Role of Zymogen and Activated Factor X as Scaffolds for the Inhibition of the Blood Coagulation Factor VIIa-Tissue Factor Complex by Recombinant Nematode Anticoagulant Protein c2
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m009116200
ISSN1083-351X
AutoresPeter W. Bergum, A. M. Cruikshank, Steven L. Maki, Curtis R. Kelly, Wolfram Ruf, George P. Vlasuk,
Tópico(s)Blood properties and coagulation
ResumoRecombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa·TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa·TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (KD = 1–10 nm), and similarly supported the inhibition of fVIIa·TF (Ki* = ∼10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa·TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TFS), or reconstituted into membranes, containing phosphatidylcholine (TFPC) or a mixture of phosphatidylcholine and phosphatidylserine (TFPCPS). In the absence of the fX derivative, inhibition of fVIIa·TF was similar for all three conditions (Ki∼1 μm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TFS, TFPC, and TFPCPS. The removal of the γ-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa·TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa·TF·fX(a) complex assembled on an appropriate phospholipid surface. Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa·TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa·TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (KD = 1–10 nm), and similarly supported the inhibition of fVIIa·TF (Ki* = ∼10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa·TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TFS), or reconstituted into membranes, containing phosphatidylcholine (TFPC) or a mixture of phosphatidylcholine and phosphatidylserine (TFPCPS). In the absence of the fX derivative, inhibition of fVIIa·TF was similar for all three conditions (Ki∼1 μm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TFS, TFPC, and TFPCPS. The removal of the γ-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa·TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa·TF·fX(a) complex assembled on an appropriate phospholipid surface. factor VIIa l-glutamyl-l-glycyl-l-arginyl chloromethyl ketone-modified factor Xa γ-carboxyglutamic acid-containing domain l-glutamyl-l-glycyl-l-arginyl chloromethyl ketone-modified factor Xa lacking the γ-carboxyglutamic acid-containing domain l-α-palmitoyloleoyl phosphatidylcholine l-α-stearoyloleoyl phosphatidylserine phospholipid vesicle 75% (w/w) phosphatidylcholine, 25% (w/w) phosphatidylserine vesicles factor X factor Xa recombinant human factor X recombinant human factor X containing the site-directed mutation of S195A recombinant human factor X containing site-directed mutations S195A and R15Q complex of factor VIIa and tissue factor recombinant nematode anticoagulant protein c2 rNAPc2 with an additional 8-amino acid sequence (DYKDDDDK) at the NH2terminus recombinant tick anticoagulant peptide human full-length tissue factor detergent-solubilized tissue factor tissue factor reconstituted into 100% (w/w) phosphatidylcholine vesicles tissue factor reconstituted into 75% (w/w) phosphatidylcholine, 25% (w/w) phosphatidylserine vesicles human tissue factor pathway inhibitor high performance liquid chromatography 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid bovine serum albumin phosphate-buffered saline monoclonal antibody polyacrylamide gel electrophoresis enzyme-linked immunosorbent assay The blood coagulation response to vascular injury or inflammation results from a series of amplified reactions, in which several specific zymogens of serine proteases in plasma are sequentially activated by limited proteolysis (1Mann K.G. Thromb. Haemostasis. 1999; 82: 165-174Crossref PubMed Scopus (416) Google Scholar). The serine protease factor VIIa (fVIIa)1 present in the blood specifically binds to tissue factor (TF), a transmembrane receptor glycoprotein bound to subendothelial structures or present on the surface of monocytic or other inflammatory cells, which accumulate at the site of injury (2Martin D.M.A. Wiiger T. Prydz H. Thromb. Res. 1998; 90: 1-25Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The exposure of TF to circulating blood is the triggering event that results in the formation of a catalytic complex (fVIIa·TF) that initiates the amplified cascade of proteolytic events leading to the formation of the serine protease thrombin (3Rapaport S.I. Rao V.M. Arterioscler. Thromb. 1992; 12: 1111-1121Crossref PubMed Scopus (272) Google Scholar). The action of thrombin coupled with the particular rheological environment found in diseased or damaged vascular beds, result in thrombi with compositions that vary from platelet-rich, a characteristic of the arterial vasculature, to fibrin-rich, platelet-poor clots, typical of the venous vasculature (4Hirsh J. Weitz J.I. Semin. Hematol. 1999; 36: 118-132PubMed Google Scholar). The pathway leading from the formation of the fVIIa·TF complex to thrombin proceeds through the serine protease factor Xa (fXa). Factor Xa is formed by the proteolytic activation of the zymogen factor X (fX) either by the fVIIa·TF complex or by the catalytic complex composed of the serine protease factor IXa and its nonenzymatic cofactor factor VIIIa assembled on an appropriate phospholipid surface (5Jenny N.S. Mann K.G. Thrombosis and Hemorrhage. Williams and Wilkins, Baltimore, MD1998: 3-27Google Scholar). Factor Xa catalyzes the formation of thrombin following assembly into a macromolecular catalytic complex (prothrombinase) with the nonenzymatic cofactor factor Va (fVa) that binds to a procoagulant phospholipid surface, such as activated platelets or inflammatory cells adhered to the site of vascular damage (6Mann K.G. Krishnaswamy S. Lawson J.H. Semin. Hematol. 1992; 29: 213-226PubMed Google Scholar). The regulation of the blood coagulation involves a variety of components, most of which act to down-regulate the proteolytic response initiated following vascular injury. The primary physiological inhibitor of the fVIIa·TF complex, tissue factor pathway inhibitor (TFPI), mediates one of these crucial pathways (7Broze Jr., G.J. Annu. Rev. Med. 1995; 46: 103-112Crossref PubMed Scopus (274) Google Scholar). The efficient inhibition of fVIIa·TF by TFPI requires binding of the inhibitor to the active site of fXa via the second of its three Kunitz-like inhibitory domains followed by the formation of the final quaternary inhibitory complex with fVIIa·TF, in which the active site of fVIIa is occupied by the first Kunitz domain of the inhibitor (8Girard T.J. Warren L.A. Novotny W.F. Likert K.M. Brown S.G. Miletich J.P. Broze Jr., G.J. Nature. 1989; 338: 518-520Crossref PubMed Scopus (431) Google Scholar). A recent study suggested that the rate-limiting step governing the inhibition of fVIIa·TF by TFPI is the binding to fXa, which occurs while fXa is either bound to or remains in the near vicinity of the fVIIa·TF complex following zymogen cleavage of fX (9Baugh R.J. Broze Jr., G.J. Krishnaswamy S. J. Biol. Chem. 1998; 273: 4378-4386Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Therefore, it appears that the role of fXa in the inhibition of fVIIa·TF by TFPI is that of an "inhibitory scaffold,ȁ upon which is built the final inhibitory complex between the Kunitz-1 domain of TFPI and the active site of fVIIa. This proposed mechanism requires the ternary fVIIa·TF·fXa complex to display a limited half-life or stability. This originates, in part, from specific protein-protein interactions outside the catalytic center of fVIIa at exo-sites on fVIIa·TF, to which the substrate fX or product fXa binds (10Shobe J. Dickinson C.D. Edgington T.S. Ruf W. J. Biol. Chem. 1999; 274: 24171-24175Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 11Baugh R.J. Dickinson C.D. Ruf W. Krishnaswamy S. J. Biol. Chem. 2000; 275: 28826-28833Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Previously, we described a potent 84-amino acid, non-Kunitz-like inhibitor of the fVIIa·TF complex called nematode anticoagulant protein c2 (NAPc2) that was originally isolated from the hematophagous nematode hookworm Ancylostoma caninum (12Stanssens P. Bergum P.W. Gansemans Y. Jespers L. Laroche Y. Huang S. Maki S.L. Messens J. Lauwereys M. Cappello M. Hotez P.J. Lasters I. Vlasuk G.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2149-2154Crossref PubMed Scopus (236) Google Scholar). An 85-amino acid recombinant form of NAPc2 (rNAPc2) was shown to significantly inhibit fVIIa·TF-mediated factor IX activation, but only in the presence of fXa, or fXa that had been irreversibly inhibited with the active-site inhibitor Glu-Gly-Arg-chloromethylketone (EGR-fXa). The effectiveness of EGR-fXa as an inhibitory scaffold suggested that rNAPc2 bound to a region of fXa outside of the catalytic center. The utilization of such an exo-site by rNAPc2 distinguishes it from TFPI, which has been shown to require an unoccupied active site in fXa to allow the binding of the Kunitz 2 domain of the inhibitor (7Broze Jr., G.J. Annu. Rev. Med. 1995; 46: 103-112Crossref PubMed Scopus (274) Google Scholar). Therefore, we propose that, although rNAPc2 is functionally similar to TFPI with respect to the requirement of an inhibitory scaffold to mediate its inhibition of fVIIa·TF, it is mechanistically distinct based on the specific binding interaction with fXa. In this report, we characterize the interaction of rNAPc2 with fX derivatives that support inhibition of fVIIa·TF. We demonstrate that rNAPc2 can bind with high affinity to zymogen fX, indicating that the activation status of the inhibitory scaffold is not crucial for the inhibition of fVIIa·TF by rNAPc2. The phospholipid membrane composition and the Gla-domain of the fX/fXa play critical roles in the presentation of rNAPc2 that we then show interacts directly with the active site of fVIIa via a reactive site sequence. Together, these data support a mechanism of fVIIa·TF inhibition by rNAPc2, which utilizes an exo-site of either the product of this catalytic complex, fXa, or more uniquely, the substrate zymogen fX to form the final quaternary inhibited complex. Hepes and Tris buffers, bovine serum albumin (BSA), CHAPS, Tween 20 and all other reagents, not indicated otherwise, were from Sigma. Recombinant human factor VIIa (fVIIa) was obtained from Novo Nordisc A/S (Gentofte, Denmark). Recombinant, human full-length tissue factor (TF) was produced using a baculovirus expression system as described (13Ruf W. Biochemistry. 1994; 33: 11631-11636Crossref PubMed Scopus (51) Google Scholar). Purified human proteins factor X (fX), factor IX (fIX), glutamylglycylarginyl chloromethyl ketone (EGR-ck) modified factor Xa (EGR-fXa), and des-Gla-domain fXa, modified with EGR-ck (des-Gla-EGR-fXa) were obtained from Hematologic Technologies (Essex Junction, VT). These proteins were further purified by immunodepletion of residual fVII. Similarly, residual fX was removed by immunodepletion from the fIX preparations. The contaminating activities of the trace fVII and fX in the fIX preparations were individually assessed in a quantitative assay for the activation of fX by fVIIa·TFPCPS and estimated at ≤0.001% and ≤0.0003%, respectively. The functional, clotting-specific activity of the purified fIX was measured as 305 units/mg in a clotting assay, using fIX deficient plasma (George King, Overland Park, KS). Each preparation of a fXa derivative contained at least 85% of the α-species. Purified human factor Xa (fXa) was prepared from fX as described previously (14Bock P.E. Craig P.A. Olson S.T. Singh P. Arch. Biochem. Biophys. 1989; 273: 375-388Crossref PubMed Scopus (83) Google Scholar). Recombinant tick anticoagulant peptide (rTAP), rNAPc2, and FLAG-rNAPc2 were expressed in the methylotropic yeast Pichia pastorisand purified to homogeneity as previously described (12Stanssens P. Bergum P.W. Gansemans Y. Jespers L. Laroche Y. Huang S. Maki S.L. Messens J. Lauwereys M. Cappello M. Hotez P.J. Lasters I. Vlasuk G.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2149-2154Crossref PubMed Scopus (236) Google Scholar). FLAG-rNAPc2 contained the 8-amino acid peptidic sequence (DYKDDDDK) at the NH2 terminus of rNAPc2. The molecular mass of the recombinant proteins was confirmed by electrospray mass spectrometry, and protein concentrations determined by quantitative amino acid analysis. Mutagenesis, expression in transfected dihydrofolate reductase-deficient Chinese hamster ovary cells, and purification of recombinant factor X (rfX) derivatives and mutants has been described elsewhere (10Shobe J. Dickinson C.D. Edgington T.S. Ruf W. J. Biol. Chem. 1999; 274: 24171-24175Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Briefly, Ser195 (chymotrypsin numbering; Ref. 16Bode W. Mayr I. Baumann U. Huber R. Hofsteenge J. EMBO J. 1989; 8: 3417-3475Crossref Scopus (823) Google Scholar) of the catalytic triad of rfX was mutated to Ala (rfXS195A) to eliminate proteolytic and amidolytic activities of the resulting rfXaS195A. Activation of the recombinant fX derivative was accomplished using Russel's viper venom, followed by purification by gel permeation chromatography. To generate a fX analog that was resistant to scissile bond cleavage by fVIIa·TF, the P1 residue 2The residue nomenclature of Schecter and Berger is used (41Schecter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4783) Google Scholar).Arg15 of the activation peptide of rfXS195A was also mutated to Gln, yielding the double mutant rfXR15Q/S195A. All protein preparations were judged homogeneous (>95%), following analysis by SDS-PAGE and staining with Coomassie Brilliant Blue (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207523) Google Scholar). The concentration of human fX and fXa were determined using an extinction coefficient at A280( E2801%) of 11.6 (18Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar). The synthetic phospholipids l-α-palmitoyloleoyl phosphatidylcholine (PC) and l-α-stearoyloleoyl phosphatidylserine (PS) were obtained from Avanti Polar Lipids (Alabaster, AL). Full-length TF apoprotein was reconstituted into phosholipid vesicles (PLV), consisting of 75% PC (w/w) and 25% PS (w/w) (TFPCPS), or 100% PC (TFPC) in the presence of detergent, as previously described (19Ruf W. Miles D.J. Rehemtulla A. Edgington T.S. Methods Enzymol. 1993; 222: 209-224Crossref PubMed Scopus (13) Google Scholar), followed by dialysis into 10 mm Hepes, 150 mm NaCl, pH 6.5. The TFPC was used immediately following dialysis. The diameter of the resulting vesicles was measured by light scattering (Fine Particle Technology, Menlo Park, CA), yielding a volume-weighted Gaussian distribution centered at a mean diameter of 83 ± 20 nm (PCPS PLV) and 112 ± 20 nm (PC PLV). The concentration of phospholipids in each preparation was determined by a colorimetric assay for inorganic phosphorus, adapted from Ref. 9Baugh R.J. Broze Jr., G.J. Krishnaswamy S. J. Biol. Chem. 1998; 273: 4378-4386Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar. The p-nitroanilide-containing chromogenic peptidyl substrate C-2081 fVIIa was synthesized as a trifluoroacetic acid salt, purified to homogeneity using HPLC, and lyophilized. The resulting molecular mass was confirmed by electrospray mass spectrometry. The C-2081 substrate was reconstituted in deionized water just prior to use. The kinetic measurement of fVIIa·TF-mediated release of tritiated activation peptide from radiolabeled fIX ([3H]fIX) was performed as described. (12Stanssens P. Bergum P.W. Gansemans Y. Jespers L. Laroche Y. Huang S. Maki S.L. Messens J. Lauwereys M. Cappello M. Hotez P.J. Lasters I. Vlasuk G.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2149-2154Crossref PubMed Scopus (236) Google Scholar). Briefly, a complex of fVIIa and TFPCPS was formed in 10 mmHepes, 150 mm NaCl, 15 μm BSA, 3.0 mm CaCl2, pH 7.4 (designated assay buffer), for 10 min prior to adding increasing amounts of equimolar rNAPc2 and one of the following fX derivatives: fX, recombinant fX mutants, fXa, EGR-fXa, and a complex of fXa and rTAP (rTAP-fXa), which was formed 45 min prior to adding to the fVIIa·TF complex. Following a 30-min incubation, the reaction was initiated by the addition of the [3H]fIX, and initial velocities were measured over 10 min, as described previously (12Stanssens P. Bergum P.W. Gansemans Y. Jespers L. Laroche Y. Huang S. Maki S.L. Messens J. Lauwereys M. Cappello M. Hotez P.J. Lasters I. Vlasuk G.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2149-2154Crossref PubMed Scopus (236) Google Scholar). The final concentration of reactants in a total volume of 420 μl of assay buffer was: fVIIa (50 pm), TF (2.7 nm), PCPS PLV (6.4 μm), equimolar rNAPc2 and fX derivative (0–1 nm), and [3H]fIX (200 nm, ∼5 × Km), and, when included, rTAP (10 nm). The fX derivatives (0–1 nm) in the absence of rNAPc2 had no effect on the velocity (V0). The ratio of the inhibited reaction velocity (Vi) to the respective uninhibited velocity (V0) (without rNAPc2) was determined for each concentration of fX derivative-rNAPc2 for three to six separate experiments. These data were fit by reiterative nonlinear regression to the quadratic Equation 1 for slow, tight-binding inhibitors (20Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (736) Google Scholar, 21Morrison J.F. Trends Biochem. Sci. 1982; 7: 102-105Abstract Full Text PDF Scopus (497) Google Scholar) to give the overall equilibrium constant (Ki*), as described previously (12Stanssens P. Bergum P.W. Gansemans Y. Jespers L. Laroche Y. Huang S. Maki S.L. Messens J. Lauwereys M. Cappello M. Hotez P.J. Lasters I. Vlasuk G.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2149-2154Crossref PubMed Scopus (236) Google Scholar, 22Krishnaswamy S. Vlasuk G.P. Bergum P.W. Biochemistry. 1994; 33: 7897-7907Crossref PubMed Scopus (56) Google Scholar). Vi/Vo={(Et−It−Ki*)+[(It+Ki*−Et)2+4Ki*Et]1/2}/2EtEquation 1 All studies were performed in assay buffer at ambient temperature (23.5 ± 0.7 °C). The kinetics of hydrolysis of the chromogenic substrate C-2081 by fVIIa were measured under a number of experimental conditions, prior to examining the inhibitory effects of rNAPc2 under these conditions. Reactions were initiated by the addition of uncomplexed fVIIa (10 nm) or fVIIa (2 nm) in complex with 5 nm TF (TFS, TFPC, TFPCPS) to the individual wells of a 96-well plate (Corning), containing C-2081 (0.05–3.0 mm) in a final volume of 125 μl of assay buffer. Where indicated other reagents were added to the following final concentrations: PCPS or PC PLV (6.8 μm), EGR-fXa (20 nm with free fVIIa, or 5 nm with fVIIa·TF). Factor VIIa and TF were incubated for 10 min prior to the addition to the reaction mixture. Initial reaction velocities were measured as a linear increase in the absorbance at 405 nm (A405 nm) over 10 min at 9-s intervals, using a Thermomax kinetic microplate reader. Measurements were made under steady-state conditions, where less than 5% of the substrate was consumed. The Km was derived from the nonlinear regression fit of the averaged velocities of triplicate reactionsversus the respective concentration of C-2081, using Enzfitter software (Biosoft, Cambridge, United Kingdom). Kinetic values were averaged from three independent kinetic determinations, generating the following Km values for each experimental condition: uncomplexed fVIIa (2.6 mm), uncomplexed fVIIa + EGR-fXa (1.8 mm), fVIIa·TF (all TF preparations) (range: 262–356 μm), fVIIa·TF (all TF preparations) + EGR-fXa (range: 272–345 μm). The effect of incubation time on the inhibition of fVIIa and fVIIa·TF amidolytic activity by rNAPc2 varied depending on the particular reaction condition used. The extent to which the inhibition for the eight tested reaction conditions was either kinetically "fastȁ or time-independent, or kinetically "slowȁ or time-dependent determined how the corresponding dissociation constant (Ki orKi* ) was determined (23Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar). The relative inhibitory potency of rNAPc2 was measured over a range of concentrations for each reaction condition by two kinetic procedures, using identical concentrations of reactants: 1) fVIIa was added to a mixture of rNAPc2 and substrate to initiate reactions (no pre-incubation between enzyme and rNAPc2), and 2) rNAPc2 was first pre-incubated with fVIIa for 30 min (inhibition in the absence of substrate), followed by addition of substrate to initiate reactions. If the apparent potency of rNAPc2, measured by each of these two procedures was equivalent, then the interaction of rNAPc2 was judged time-independent or fast. The Ki was measured in subsequent experiments, as detailed below for time-independent inhibition. In contrast, if the apparent potency of rNAPc2 measured by procedure 2 was significantly greater than that measured for procedure 1, i.e. determined to be time-dependent or slow,Ki* was subsequently measured as detailed below for time-dependent inhibition. The potency of rNAPc2 was measured over a range of substrate concentrations in the presence of increasing concentrations of rNAPc2, and when included, of equimolar EGR-fXa or des-Gla EGR-fXa. Reactions were initiated by the addition of either free fVIIa (10 nm), or preformed fVIIa·TF complex (2 nm fVIIa with 5 nm TFS, TFPSPC, or TFPC) to premixed inhibitor and substrate in the wells of microtiter plates. All reactions were performed in triplicate, and contained in 125 μl of assay buffer, containing a range of six to eight inhibitor concentrations [I] and six substrate concentrations [S]. For reactions with free fVIIa, the concentrations were 1–20 μm ([I]) and 0.1–3.0 mm ([S]), and those with fVIIa·TF complexes were 0.5–10 μm ([I]) and 0.1–2.0 mm([S]). The initial velocities measured over 10 min under steady-state conditions for three separate experiments were fit by reiterative nonlinear regression to Equation 2, describing a time-independent, classical, reversible competitive inhibitor, to derive theKi value. V=Vmax/1+(Km/[S])(1+[I]/Ki)Equation 2 Varying concentrations of rNAPc2 and equimolar EGR-fXa (0.00025–1 μm) were pre-incubated with a complex of fVIIa (2 nm) and TFS, TFPC, or TFPSPC (5 nm) for 30 min. Initial velocities were measured, following the addition of substrate (650 μm). Preliminary experiments showed that EGR-Xa in the absence of rNAPc2 had no effect on V0. Ratios of the inhibited reaction velocity (Vi) to the uninhibited velocity (V0) for each concentration of rNAPc2 were fit to the quadratic Equation 1 for slow, tight-binding inhibitors for three separate experiments to give the apparent dissociation constant (Ki*). The rate constants for inhibition of fVIIa·TF by rNAPc2 for each reconstituted TF condition were measured as previously described (25Cha S. Biochem. Pharmacol. 1975; 24: 2177-2185Crossref PubMed Scopus (455) Google Scholar). Briefly, the chromogenic substrate C-2081 (800 μm) was added to the wells of a microtiter plate, containing a range of concentrations of rNAPc2 and equimolar EGR-fXa (0–100 nm) in assay buffer. The reactions were initiated by the addition of a preformed complex of fVIIa (2 nm) and TFS, TFPC, or TFPSPC (5 nm). Progress curves generated over 60 min were analyzed using Equation 3 as described by Cha (25Cha S. Biochem. Pharmacol. 1975; 24: 2177-2185Crossref PubMed Scopus (455) Google Scholar) and Williams (26Williams J.W. Morrison J.F. Methods Enzymol. 1979; 63: 437-467Crossref PubMed Scopus (663) Google Scholar) and detailed in ref. 24Håkansson K. Tulinsky A. Ableman M.M. Miller T.A. Vlasuk G.P. Bergum P.W. Lim-Wilby M.S.L. Brunck T.K. Biorg. Med. Chem. 1995; 3: 1009-1017Crossref PubMed Scopus (23) Google Scholar, where P is the measured absorbance defined as a function of initial (Vo) and final (Vs) steady state velocities and the apparent first-order rate constant,kobs, which describes the equilibrium from the initial to the final state. P=Vst+(Vo−Vs)(1−e−kobst)/kobsEquation 3 The derived, apparent first-order rate constants(kobs), from three separate experiments derived using Equation 3, were fit along with the corresponding (rNAPc2·EGR-fXa) by re-iterative nonlinear regression to Equation 4, which describes a one-step mechanism for slow-binding inhibitors to give the measured constants of k1, andk−1. The derived dissociation inhibitory constant (Ki) was calculated from the ratio ofk−1/k1 . kobs=k−1+k1[I]/(1+[S]/Km)Equation 4 Binding kinetics for derivatives of fX to immunocaptured FLAG-rNAPc2 were determined by surface plasmon resonance, using a BIAcore 2000 instrument (Pharmacia Biosensor). The mAb to the FLAG epitope (Anti-FLAG M1, Eastman Kodak Co.) was immobilized on the surface of a CM5 sensor chip by amine coupling, according to manufacturer's recommendations. Recombinant FLAG-rNAPc2 (0.1 mg/ml) was injected onto the sensor chip to saturate the immobilized antibody in Hepes-buffered saline containing 1 mm CHAPS, 0.005% surfactant P20, 5 mmCaCl2, pH 7.4. The kinetics of binding of various derivatives of fX were measured, following the injection of each protein at different concentrations (3–300 nm). Between runs, the Ca2+-dependent mAb was regenerated by eluting bound FLAG-rNAPc2 with 0.1 m EDTA. The kinetic binding constants (ka,kd, and KD) were determined by nonlinear regression analysis of the data from three separate experiments using software provided by the manufacturer. The association rate constant (ka) was calculated from multiple sensorgrams, representing at least five different concentrations of ligand for each experiment. The dissociation rate constant (kd) was calculated from the initial dissociation phase of the binding curves, and the equilibrium dissociation constant (KD) equaled the ratio ofkd/ka (27Kelly C.R. Dickinson C.D. Ruf W. J. Biol. Chem. 1997; 272: 17467-17472Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The binding of FLAG-rNAPc2 to immunocaptured fX derivatives was measured by modified enzyme-linked immunoassay detection method. A murine monoclonal antibody (mAb 10) 3T. S. Edgington, unpublished data.produced against human fX, which was shown to recognize all fX derivatives equally well, was used for solid phase immobilization of the various fX-derived inhibitory scaffolds. mAb 10 was bound to microtiter plate wells following an overnight incubation in phosphate-buffered saline (PBS) at 4 °C followed by washing with PBS containing 0.05% Tween 20 (v/v). Blocking with PBS containing 1% BSA (w/v), and 2% mannose (w/v) was followed by washing, and the addition of a saturating amount of one of the fX derivatives (final concentration 50 nm) in Hepes-buffered saline containing 15 μm BSA. Following a 1-h incubation and washing, varying concentrations of FLAG-rNAPc2 (0–100 nm) were added and incubated for 1 h followed by washing and addition of the HRP-anti-FLAG mAb M1 conjugate. Following a 1-h incubation and washing, bound peroxidase activity was visualized using 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate and 1n H2SO4. The end point absorbance (A450–650 nm) was read, and the data were analyzed by fitting A450–650 nm versus [fX derivative] using reiterative nonlinear regression to the binding equation Y = Bmax* [L]/(KD + [L]), where [L] represents the concentration of fX derivative,Bmax the maximal binding, andKD the measured dissociation constant. Three separate binding experiments were performed for each fX derivative, and the reported KD value represented the mean of the three resolved KD values from each of those experiments. rNAPc2 was added to both a preformed complex of fVIIa·TFPCPS, or TFPCPS alone (control), and incubated for 0–5 h at 37 °C. The final concentration of reactants in 300 μl of 25 mm Hepes, 150 mm NaCl, 5 mm CaCl2, pH 7.4, was 90 μmrNAPc2, 1.9 μm TFPCPS, 966 μmPCPS PLV, and 0.9 μm rfVIIa. At various time intervals (0–5 h), aliquots were quenched with EDTA and submitted to SDS-PAGE followed by Coomassie Blue staining, which demonstrated that rNAPc2 was completely cleaved by the fVIIa·TF into two distinct bands by 5 h. The control sample ran as a single band, comparable to the starting material. The remaining sample was reduced and carboxymethylated by adding dithiothreitol (10 mm) and heating at 100 °C for 2 min in denaturing buffer (final concentration 0.1 m Tris, 6m guanidine HCl, pH 8.0) followed by the addition of sodium iodoacetamide (NaIOAc) in denaturing buffer (1 mm) and an additional 30-min incubation in the dark at 23 °C. Additional NaIOAc (40 mm) and dithiothreitol (43 mm) were ad
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