position 49 in hPS and mPS is occupied by Arg, whereas bPS and pPS have Gly; at position 52, hPS and mPS have Gln, whereas bPS and pPS have Arg; at position 61, hPS and mPS have Gln, whereas Leu is found in both bPS and pPS. In the EGF1 module, the following six positions have a consensus sequence for the human/monkey pair that is different from that of the bovine/porcine one: Ser⁸¹  Asn, Tyr⁹⁰  Phe, Ser⁹²  Thr, Lys⁹⁷  Gln, Thr¹⁰³  Ile, and Pro¹⁰⁶  Ser. In addition, at positions 77 and 99 there are some differences between the four sequences which, however, do not obey the human/monkey and bovine/porcine pairing rule. Thus, at position 77, Pro is found in hPS, mPS, and pPS, whereas bPS has Ser, and at position 99 hPS and mPS have Ser, and bPS has Thr, and pPS has Met.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Comparison of the TSR and EGF1 amino acid sequences of protein S. A, schematic diagram of amino acid sequences of the human TSR and EGF1 modules. The shaded residues indicate amino acid residues that are different in human and bovine PS, and the arrows point to the amino acid residues present in bovine PS. B, alignment of the amino acid sequences (TSR and EGF1) from human, monkey, bovine, porcine, rabbit, rat, and mouse origins. Asterisks denote positions that are different between human/monkey and bovine/porcine.

Expression and Characterization of Recombinant Wild-type and Mutant PS-- In addition to the discussed four primary structures, amino acid sequences of rabbit, rat, and mouse PS are known, and the sequences of the TSR and EGF1 of these species are also given in Fig. 1. It has been demonstrated that rabbit PS potentiates the anticoagulant activity of human APC, whereas rabbit PS is a poor cofactor to bovine APC. In this context it is interesting to note that the rabbit sequence is identical to the bovine/porcine pair at positions 49, 52, 53, 81, 97, and 103. However, it is identical to the human/monkey pair in having a Pro at position 106. Moreover, it is identical to hPS, mPS, and pPS in having a Pro at position 77, a position at which there is a Ser in bPS. In addition, rabbit PS is different from both human and bovine PS at five positions in the TSR (positions 54, 59, 61, 63, and 65) and four in the EGF1 (positions 86, 88, 91, and 92). The rat and mouse sequences are not discussed in detail as they have not been fully characterized with respect to their cofactor activity to human and bovine APC.

APC Cofactor Activity of Recombinant Wild-type and Mutant PS-- The APC cofactor activity of the recombinant proteins was tested in an APTT-based assay using human or bovine PS-deficient plasma and human or bovine APC. The use of bovine and human PS-deficient plasma yielded similar results demonstrating that the species of factor V(a) did not influence the results. Only the results obtained with human PS-deficient plasma will be shown. In the absence of added PS, both human and bovine APC yielded approximately 10 s prolongation of the clotting time. In the absence of APC, the addition of protein S did not prolong the clotting time. In the presence of APC, wild-type human and bovine PS (and the plasma-derived proteins) yielded results in accordance with those on record, i.e. both human and bovine PS potentiated the anticoagulant activity of human APC, whereas only bovine PS functioned as cofactor to bovine APC. In this system, bovine PS was found to be more potent than its human counterpart even in the presence of human APC. The plasma-derived human and bovine PS gave results similar to those of their recombinant counterparts (results not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   APC cofactor activity of protein S mutants. Increasing concentrations of selected PS mutants (final concentrations of 0-10 µg/ml) were included together with human APC (A) or bovine APC (B) (both at final concentration of 0.3 µg/ml) in an APTT-based assay using PS-deficient human plasma, and the clotting time was measured. The values are the means of triplicate measurements. The clotting time without added APC was 34 s. , wt hPS; , wt bPS; +, mutant 1; ×, mutant 7; , mutant 8; , mutant 9; , mutant 10; , mutant 32; , mutant 33; , mutant 40.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Regression slope coefficient histogram of mutants versus wt human protein S using bovine APC (A) and human APC (B). The clotting times obtained with wt-hPS were used as reference in regression analysis of clotting times obtained with the different mutants. The slopes of the regression equations are plotted. They indicate the potency of the different mutant in relation to that of wt hPS. The mutants are listed as in Table II and the mutations in TSR and EGF1 are indicated by the one-letter amino acid residue code.

GRA

L

L

Increased Binding of Bovine APC to Phospholipid in the Presence of Bovine Protein S and Some Human Protein S Mutants-- As described above, bovine protein S and several of the human protein S mutants expressed cofactor activity to bovine APC. A likely explanation for this phenomenon was that these protein S variants stimulate binding of bovine APC to the phospholipid. To investigate this possibility, binding of bovine APC (fluorescein-labeled in its active site) to phospholipid vesicles was measured using FACScan flow cytometry. In the absence of protein S, binding of bovine APC to the vesicles was characterized by a K_d of 626 nM (Table III). Bovine protein S yielded a 3.5-fold stimulation of the binding of bovine APC (K_d was 177 nM). In contrast, human protein S did not affect the binding of bovine APC. Nine of the mutants were tested for their ability to stimulate binding of bovine APC to the phospholipid. Mutant 1 (human TSR + bovine EGF1) did not stimulate binding of bovine APC. In contrast, mutant 2 (bovine EGF1 and the double mutant R49Q,Q52R in the TSR) yielded a 3-fold stimulation of bovine APC binding. The individual Q52R and Q61L mutations in TSR combined with bovine EGF1 (mutants 3 and 4) were not sufficient to stimulate binding of bovine APC. In mutants 9, 10, and 11, the TSR carried the triple mutation R49Q,Q52R,T53A (GRA-) in combination with point mutations at one or more of residues at positions 92, 97, 99, 103, and 106. These mutants all stimulated the binding of bovine APC. The mutant 35 yielded a 1.9-fold increase in bovine APC binding, which was somewhat lower than expected.

Structural Interpretation of Mutations Using a Model for the Gla-TSR-EGF1 Region-- Structural studies of PS using x-ray crystallography or NMR have so far been unsuccessful. However, it is possible to use some of the tools provided by computational biochemistry research to investigate a protein of unknown structure. We have used comparative model building and structural prediction analysis to develop a model for the Gla-TSR-EGF1 region of PS (54). Recently, the model has been refined based on experimental data, including monoclonal antibody epitope mapping (55), new information regarding sensitivity to proteolytic enzymes (56), and the description of a thrombosis patient carrying a truncated protein S molecule lacking EGF1 (32). These data support the conclusion that Arg⁴⁹, Gln⁵², and Arg⁶⁰ are solvent-exposed because Arg⁴⁹ and Gln⁵² are included in the epitope of the calcium-dependent monoclonal antibody HPS67, whereas factor Xa cleaves the TSR at Arg⁶⁰ (55, 56). Lys⁹⁷ is part of an epitope for HPS54 suggesting also this residue to be solvent-exposed (55). Moreover, as deletion of EGF1 from the protein S molecule does not affect the synthesis or stability of the protein, we suggest that EGF1 has no specific or very limited contact with the Gla, TSR, and (possibly) EGF2 domains.

View larger version (133K): [in this window] [in a new window]   Fig. 4.   Three-dimensional model for the Gla-TSR-EGF1 region of protein S. The Gla module (residues 1-46), with the -loop (green, residues 1-11, important for membrane binding), the disulfide loop (pink, Cys17-Cys22), and the helix forming part of the aromatic stack (red) are shown using a ribbon representation. The remaining part of the Gla module is in yellow. The TSR (white) is followed by the EGF1 (magenta). Some hydrophobic and ionic interactions between the Gla module and the TSR are expected at least in a calcium-loaded conformation (not shown to simplify the figure). The EGF-1 -sheets are colored orange (major -sheet) and green (minor -sheet). The seven calcium ions are magenta and disulfide bridges blue (ball and stick representation). In EGF1, the disulfide bridges involve Cys80-Cys93, Cys85-Cys102, and Cys104-Cys113. The side chains of some important residues discussed in this paper are shown as stick model, with a color code not related to the one used for the ribbon trace. The residues found essential for the species-specific interaction by our site-directed mutagenesis study are Arg49, Gln52, Ser99, Lys97, and Pro106. Inset, the model has been rotated when compared with the main figure in order to see the molecule from the opposite face. The color code for the ribbon trace is unchanged. Only a few key residues are labeled to simplify the figure. The cell membrane is drawn in white, and the short arrow indicates that the orientation of the membrane plane is approximate. Clearly, the areas Arg49, Lys97, and Pro106 could form one APC-interacting site, whereas Gln61 and Ser81 are definitely on opposite faces. The overall structure of the model is in good agreement with the experimental data.

L