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• W2094605213 abstract "Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, α1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC. Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, α1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC. activated protein C protein C mutant with E60aS plus S61R replacements polyacrylamide gel electrophoresis wild type protein C inhibitor basic fibroblast growth factor dissociation constant morpholinoethanesulfonic acid streptavidin sensor chips The protein C pathway is a functionally important anticoagulant system that regulates blood coagulation in vivo. The key component of this pathway is the vitamin K-dependent protein C (1Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1624) Google Scholar, 2Esmon C.T. Ding W. Yasuhiro K. Gu J.M. Ferrell G. Regan L.M. Stearns-Kurosawa D.J. Kurosawa S. Mather T. Laszik Z. Esmon N.L. Thromb. Haemostasis. 1997; 78: 70-74Crossref PubMed Scopus (108) Google Scholar, 3Dahlbäck B. Lancet. 2000; 355: 1627-1632Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). Protein C circulates in plasma as a zymogen to a serine protease that has anticoagulant properties. Protein C is a multidomain molecule that is composed of two disulfide-linked chains. The light chain comprises a γ-carboxyglutamatic acid domain and two epidermal growth factor (EGF)-like domains, whereas the heavy chain is composed of the activation peptide and a serine protease domain (4Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (989) Google Scholar). Protein C is activated on endothelial cells by thrombin bound to thrombomodulin. Activated protein C (APC)1 regulates blood coagulation by cleaving and inhibiting two cofactors, activated factor V (FVa) and activated factor VIII (FVIIIa) (5Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: 31869-31880Abstract Full Text PDF PubMed Google Scholar), which serve as phospholipid-membrane-bound cofactors to factor Xa (FXa) and factor IXa (FIXa), respectively. FXa is the enzyme that activates prothrombin to thrombin, whereas FIXa converts FX to its active form (1Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1624) Google Scholar, 6Rosing J. Tans G. Thromb. Haemostasis. 1997; 78: 427-433Crossref PubMed Scopus (73) Google Scholar). In vivo, the proteolytic activity of APC is regulated by two serpins, namely α1-antitrypsin and protein C inhibitor (PCI) (7Heeb M.J. Griffin J.H. J. Biol. Chem. 1988; 263: 11613-11616Abstract Full Text PDF PubMed Google Scholar, 8Hermans J.M. Stone S.R. Biochem. J. 1993; 295: 239-245Crossref PubMed Scopus (52) Google Scholar). Inhibition of APC by PCI is potentiated by the glycosaminoglycan heparin, whereas the inhibition by α1-antitrypsin is insensitive to the presence of heparin. Structurally, heparin is heterogeneous in nature and is composed of long, highly negatively charged, unbranched polysaccharide chains. It is hypothesized that heparin binds to both PCI and APC during PCI-mediated inhibition of APC, thus guiding the encounter of these proteins via a template mechanism (9Aznar J. Espana F. Estelles A. Royo M. Thromb. Haemostasis. 1996; 76: 983-988Crossref PubMed Scopus (18) Google Scholar, 10Neese L.L. Wolfe C.A. Church F.C. Arch. Biochem. Biophys. 1998; 355: 101-108Crossref PubMed Scopus (33) Google Scholar). We therefore expect the formation of a ternary complex similar to the one suggested between antithrombin, heparin, and thrombin (11Olson S.T. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar, 12Olson S.T. Bjork I. Semin. Thromb. Hemostasis. 1994; 20: 373-409Crossref PubMed Scopus (129) Google Scholar). Recently some residues in APC were implied to interact directly with heparin during the PCI-induced inhibition of APC (13Shen L. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 72-79Crossref PubMed Scopus (32) Google Scholar, 14Shen L. Dahlbäck B. Villoutreix B.O. Biochemistry. 2000; 39: 2853-2860Crossref PubMed Scopus (17) Google Scholar), but a more complete definition of a heparin-binding site(s) in APC and energetic characteristics of the heparin interaction with APC were lacking. Binding of heparin to proteins is usually ionic in nature, involving the side chains of clustered basic amino acids on the protein and negatively charged groups on the heparin molecule. Amino acid sequence patterns such as XBBXBX and XBBBXXBX (B denotes basic, and X denotes nonbasic residues) are potential heparin recognition sites (15Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar, 16Montserret R. Aubert-Foucher E. McLeish M.J. Hill J.M. Ficheux D. Jaquinod M. van der Rest M. Deleage G. Penin F. Biochemistry. 1999; 38: 6479-6488Crossref PubMed Scopus (29) Google Scholar). Alternatively, the basic residues may be located far apart in the linear sequence but are topological neighbors in the three-dimensional structure (17Sali A. Matsumoto R. McNeil H.P. Karplus M. Stevens R.L. J. Biol. Chem. 1993; 268: 9023-9034Abstract Full Text PDF PubMed Google Scholar, 18Matsumoto R. Sali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 19Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 20Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J.F. Sali A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar). In the three-dimensional structure of APC, a basic cluster of amino acids is located on loops 37, 60, and 70 (chymotrypsinogen nomenclature) (see Fig. 1) (21Wacey A.I. Pemberton S. Cooper D.N. Kakkar V.V. Tuddenham E.G. Br. J. Haematol. 1993; 84: 290-300Crossref PubMed Scopus (27) Google Scholar, 22Fisher C.L. Greengard J.S. Griffin J.H. Protein Sci. 1994; 3: 588-599Crossref PubMed Scopus (40) Google Scholar, 23Mather T. Oganessyan V. Hof P. Huber R. Foundling S. Esmon C. Bode W. EMBO J. 1996; 15: 6822-6831Crossref PubMed Scopus (191) Google Scholar). Heparin-binding sites sharing similar distribution of charged amino acids are present on the surface of many proteins that bind heparin, e.g.hepatocyte growth factor (24Zhou H. Casas-Finet J.R. Heath Coats R. Kaufman J.D. Stahl S.J. Wingfield P.T. Rubin J.S. Bottaro D.P. Byrd R.A. Biochemistry. 1999; 38: 14793-14802Crossref PubMed Scopus (51) Google Scholar). Multiple-sequence and structural alignments of APC with other serine proteases formed the basis for our mutagenesis strategy, aimed at identification of the APC heparin-binding site. In the present study, site-directed mutagenesis and recombinant human protein C expression were used to localize the heparin-binding site in APC. A cluster of lysines located on loops 37 and 60 was found to be crucial for heparin binding. Characterization of the binding of heparin to APC under different salt concentrations provided evidence for the electrostatic signature of the interaction. Reduction of the electropositive character of these loops resulted in lost heparin binding and reduced heparin stimulation of APC inhibition by PCI. Restriction endonucleases were obtained from New England Biolabs or Fermentas. Dulbecco's modified Eagle's medium, Waymouth's medium, and fetal calf serum were obtained from Life Technologies, Inc. Cell culture ware was purchased from Falcon, vitamin K1 was from Hoffmann La Roche, hygromycin B was from Calbiochem. Q-Sepharose Fast flow, sulfopropyl-Sepharose, heparin-Sepharose, and PD-10 columns were from Amersham Pharmacia Biotech. The chromogenic substrate S-2366 (l-pyroglutamyl-l-prolyl-l-arginine-p-nitroanilin), specific for protein C, was obtained form Chromogenix (Sweden), and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT) were from ICN. Human α-thrombin was prepared from plasma-purified prothrombin, as described (25Dahlbäck B. Hildebrand B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1396-1400Crossref PubMed Scopus (341) Google Scholar). Basic fibroblast growth factor (bFGF) was from Sigma. Heparan sulfate (bovine kidney) was from Seikagaku (Japan). The EZ-link biotin hydrazide was from Pierce. Streptavidin sensor chips (SA-chip) were from Biacore (Sweden). Three new recombinant human protein C variants, K37S/K38Q/K39Q, K37S/K38Q/K39Q/K62N/K63D, and R74Q, were created by site-directed in vitro mutagenesis using the polymerase chain reaction technique. In brief, full-length human protein C WT cDNA (1425 base pairs) was inserted into theBcII site of the eukaryotic expression vector pGT-hyg (Eli Lilly). The following primers were used to introduce the mutations into the human protein C WT cDNA: sense primer, 5′-CTG CTG GAC TCAAGC CAG CAG CTG GCC TGC GGG for variant K37S/K38Q/K39Q; sense primer, 5′-GAG TAT GAC CTGCAG CGC TGG GAG AAG for variant R74Q. The construction of variants E60aS/S61R and K62N/K63D have been previously described (13Shen L. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 72-79Crossref PubMed Scopus (32) Google Scholar, 14Shen L. Dahlbäck B. Villoutreix B.O. Biochemistry. 2000; 39: 2853-2860Crossref PubMed Scopus (17) Google Scholar). The human protein C variant K37S/K38Q/K39Q-cDNA and sense primer 5′-TGC ATG GAT GAG TCCAAC GAC CTC CTT GTC AGG CTT was used to construct mutant K37S/K38Q/K39Q/K62N/K63D. Exchanged nucleotides as listed above are marked in bold, and codons corresponding to the exchanged amino acids are underlined. The sequence of cDNAs coding for WT protein C and all variants were confirmed using automated DNA sequencing (PerkinElmer Life Sciences) before transfection. Adenovirus-transformed human kidney cell line 293 (HU 293, CRL-1573 ATCC) was cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. The complete human protein C-coding cDNA sequences corresponding to WT and variants K37S/K38Q/K39Q, K37S/K38Q/K39Q/K62N/K63D, and R74Q, all inserted into the eukaryotic expression vector pGT-hyg, were transfected into HU 293 cells using Lipofectin (Life Technologies). Hygromycin B was used for selection of transfected cells. Colonies characterized by the highest protein expression, as judged by immunoblotting, were cultured. Variants E60aS/S61R, and K62N/K63D were expressed as previously described (13Shen L. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 72-79Crossref PubMed Scopus (32) Google Scholar, 14Shen L. Dahlbäck B. Villoutreix B.O. Biochemistry. 2000; 39: 2853-2860Crossref PubMed Scopus (17) Google Scholar). Cells were grown in Optimem Glutamax (Life Technologies, Inc.) medium supplemented with 10 μg/ml vitamin K1. After 72 h, the conditioned medium was collected, and the recombinant expressed human protein C was purified from the supernatant by anion-exchange chromatography and barium citrate adsorption as previously described (26Friedrich U. Potzsch B. Preissner K.T. Muller-Berghaus G. Ehrlich H. Thromb. Haemostasis. 1994; 72: 567-572Crossref PubMed Scopus (8) Google Scholar). The purity and integrity of the isolated protein C was evaluated by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Protein C concentrations were quantified by measurement of absorbance at 280 nm using an extinction coefficient of 14.5 (280 nm, 1%, 1 cm). In addition, activated protein C concentrations were determined by a chromogenic assay measuring the rate of S-2366 (Chromogenix) hydrolysis. Proteins subjected to 10% SDS-PAGE were either detected by silver staining or transferred to polyvinylidene difluoride membranes (Millipore) for Western blotting. After transfer of the proteins to the polyvinylidene difluoride, membranes were quenched with Tris-buffered saline containing 3% (v/v) fish gelatin and 0.1% (v/v) Tween 20 and then incubated with antibodies. The proteins were detected using a polyclonal antibody against human protein C (No. 370 DAKO) followed by an alkaline phosphatase-conjugated swine anti-rabbit antibody (No. 306 DAKO), and BCIP (5-bromo-4-chloro-3-indolyl phosphate)/nitro blue tetrazolium chloride (NBT) substrate was used for development. Purified WT protein C or protein C variants were incubated with human α-thrombin (10:1 mol/mol) in 20 mmTris-HCl, 150 mm NaCl, pH 7.4, at 37 °C for 2 h in the presence of 5 mm EDTA. A sulfopropyl-Sepharose column was used to remove thrombin from the reaction mixture. APC concentrations were estimated by measurement of absorbance at 280 nm. Amidolytic activities were determined using chromogenic substrate S-2366 (20 mm Tris-HCl, 150 mm NaCl, pH 7.4 at 37 °C) in a microplate reader (ELX808IU from Bio-TEK instruments) and expressed in absorbance change at 405 nm (ΔA/min). The concentrations of chromogenic substrate S-2366 ranged between 0.015 and 3 mm, and the concentrations of APC ranged between 1 and 50 nm.Km and Vmax values were obtained from Lineweaver-Burk plots, and kcatwas calculated from the Michaelis-Menten equation (27Pfleiderer G. Methods Enzymol. 1970; 19: 514-521Crossref Scopus (168) Google Scholar, 28Cleland W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1930) Google Scholar). Heparin or heparan sulfate (0.5 mg of each) were dissolved in 200 μl of 0.1m MES, pH 5.6. EZ-link biotin hydrazide andN-ethyl-N′-(dimethylaminopropyl)carbodiimide were added to a final concentration of 2.5 and 0.5 mm, respectively. After a 6-h incubation at room temperature with constant shaking, the biotinylated glycosaminoglycans were desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated in water, then freeze-dried, and dissolved in 0.3 m NaCl. Binding of WT and variant APCs to heparin and heparan sulfate was analyzed by surface plasmon resonance using BIAcore 2000. Biotinylated, unfractionated heparin and heparan sulfate were immobilized in flow cells 2 and 3, respectively, of a streptavidin sensor chip. Functional integrity of the chip-bound heparan sulfate was shown by the fact that it bound bFGF with high affinity. APC (5–150 μg/ml) was injected at a flow rate of 30 μl/min at various NaCl concentrations (80–300 mm) into flow cells containing heparin. In addition, APC (150 μg/ml) was injected at 30 μl/min into a flow cell containing immobilized heparan sulfate. Flow cell 1 without any immobilized glycosaminoglycan was used as control. To investigate whether fluid phase heparan sulfate could compete with immobilized heparin for the binding to APC, 0.5 mg/ml heparan sulfate was included in the flow buffer. The derived BIAcore sensograms were evaluated with BIAevaluation 3.0 software to calculate association (kon) and dissociation (koff) rate constants. WT and variant APCs in 20 mm Tris-HCl, 50 mm NaCl, pH 7.4 buffer were applied on heparin-Sepharose, and bound APC was eluted with a linear NaCl gradient from 0.05 to 0.4 m. APC and NaCl concentrations were measured by hydrolysis of chromogenic substrate S-2366 hydrolysis and flame photometry, respectively (13Shen L. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 72-79Crossref PubMed Scopus (32) Google Scholar). The side chains of variant APC molecules were replaced interactively using InsightII and Biopolymer, and all structures were energy-minimized using the simulation program Discover (Biosym-MSI). The electrostatic potential isosurfaces were computed with DelPhi as part of the Biosym-MSI-modeling suite (inner/outer dielectrics 4/80; physiological ionic strength, formal charges) for APC molecules and for the highly negatively charged heparin molecule. The rate of inhibition of WT and mutant APCs by PCI in the absence of heparin or heparan sulfate was measured under pseudo-first-order conditions. In brief, WT and variant APCs were incubated (between 0 to 120 min) with a 10-fold molar excess of human plasma PCI at room temperature in 20 mm Tris-HCl, 0.10 m NaCl, 5 mmCaCl2, pH 7.5, containing 0.1% bovine serum albumin. Chromogenic substrate S-2366 was added to a final concentration of 0.2 mm, and the rate of substrate hydrolysis was measured with a Vmax kinetic microplate reader (Bio-TEK instruments). First-order rate constants (k) were calculated as −ln a/t [I], where a is the fractional protease activity remaining relative to the uninhibited control, t is the time of incubation, and [I] is the PCI concentration. In the presence of unfractionated heparin (Mrfrom 5,000 to 30,000; Leo) the rate of APC inactivation was too fast to be measured under the conditions that are described above. Therefore, PCI (7.5–200 nm at final concentration) was mixed with APC (30 nm) and 1 IU/ml (equal to 6.6 μg/ml) unfractionated heparin in a final volume of 100 μl. After defined time points, the reaction was stopped by the addition of 2 mg/ml Polybrene. The residual APC activities were determined by adding 50 μl of S-2366 (final concentration was 0.2 mm), and the ΔA at 405 nm was measured. The following equation was used to calculate the k2 value. k2=1[A]0−[B]0ln[B]0[A]0−χ[A]0[B]0−χ/tM−1s−1Equation 1 [A]0 and [B]0 were the initial concentrations of APC and PCI, respectively, and χ was the molar concentration of APC·PCI complexes formed after timet; k2 was calculated from data points demonstrating 15–85% proteinase inhibition. All experiments were performed in triplicates. To determine the effect of heparan sulfate on the rate of inhibition of APC by PCI, PCI (60 nm final concentration) was mixed with APC (30 nm final concentration) and increasing concentrations of heparan sulfate (0–100 μg/ml) in a final volume of 100 μl. Under these conditions, maximum stimulation of inhibition was observed at concentrations ≥50 μg/ml. At intervals, the reactions were terminated by the addition of 2 mg/ml Polybrene. Residual activity of APC was determined by the addition of 50 μl of S-2366, and the ΔA at 405 nm was measured. Thek2 values were calculated as described for heparin from data points demonstrating 15–85% proteinase inhibition. All experiments were performed in triplicate. To evaluate the involvement of positively charged amino acid residues in loops 37, 60, and 70 in the binding of heparin to APC, five protein C variants were constructed by site-directed mutagenesis (Fig.1). According to our structural analysis, four of the variants were expected to have reduced affinity toward heparin (K37S/K38Q/K39Q, K62N/K63D, K37S/K38Q/K39Q/K62N/K63D, R74Q), whereas one mutant was created with the intention of increasing the heparin affinity (E60aS/S61R). Since the target amino acid residues are solvent-exposed on loop structures and naturally occurring in related serine proteases, it should be expected that the above amino acid substitutions will not alter the folding of APC or damage its catalytic machinery. Furthermore, because the newly introduced residues are similar in size to the original ones, these mutations can be considered as conservative, and they should be better tolerated in the structure than, for instance, small hydrophobic alanine substitutions. Clustering of three to five alanine residues could create a destabilizing solvent-exposed hydrophobic patch no longer able to form hydrogen bonds with water molecules or could induce misfolding due to the high helical propensity of this amino acid. The various protein C cDNA variants were used to transfect the eukaryotic cell line HU293, and stable cell lines were established. The different protein C variants demonstrated similar expression levels (2.7 to 6.7 mg/liter). The proteins were purified with overall recoveries of 35–40%, and the isolated proteins were more than 90% pure, as estimated by SDS-PAGE. On SDS-PAGE, the protein C variants migrated at similar positions as WT protein C (Fig.2). Under nonreducing conditions, the different protein C variants migrated as single bands. After reduction of disulfide bridges, the 41-kDa heavy chains and the 21-kDa light chains were observed in addition to the 62-kDa single chain forms of protein C. Under reducing conditions, for all recombinant proteins a small amount of single chain protein C remained. After activation, the different recombinant protein variants demonstrated similar amidolytic activities (Km and kcat) (Table I). The equally high expression levels observed for all the different protein C variants and their full amidolytic activities after activation together with the above structural considerations suggest that the recombinant proteins were correctly folded and could be used for further functional characterizations.Table ISteady-state kinetic analysis of chromogenic substrate S-2366 hydrolysis by WT APC and the APC variantsEnzymeKmkcatkcat/Kmμms−1μm−1s−1Wild type0.91 ± 0.0270 ± 176.9K62N/K63D0.95 ± 0.0178 ± 582.1K37S/K38Q/K39Q0.98 ± 0.0475 ± 776.5K37S/K38Q/K39Q/K62N/K63D0.93 ± 0.0171 ± 176.3E60aS/S61R0.93 ± 0.0870 ± 675.3R74Q0.90 ± 0.0276 ± 884.4 Open table in a new tab Binding of WT APC and APC variants to immobilized heparin was analyzed with surface plasmon resonance using a BIAcore 2000 (TableII). At physiological NaCl concentration, the dissociation constant (Kdobs) of WT APC for heparin was 0.32 μm. The R74Q variant showed slightly lower affinity (Kdobs = 0.54 μm), whereas E60aS/S61R bound more tightly (Kdobs = 0.19 μm) to heparin than WT APC (Table II, Fig. 3). There was no detectable binding of K37S/K38Q/K39Q, K62N/K63D, and K37S/K38Q/K39Q/K62N/K63D to immobilized heparin molecules. To qualitatively confirm the results obtained by BIAcore, WT APC and the five APC variants were applied on heparin-Sepharose, and bound protein was eluted with a linear NaCl gradient. The results agreed well with those obtained by the BIAcore analysis. Thus, removal of the positively charged residues at positions 37/38/39 or 62/63 or 37/38/39/62/63 yielded diminished or undetectable binding to the heparin-Sepharose (results not shown). In contrast, the E60aS/S61R variant bound tighter to the heparin column than WT APC and was eluted at higher NaCl concentrations. These results strongly suggest that the heparin-binding site of APC is entirely dependent on the presence of a few positively charged residues on loops 37 and 60 (Fig.4).Table IIAffinity and rate constants for interaction of APC with heparinAPCKd obskonkoffμmm−1s−1s−1Wild type0.322.6 × 1048.4 × 10−3[NaCl] = 150 mmΔG = −8.8 (kcal/mol)R74Q0.546.1 × 1033.3 × 10−3[NaCl] = 150 mmΔG = −8.5 (kcal/mol)E60aS/S61R0.197.8 × 1031.5 × 10−3[NaCl] = 150 mmΔG = −9.1 (kcal/mol)The binding of APC to immobilized heparin was measured with surface plasmon resonance, and BIAevaluation 3.0 software was used to calculate association (kon) and dissociation (koff) rate constants from the sensograms. APC variants K37S/K38Q/K39Q, K62N/K63D, and K37S/K38Q/K39Q/K62N/K63D demonstrated no binding to the heparin-coated SA-chip. Therefore, nokon and koff rate constants could be calculated for these APC variants. Open table in a new tab Figure 4Electrostatic surface potential of APC. A model for heparin containing 7 monosaccharide units was aligned onto APC loops 37 and 60. Electrostatic potential isosurfaces are shown at +1 (blue) and −1 (red) kcal/mol/e for WT and variant APC molecules and at −4 (magenta) kcal/mol/e for heparin. The electropositive characters of loops 37 and 60 in WT, E60aS/S61R, and R74Q are consistent with normal or enhanced binding of heparin. This energy field is partially or fully destroyed in variants K37S/K38Q/K39Q, K62N/K63D, and K37S/K38Q/K39Q/K62N/K63D, which bind heparin weakly or not at all.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The binding of APC to immobilized heparin was measured with surface plasmon resonance, and BIAevaluation 3.0 software was used to calculate association (kon) and dissociation (koff) rate constants from the sensograms. APC variants K37S/K38Q/K39Q, K62N/K63D, and K37S/K38Q/K39Q/K62N/K63D demonstrated no binding to the heparin-coated SA-chip. Therefore, nokon and koff rate constants could be calculated for these APC variants. Heparin is derived from mast cells, and it is unlikely that APC interacts with heparin under normal physiological conditions if heparin is not administered as a therapeutic. Heparan sulfate present on the surface of endothelial cells is the glycosaminoglycan that is more likely to interact with circulating APC. For this reason, we tested the binding of APC to immobilized heparan sulfate in the BIAcore under similar conditions as for heparin. In contrast to the clear binding curves observed with heparin, no binding of APC to heparan sulfate could be detected. In this context, it should be considered that BIAcore is only able to detect interactions characterized by relatively high association rate constants. Therefore, many physiologically relevant interactions cannot be detected by this method. To further investigate whether heparan sulfate was able to interact with APC, 0.5 mg/ml fluid phase heparan sulfate was co-injected with APC in the flow cell containing immobilized heparin. Under these conditions, complete inhibition of binding of APC to heparin was observed, suggesting that APC may interact with heparan sulfate, albeit weaker than with heparin. BIAcore was used to determine the kinetic characteristics of the binding of WT APC to heparin at various salt concentrations, the purpose being to gain insights into the relative contributions of ionic versus nonionic components to the interaction (Table III). The results were evaluated using the counter-ion condensation theory, assuming that heparin behaves as a polyelectrolyte surrounded by a counter-ion condensation volume (29Record Jr., M.T. Anderson C.F. Lohman T.M. Q. Rev. Biophys. 1978; 11: 103-178Crossref PubMed Scopus (1532) Google Scholar, 30Manning G.S. Q. Rev. Biophys. 1978; 11: 179-246Crossref PubMed Scopus (2630) Google Scholar). Counter-ions (e.g.Na+) can interact with heparin by delocalized, long range electrostatic interactions and by direct binding to ionic sites. Displacement of counter-ions upon heparin binding to protein has been experimentally demonstrated (31Hari S.P. McAllister H. Chuang W.L. Christ M.D. Rabenstein D.L. Biochemistry. 2000; 39: 3763-3773Crossref PubMed Scopus (18) Google Scholar). In cases where a plot of logKdobs against log [salt] is linear, the magnitude of the slope corresponds to the effective number of heparin counter-ions released upon its binding to a protein. This approach has been used successfully to study many interactions such as the ones between heparin and thrombin or bFGF (11Olson S.T. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar, 32Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (277) Google Scholar). The bFGF predictions were remarkable, since it was reported that ∼30% of the binding energy between bFGF and heparin resulted from purely ionic interactions. In this case, the plot of logKdobs against log [salt] was linear (slope = 1.95), and it was estimated that only 2–3 net ionic interactions were involved between the two molecules. This result was fully supported at a later stage by x-ray analysis of the bFGF-heparin complex (33Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (736) Google Scholar).Table IIIInfluence of [NaCl] on the Kd of APC-heparin complex formationAPCKd obskonkoffμmm−1s−1s−1Wild type0.0755.7 × 1044.3 × 10−3[NaCl] = 80 mmΔG = −9.6 (kcal/mol)Wild type0.322.6 × 1048.4 × 10−3[NaCl] = 150 mmΔG = −8.8 (kcal/mol)Wild type1.90.6 × 10411.6 × 10−3[NaCl] = 200 mmΔG = −7.7 (kcal/mol)Wild type470.1 × 10447.7 × 10−3[NaCl] = 300 mmΔG = −6 (kcal/mol)Under various NaCl concentrations, binding of 5–150 μg/ml WT APC to immobilized heparin was measured with surface plasmon resonance.kon and koff rate constants were calculated by global fitting of the BIAcore sensograms in BIAevaluation 3.0. Open table in a new tab Under various NaCl concentrations, binding of 5–150 μg/ml WT APC to immobilized heparin was measured with surface plasmon resonance.kon and koff rate constants were calculated by global fitting of the BIAcore sensograms in BIAevaluation 3.0. To evaluate the APC-heparin interaction, the following scheme was used. The observed Kd(Kdobs) is related to the nonionic equilibrium dissociation constant (Kdnonionic) through the relationship log Kdobs = logKdnonionic + ψZ[Na+]. The plot of logKdobs versus log [Na+] is linear, and the value of Z can be calculated from the slope. The formation of Z electrostatic interactions between WT APC and heparin is accompanied by the displacement of ΨZ-condensed counter-ions from the heparin. Ψ is the effective fraction of counter-ion bound per heparin negative charge, which has been estimated to be 0.8 (11Olson S.T. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar). The slope (0.8 × Z = 4.86) provides the effective number of purely ionic interactions formed between WT APC and heparin. Thus, about 4–6 ionic interactions seem to be involved in this process. At 1.0 m NaCl, ionic interactions were neutralized, and logKdobs = log Kdnonionic, which we estimate to be 5600 μm. Because the Kd for the APC-heparin interaction at physiological ionic strength was 0.32 μm, 66% of the binding free energy (ΔG) was estimated to be ionic, with the remaining 34% nonionic. The ΔG of WT APC binding to heparin at physiological salt concentration (150 mm) was estimated to be −8.8 kcal/mol, with the nonionic and ionic contributions to this interaction −3 kcal/mol and −5.7 kcal/mol, respectively. Thus, the WT APC binding to heparin is mainly electrostatic in nature just like the binding of heparin to thrombin (Kdobs = 6–10 μm, about 80% ionic) (11Olson S.T. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar) or to mucus proteinase inhibitor (Kdobs = 0.05 μm; >80% ionic) (34Faller B. Mely Y. Gerard D. Bieth J.G. Biochemistry. 1992; 31: 8285-8290Crossref PubMed Scopus (69) Google Scholar). There is a considerable effect of salt on Kdobsfor the APC-heparin interaction (Table III), and the corresponding ΔG values run from −9.6 kcal/mol at 80 mmNaCl to −6 kcal/mol at 300 mm NaCl (the Kdobs increases 626-fold). Salt influences mostlykon, whereas the koff is not dramatically affected, suggesting that long range electrostatic interactions are important for the formation of an initial encounter complex. Once salt bridges are formed, NaCl does not significantly weaken the short range ionic interactions. We note also that although the APC-heparin interaction is mainly electrostatic, the rate of association is well below the maximum rate for collision of molecules in solution, which is around 109—1010m−1 s−1(35Smoluchowski M.V. Z. Phys. Chem. 1918; 92: 129-168Crossref Google Scholar). During inhibition of APC by PCI, heparin or heparan sulfate may function as templates to guide PCI into the active site of APC during the formation of the Michaelis-like complex. Assuming the identified heparin/heparan sulfate-binding site on APC to be functionally important, the recombinant APC modifications are expected to affect the heparin/heparan sulfate stimulation of inhibition by PCI. This was indeed found to be the case. Heparin (at 1 IU/ml, equal to 6.6 μg/ml) accelerated the rate of PCI-mediated inhibition of WT APC 800-fold, whereas heparin only yielded an 11-fold increase in the rate of inhibition of the K37S/K38Q/K39Q/K62N/K63D variant (Table IV). The E60aS/S61R variant, which bound heparin with higher affinity than WT APC, demonstrated even higher stimulation of inhibition by heparin (11,000-fold). Heparan sulfate (at 50 μg/ml) was tested in the same experimental setup and was found to give a 350-fold increased rate of inhibition of WT APC and a 2200-fold increased inhibition of the E60aS/S61R variant but only a 40-fold increased rate of inhibition of K62N/K63D and a 6-fold increased rate of inhibition of the K37S/K38Q/K39Q/K62N/K63D variant (results not shown) . These results demonstrate that heparan sulfate is able to interact with the same site as heparin on APC and that heparin and heparan sulfate yield similar functional effects on the rate of inhibition by PCI. However, based on both BIAcore results and the PCI inhibition data, heparin appears to bind APC with higher affinity than does heparan sulfate.Table IVSecond-order rate constants for PCI-mediated inhibition of APC variants in the presence and absence of heparinAPCAbsence of heparin, k2Presence of heparin, k2Acceleration of APC inhibition× 102m−1 s−1× 106m−1 s−1Wild type8.7 ± 0.10.7 ± 0.1805R74Q7.4 ± 0.90.4 ± 0.01541E60aS/S61R7.9 ± 1.58.7 ± 0.511,012K62N/K63D46.5 ± 0.20.4 ± 0.186K37S/K38Q/K39Q16.1 ± 0.10.2 ± 0.2124K62N/K63D/K37S/K38Q/K39Q175 ± 0.10.2 ± 0.111 Open table in a new tab In the absence of heparin/heparan sulfate, E60aS/S61R and R74Q variants were inhibited by PCI equally fast as WT APC, whereas the rate of PCI inhibition was increased for the K37S/K38Q/K39Q, K62N/K63D, and K37S/K38Q/K39Q/K62N/K63D variants (Fig.5 A). The biggest effect was shown for the variant K37S/K38Q/K39Q/K62N/K63D. Already after 10 min more than 90% of the variant was inhibited, whereas WT APC activity was reduced only by 10% (Fig. 5 A). This suggests that the lysine cluster in loops 37 and 60 repulses PCI in the absence of heparin. The poor heparin-mediated stimulation of PCI inhibition of the K37S/K38Q/K39Q, K62N/K63D and K37S/K38Q/K39Q/K62N/K63D (Fig.5 B) is in agreement with the bridging theory involving heparin or heparan sulfate binding to the lysine cluster of APC as well as to PCI. In line with this theory, heparin and heparan sulfate were found to provide increased stimulation to the PCI inhibition of the E60aS/S61R variant, which demonstrated increased affinity for heparin (Fig. 5 B). It is noteworthy that the now demonstrated heparin/heparan sulfate-binding site in APC is located on another part of the protease domain as compared with the heparin binding exosite II of thrombin (Fig. 1). Similarly, PCI binds heparin on the other side (around helix H) as compared with the heparin binding D-helix of antithrombin (10Neese L.L. Wolfe C.A. Church F.C. Arch. Biochem. Biophys. 1998; 355: 101-108Crossref PubMed Scopus (33) Google Scholar,36Sheehan J.P. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5518-5522Crossref PubMed Scopus (181) Google Scholar, 37Ersdal-Badju E. Lu A. Zuo Y. Picard V. Bock S.C. J. Biol. Chem. 1997; 272: 19393-19400Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Therefore, the helix H area of PCI should be the topological neighbor of APC loops 37 and 60 in the Michaelis-like complex. In conclusion, we have characterized the structural and energetic basis of a functionally important heparin-binding site in the serine protease domain of APC. This binding site is functionally important in the heparin/heparan sulfate-stimulated inhibition of APC by its serpin inhibitor PCI. We thank Astra Andersson for technical assistance, Prof. Johan Stenflo for the kind gift of purified PCI, Dr. Markku Salmivirta for providing the biotinylated heparin, and Dr. Sassan Hafizi for language revision." @default.
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