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• W2091215711 abstract "Previously we have determined that residues 88–109 (but not Arg94) in the second epidermal growth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) activating complex on phospholipid vesicles (Wilkinson, F. H., London, F. S., and Walsh, P. N. (2002) J. Biol. Chem. 277, 5725–5733). Here we report that these residues are important for platelet binding affinity, stoichiometry, and assembly of the FX activating complex. We prepared several chimeric FIXa proteins using homologous sequences from factor VII (FVII): FIXaFVIIEGF2 (FIXΔ88–124,∇FVII91–127), FIXaloop1 (FIXΔ88–99,∇FVII91–102), FIXaloop2 (FIXΔ95–109,∇FVII98–112), and FIXaloop3 (FIXΔ111–124,∇FVII114–127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (K d values in 10−9m) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXaN (0.55 ± 0.06, 2.9 ± 0.45), FIXaWT (0.80 ± 0.08, 3.5 ± 0.5), FIXaloop1 (19 ± 4.0, 27 ± 5.0), FIXaloop2 (35 ± 9.0, 65 ± 12.0), and FIXaloop3 (1.1 ± 0.09, 5.0 ± 0.90). TheseK d values are in good agreement withK d (app) values (in 10−9m) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXaN (0.46 ± 0.05, 1.40 ± 0.14), FIXaWT (0.72 ± 0.08, 3.8 ± 0.08), FIXaloop1 (3.2 ± 0.72, 14.0 ± 1.60), FIXaloop2 (18.4 ± 1.60, 26.3 ± 3.40), and FIXaloop3 (0.7 ± 0.05, 3.0 ± 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with V max of FX activation and was reduced in those proteins that also showed a decreased platelet binding affinity. A peptide corresponding to the FIX EGF2 domain (Leu84-Val128) was an effective inhibitor of FIXa binding to platelets in both the presence (K i = 0.7 × 10−6m) and the absence (K i = 1.5 × 10−6m) of FVIIIa and FX. We conclude that residues 88–109 of the FIXa EGF2 domain mediate binding to platelets and assembly of the FX activating complex. Previously we have determined that residues 88–109 (but not Arg94) in the second epidermal growth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) activating complex on phospholipid vesicles (Wilkinson, F. H., London, F. S., and Walsh, P. N. (2002) J. Biol. Chem. 277, 5725–5733). Here we report that these residues are important for platelet binding affinity, stoichiometry, and assembly of the FX activating complex. We prepared several chimeric FIXa proteins using homologous sequences from factor VII (FVII): FIXaFVIIEGF2 (FIXΔ88–124,∇FVII91–127), FIXaloop1 (FIXΔ88–99,∇FVII91–102), FIXaloop2 (FIXΔ95–109,∇FVII98–112), and FIXaloop3 (FIXΔ111–124,∇FVII114–127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (K d values in 10−9m) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXaN (0.55 ± 0.06, 2.9 ± 0.45), FIXaWT (0.80 ± 0.08, 3.5 ± 0.5), FIXaloop1 (19 ± 4.0, 27 ± 5.0), FIXaloop2 (35 ± 9.0, 65 ± 12.0), and FIXaloop3 (1.1 ± 0.09, 5.0 ± 0.90). TheseK d values are in good agreement withK d (app) values (in 10−9m) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXaN (0.46 ± 0.05, 1.40 ± 0.14), FIXaWT (0.72 ± 0.08, 3.8 ± 0.08), FIXaloop1 (3.2 ± 0.72, 14.0 ± 1.60), FIXaloop2 (18.4 ± 1.60, 26.3 ± 3.40), and FIXaloop3 (0.7 ± 0.05, 3.0 ± 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with V max of FX activation and was reduced in those proteins that also showed a decreased platelet binding affinity. A peptide corresponding to the FIX EGF2 domain (Leu84-Val128) was an effective inhibitor of FIXa binding to platelets in both the presence (K i = 0.7 × 10−6m) and the absence (K i = 1.5 × 10−6m) of FVIIIa and FX. We conclude that residues 88–109 of the FIXa EGF2 domain mediate binding to platelets and assembly of the FX activating complex. Factor IXa (FIXa) 1FIXafactor IXaFXfactor XHTHEPES-Tyrodes bufferFIXaNnormal FIXaBSAbovine serum albuminGlaγ-carboxyglutamic acidEGF2second epidermal growth factorFVIIafactor VIIaFVIIIafactor VIIIaFIXaWTwild type FIXa is a serine protease that participates in the intrinsic phase of blood coagulation (1Aggeler P. Proc. Soc. Exp. Biol. Med. 1952; 79: 692Crossref PubMed Scopus (72) Google Scholar, 2Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1636) Google Scholar). FIXa is derived from the zymogen FIX by limited proteolytic cleavage at Arg145-Ala146 and Arg180-Val181 mediated either by factor XIa (FXIa) or by factor VIIa/tissue factor (FVIIa/TF). FIXa catalyzes the activation of factor X (FX) by enzymatic hydrolysis of the Arg52-Ile53 bond in the heavy chain. Maximal catalytic efficiency (k cat/K m ) of this reaction results from assembly of the FX activating complex (3Rawala-Sheikh R. Ahmad S.S. Ashby B. Walsh P.N. Biochemistry. 1990; 29: 2606-2611Crossref PubMed Scopus (92) Google Scholar), which consists of FIXa, factor VIIIa (FVIIIa) (a nonenzymatic cofactor), and FX assembled on a procoagulant surface such as the activated platelet membrane. Assembly of the FX activating-complex is a result of surface localization of each of the protein components. factor IXa factor X HEPES-Tyrodes buffer normal FIXa bovine serum albumin γ-carboxyglutamic acid second epidermal growth factor factor VIIa factor VIIIa wild type FIXa Surface localization of FIXa, FVIIIa, and FX results from interactions with high-affinity, specific binding sites on the procoagulant surface. A three-receptor model has been proposed whereby platelets expose distinct binding sites for FIXa, FVIIIa, and FX (4Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar, 5Ahmad S.S. Scandura J.M. Walsh P.N. J. Biol. Chem. 2000; 275: 13071-13081Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Nesheim M.E. Pittman D.D. Wang J.H. Slonosky D. Giles A.R. Kaufman R.J. J. Biol. Chem. 1988; 263: 16467-16470Abstract Full Text PDF PubMed Google Scholar, 7Scandura J.M. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: 8890-8902Crossref PubMed Scopus (64) Google Scholar). Occupancy of these binding sites is thought to promote the interactions among these proteins and facilitate assembly of functional FX activating complexes. In support of this model, FIXa has been found to bind to thrombin-activated platelets (4Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar), as have FVIIIa (5Ahmad S.S. Scandura J.M. Walsh P.N. J. Biol. Chem. 2000; 275: 13071-13081Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Nesheim M.E. Pittman D.D. Wang J.H. Slonosky D. Giles A.R. Kaufman R.J. J. Biol. Chem. 1988; 263: 16467-16470Abstract Full Text PDF PubMed Google Scholar) and FX (7Scandura J.M. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: 8890-8902Crossref PubMed Scopus (64) Google Scholar). FIXa has been found to bind to ∼600 sites per platelet in the presence of FVIIIa and FX (K d = 0.5 nm) as well as in their absence (K d = 3 nm). Thus, one consequence of FIXa-FVIIIa interactions is an enhancement of FIXa surface binding affinity. In contrast, the FIX zymogen binds to a reduced number of sites (250–300 sites per platelet, K d = 3 nm), and neither its affinity nor stoichiometry is enhanced by the presence of FVIIIa and FX. FX binds to platelets (∼16000 sites per platelet,K d = 300 nm) at a site shared with prothrombin (7Scandura J.M. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: 8890-8902Crossref PubMed Scopus (64) Google Scholar). FVIIIa has been found to bind to ∼750 sites per platelet (K d = 1.5 nm) with an increase in stoichiometry (1200 sites per platelet, K d = 0.8 nm) upon addition of FIXa and FX (5Ahmad S.S. Scandura J.M. Walsh P.N. J. Biol. Chem. 2000; 275: 13071-13081Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Nesheim M.E. Pittman D.D. Wang J.H. Slonosky D. Giles A.R. Kaufman R.J. J. Biol. Chem. 1988; 263: 16467-16470Abstract Full Text PDF PubMed Google Scholar). The rate of FX activation is closely correlated with the amount of surface-bound FIXa (8Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 20012-20016Abstract Full Text PDF PubMed Google Scholar). Further, surface-bound FX has been found to be the preferred substrate for the FX activating complex (9Scandura J.M. Walsh P.N. Biochemistry. 1996; 35: 8903-8913Crossref PubMed Scopus (37) Google Scholar). While informative with respect to the consequences of surface binding, these observations do not provide details regarding the mechanism of surface binding. Investigations into the mechanism of FIXa surface binding have identified structural regions of FIXa that mediate surface interactions. Residues at the amino terminus of FIXa (the ω loop, residues 1–12) have been found to contain an interactive site that promotes binding to platelets (10Ahmad S.S. Rawala-Sheikh R. Cheung W.F. Jameson B.A. Stafford D.W. Walsh P.N. Biochemistry. 1994; 33: 12048-12055Crossref PubMed Scopus (34) Google Scholar, 11Ahmad S.S. Wong M.Y. Rawala R. Jameson B.A. Walsh P.N. Biochemistry. 1998; 37: 1671-1679Crossref PubMed Scopus (22) Google Scholar), phospholipid vesicles (12Freedman S.J. Blostein M.D. Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and endothelial cells (13Cheung W.F. Hamaguchi N. Smith K.J. Stafford D.W. J. Biol. Chem. 1992; 267: 20529-20531Abstract Full Text PDF PubMed Google Scholar). Experiments from our laboratory suggest that residues distinct from those contained within 1–12 are also important for binding to platelets and phospholipids (11Ahmad S.S. Wong M.Y. Rawala R. Jameson B.A. Walsh P.N. Biochemistry. 1998; 37: 1671-1679Crossref PubMed Scopus (22) Google Scholar, 14Rawala-Sheikh R. Ahmad S.S. Monroe D.M. Roberts H.R. Walsh P.N. Blood. 1992; 79: 398-405Crossref PubMed Google Scholar). Residues contained within the second epidermal growth factor (EGF2)-like domain have been found to be important for surface binding and optimal rates of FX activation (15Ahmad S.S. Rawala R. Cheung W.F. Stafford D.W. Walsh P.N. Biochem. J. 1995; 310: 427-431Crossref PubMed Scopus (22) Google Scholar). A chimeric protein containing the FX EGF2 domain in place of that of FIXa (FIXaFXEGF2) was found to bind to a reduced number of sites (175 sites per platelet) and with a reduced affinity K d (app) = 65 nm) compared with wild type FIXa (FIXaWT) (600 sites per platelet, K d = 3 nm) (16Wong M.Y. Gurr J.A. Walsh P.N. Biochemistry. 1999; 38: 8948-8960Crossref PubMed Scopus (15) Google Scholar). FIXaFXEGF2 was also deficient in its FX activating activity (V max = 3 pm FXa per min) compared with FIXaWT (V max = 38 pm FXa per min) (16Wong M.Y. Gurr J.A. Walsh P.N. Biochemistry. 1999; 38: 8948-8960Crossref PubMed Scopus (15) Google Scholar). These results highlight the important contribution of the EGF2 domain to the surface-localization and catalytic properties of FIXa. Our interest has been to further investigate the mechanism by which FIXa binds to surfaces and to understand the functional consequences of these interactions. We have prepared several chimeric proteins substituting various loops of the FIXa EGF2 domain with those of FVII. Our objective is to determine which loops of the FIXa EGF2 domain are important for platelet binding properties and for FX activation. We have previously described the purification, characterization, and the kinetic properties of FIXa EGF2 chimeric proteins in the presence of phospholipid vesicles (45Wilkinson F.H. London F.S. Walsh P.N. J. Biol. Chem. 2002; 277: 5725-5733Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In this report, we describe the platelet binding and kinetic properties of FIXa EGF2 chimeric proteins. We have identified residues contained within the sequence Cys88 to Cys109 that mediate surface binding and assembly of FX activating complexes on the platelet membrane. Grade VII apyrase was purchased from Sigma. Pentex density gradient medium (35% w/v bovine serum albumin) was purchased from Bayer (Kankakee, IL). Sepharose CL-2B, fatty acid-free bovine serum albumin (BSA),para-aminobenzamidine, and heparin from porcine intestinal mucosa were purchased from Sigma. Carrier-free Na125I was obtained from Amersham Biosciences, Inc. Human FIX, human FX, human antithrombin III, and the FX activator from Russell's Viper (Vipera russeli) venom were purchased from Enzyme Research Laboratories (South Bend, IN). FXIa was purchased from Hematologic Technologies (Essex Junction, VT). High-purity recombinant human FVIII was obtained as a generous gift from Baxter Healthcare Corp. (Duarte, CA). Thrombin was purchased from Sigma. The chromogenic substrate S-2765 (N-benzlyoxy-carbonyl-d-arginyl-glycyl-l-arginine-para-nitroanalide-dihydrochloride) was purchased from Dia Pharma Group (Stockholm, Sweden). The following peptides were synthesized by the Protein Chemistry Laboratory (Dr. John Lambris) of the University of Pennsylvania (Philadelphia, PA) as described previously using 9-fluorenyl-methyloxycarbonyl chemistry on an Applied Biosystems 430A Synthesizer and reverse-phase high pressure liquid chromatography purified to >99% homogeneity (7Scandura J.M. Ahmad S.S. Walsh P.N. Biochemistry. 1996; 35: 8890-8902Crossref PubMed Scopus (64) Google Scholar, 11Ahmad S.S. Wong M.Y. Rawala R. Jameson B.A. Walsh P.N. Biochemistry. 1998; 37: 1671-1679Crossref PubMed Scopus (22) Google Scholar): thrombin receptor agonist peptide (SFLLRN-amide), a conformationally constrained γ-carboxyglutamic acid (“Gla”) peptide (CPGKLDEFVQPC) comprising Gly4-Gln11 of FIX, a “scrambled” Gla peptide, and the EGF2 peptide (LDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAV) comprising Leu84 to Val128 of FIX. The EGF2 domain peptide contained 45 residues including six cysteines involved in disulfide bridges formed as in the native protein using a selective protection/deprotection strategy as previously reported by Yang,et al. (17Yang Y. Sweeney W.V. Schneider K. Chait B.T. Tam J.P. Protein Sci. 1994; 3: 1267-1275Crossref PubMed Scopus (46) Google Scholar). Peptides were dissolved in deionized water to a final concentration of 100 μg/ml in a flask containing a stir bar in order to refold the peptides containing cysteine residues. After the pH value was adjusted to 8.5 with NH4OH, the solution was allowed to stir at 5 °C for at least 3 days. The solution was then lyophilized or, alternatively, peptides were reduced with dithiothreitol (1 mm) and alkylated with iodoacetamide (5 mm) as described previously (18Baglia F.A. Jameson B.A. Walsh P.N. J. Biol. Chem. 1993; 268: 3838-3844Abstract Full Text PDF PubMed Google Scholar). All peptides used in this study were examined by high-performance liquid chromatography (both reverse-phase and gel filtration) and all demonstrated a single homogenous peak. This demonstrates the presence of a single homogenous mixture of refolded peptides and not a mixed population of diverse polymers. The results are the same after reduction and alkylation of the same peptides. In addition, all peptides were examined for the presence of free sulfhydryl groups using the Ellman reagent, 5,5′-dithiobis-(2-nitrobenzoic acid). It was determined that there was less than 0.02 mol of free sulfhydryl per mol of peptide, which further verifies that these peptides were homogenous preparations. HEPES-Tyrodes buffer (HT): 15 mm HEPES (N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid), 126 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, 375 μmNaH2PO4, and 5.6 mm glucose. The pH of HT for platelet isolation was 6.5, for enzyme assays the pH was 7.4. HT was supplemented with BSA (2 mg/ml or 0.5 mg/ml) and CaCl2 (5 mm or 10 mm) where indicated. Preparation and purification of FIXWT, the FVII chimeric proteins, FIXFVIIEGF2 (FIXΔ88–124,∇FVII91–127), FIXloop1 (FIXΔ88–99,∇FVII91–102), FIXloop2 (FIXΔ95–109,∇FVII98–112), FIXloop3 (FIXΔ111–124,∇FVII114–127), and point mutant FIXR94D and FIXloop1G94R proteins has been described previously (45Wilkinson F.H. London F.S. Walsh P.N. J. Biol. Chem. 2002; 277: 5725-5733Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The symbol “Δ” denotes a deletion of FIX residues and “∇” denotes the insertion of FVII residues. FIX proteins were diluted in HT, pH 7.4 and supplemented with 10 mm CaCl2, and FXIa was added at a 1/200 molar ratio. The reaction was incubated at 37 °C for 90 min. Complete activation was judged by SDS-PAGE and active site titration with antithrombin III. No abnormalities of the chimeric or point mutant proteins were observed under these conditions (i.e. all of the FIXa proteins were completely cleaved and yielded the predicted number of active sites). For binding assays,125I-labeled FIX was prepared by the iodogen method and had a specific radioactivity of ∼2.5 × 106 cpm/mg (19Tuszynski G.P. Knight L. Piperno J.R. Walsh P.N. Anal. Biochem. 1980; 106: 118-122Crossref PubMed Scopus (108) Google Scholar). Following activation with FXIa (1/200 molar ratio) and SDS-PAGE, autoradiography of the sample was carried out to provide structural characterization of 125I-labeled protein. Under reducing conditions, 125I-FIXa migrated as two polypeptide chains ofM r = 27 kDa and 17 kDa representing the heavy chain and the light chain. 125I-labeled FIXa migrated indistinguishably from unlabeled, plasma-derived FIXa under identical conditions (not shown) thus confirming the purity and chain composition of both labeled and unlabeled FIXa molecules. Washed, gel-filtered platelets were prepared according to the following protocol (20Walsh P.N. Br. J. Haematol. 1972; 22: 205-217Crossref PubMed Scopus (152) Google Scholar, 21Walsh P.N. Adv. Exp. Med. Biol. 1972; 34: 245-256Crossref PubMed Scopus (12) Google Scholar, 22Walsh P.N. Mills D.C. White J.G. Br. J. Haematol. 1977; 36: 287-296Crossref PubMed Scopus (96) Google Scholar). Donor blood was collected into tubes (50 ml) containing 7 ml of the following buffer: trisodium citrate (25 g/L), citric acid (15 g/L), and glucose (20 g/L). The blood was centrifuged (200 × g) to separate platelet-rich plasma. The platelet-rich plasma was collected and supplemented with apyrase to a final concentration of 0.5 units/ml with incubation at 37 °C for 15 min. The platelet-rich plasma was layered gently over a 17.5% (w/v) albumin solution (0.8 ml prepared by addition of 0.4 ml each of HT, pH 6.5, supplemented with BSA (2 mg/ml) and Pentex density gradient medium (35% w/v BSA)) containing apyrase (2.5 units/ml) in a 15-ml polystyrene tube (Becton Dickinson, Franklin Lakes, NJ). The platelets were pelleted by centrifugation (800 ×g), resuspended in 10 ml of HT (pH 6.5) supplemented with BSA (2 mg/ml). The platelet suspension was layered gently over 1 ml of the 17.5% albumin solution described above, and the platelets pelleted again (800 × g). The platelets were resuspended in 2 ml of HT (pH 6.5) supplemented with BSA (2 mg/ml) and loaded onto a Sepharose CL-2B column (50 ml) pre-equilibrated with HT supplemented with BSA (2 mg/ml). Platelets were eluted from the column with HT supplemented with BSA (2 mg/ml). The most platelet-rich fractions as judged by visual inspection were pooled, and the platelet concentration was determined electronically using a Coulter Counter (Coulter Electronics, model ZBI, Hialeah Fl). Platelets were maintained at 37 °C and used within 3–4 h after isolation. In a typical binding experiment, platelets (3–4 × 108 platelets/ml) were incubated in HT supplemented with BSA (2 mg/ml), 5 mmCaCl2, and mixtures of labeled and unlabeled FIXa proteins or peptides as described previously (4Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar, 11Ahmad S.S. Wong M.Y. Rawala R. Jameson B.A. Walsh P.N. Biochemistry. 1998; 37: 1671-1679Crossref PubMed Scopus (22) Google Scholar). In brief, all binding experiments were performed in 1.5-ml Eppendorf plastic centrifuge tubes at 37 °C for 20 min. After incubation, aliquots (100 μl) were removed and centrifuged (at 12,000 × g in a Beckman Microfuge E through a mixture of silicone oils (Dow-Corning 500 and Dow Corning 200 mixed 4:1, vol:vol) to separate platelets from unbound proteins (4Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar). The 125I content in both the platelet pellets and the supernatants was determined by counting γ-emission in a Wallac γ counter (Gaithersburg, MD) using 125I energy window. The data were analyzed, and the number of binding sites andK d values were calculated from the means of four independent determinations each done in duplicate using a Macintosh Quadra 900 computer (Apple Computer, Cupertino, CA) and the ligand program as modified by G. A. McPherson Elsevier Science Publishers BV, The Netherlands). The IC50 method of Cha (23Cha S. Biochem. Pharmacol. 1975; 24: 2177-2185Crossref PubMed Scopus (455) Google Scholar) was used to determine the inhibition constants as previously published (18Baglia F.A. Jameson B.A. Walsh P.N. J. Biol. Chem. 1993; 268: 3838-3844Abstract Full Text PDF PubMed Google Scholar). Binding experiments were performed both in the absence and presence of FVIIIa and FX. FXa was assayed by hydrolysis of the FXa-specific chromogenic substrate S-2765. FXa (50 μl) was added to wells of a microtiter plate, and S-2765 (50 μl) was added to a final concentration of 350 μm. Change in absorbance at 405 nm was monitored immediately. Unknown FXa concentrations were determined by comparison to a standard curve prepared with known dilutions of FXa. FIXa proteins were added to 5 nm in reaction vessels containing platelets (3 × 108 platelets/ml) in HT supplemented with BSA (2 mg/ml) and 5 mm CaCl2. Platelets were activated by addition of the peptide SFLLRN-amide to 5 μm. FIXa proteins were preincubated with the activated platelets at 37 °C for 10 min. The reactions were initiated by addition of FX to the indicated final concentrations. After 20 min at 37 °C, the reactions were stopped by addition of EDTA to 10 mm. FXa was determined as described above. Velocity of FXa generation (nm FXa/min) was plotted as a function of input FX concentration (nm). Kinetic constants were determined as described below. FIXa proteins were added to 1 nmin reaction vessels containing platelets (5 × 107platelets/ml) in HT supplemented with BSA (2 mg/ml) and 5 mm CaCl2. Platelets were activated by addition of the peptide SFLLRN-amide to 5 μm. FIXa proteins were incubated with the activated platelets at 37 °C for 6 min. FVIII was activated to FVIIIa (5 units/ml final) by thrombin (0.1 units/ml) for 1 min at 37 °C immediately before addition to the reaction vessels. The reactions were initiated by addition of FX to the indicated final concentrations. After 2 min at 37 °C, the reaction was stopped by addition of EDTA to 10 mm. FXa was determined as described above. Kinetic constants were determined as described below. FIXa proteins were titrated into reaction vessels (to the indicated final concentrations) containing platelets (3 × 108platelets/ml) in HT supplemented with BSA (2 mg/ml) and 5 mm CaCl2. Platelets were activated by addition of the peptide SFLLRN-amide to 5 μm. FIXa proteins were preincubated with the activated platelets at 37 °C for 10 min. The reactions were initiated by addition of FX to 250 nm. After 20 min at 37 °C, the reactions were stopped by addition of EDTA to 10 mm. FXa was determined as described above. Kinetic constants were determined as described below. FIXa proteins were titrated into reaction vessels (to the indicated final concentrations) containing platelets (5 × 107platelets/ml) in HT supplemented with BSA (2 mg/ml) and 5 mm CaCl2. Platelets were activated by addition of the peptide SFLLRN-amide to 5 μm. FIXa proteins were incubated with the activated platelets at 37 °C for 6 min. FVIII was activated to FVIIIa (5 units/ml final concentration) by thrombin (0.1 units/ml) for 1 min at 37 °C immediately before addition to the reaction vessels. The reactions were initiated by addition of FX to 250 nm. After 2 min at 37 °C, the reactions were stopped by addition of EDTA to 10 mm. FXa was determined as described above. Kinetic constants were determined as described below. ForK m (app) and V maxdeterminations, velocity of FXa generation (nm FXa/min) was plotted as a function of input FX concentration (nm).K m (app) and V maxwere calculated by fitting the data to the Michaelis-Menten equation. For K d (app) andV max9 determinations, velocity of FXa generation (nm FXa/min) was plotted as a function of input FIXa concentration (nm).K d (app) andV max9 were calculated from the following equation modified from the Michaelis-Menten equation:V = V max9 × [FIXa]/([FIXa] + K d (app)), whereV is velocity, K d (app) is the apparent dissociation constant, andV max9 is the velocity observed at saturating FIXa concentration. Turnover number (k cat) is defined as mol FXa generated per mol of surface-bound FIXa per second. Mol FXa generated was calculated from the V max (nm FXa/min), mol surface-bound FIXa was calculated from the determinedK d values for the given set of reaction conditions and the input FIXa protein concentration. Bmax was determined from the platelet concentration and the determined binding stoichiometry for each FIXa protein. Between group differences were tested for statistical significance using analysis of variance followed by pair-wise comparisons with the Bonferroni adjustment procedure for multiple comparisons maintaining an experiment-wise Type 1 error level of 0.05(24). We were interested to determine the contribution of residues within the EGF2 domain of FIXa to platelet binding and activation of FX. Chimeric proteins (FIXaFVIIEGF2, FIXaloop1, FIXaloop2, and FIXaloop3) were prepared by substituting residues of the FIXa EGF2 domain with the homologous residues of FVII. Point mutant proteins (FIXaR94D and FIXaloop1G94R) were designed to address the contribution of Arg94 present within loop 1 of the EGF2 domain. These proteins were purified and characterized as described previously (45Wilkinson F.H. London F.S. Walsh P.N. J. Biol. Chem. 2002; 277: 5725-5733Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Kinetic analyses of the various FIXa proteins identified loop 1 (residues 88–99) and loop 2 (residues 95–109) as important for binding affinity to phospholipid vesicles. Arg94 was found to be unnecessary for the kinetic properties assayed. In the present study, we characterized these residues with respect to their contribution to platelet binding affinity and stoichiometry, and activation of FX on the surface of activated platelets. The chimeric proteins FIXaloop1 and FIXaloop2were previously found to be deficient in their affinity for phospholipid vesicles as measured by an increasedK d (app) (45Wilkinson F.H. London F.S. Walsh P.N. J. Biol. Chem. 2002; 277: 5725-5733Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Here, we determined the platelet binding properties of FIXaloop1, FIXaloop2, and FIXaloop3. FIX proteins were labeled with 125I, activated with FXIa, and tested for their ability to bind to platelets in the absence of FVIIIa and FX using equilibrium binding assays. As shown in Fig.1 and TableI, FIXaloop1 and FIXaloop2 were deficient in platelet binding that was manifested in both a decreased affinity (increasedK d ) and a reduced stoichiometry. FIXaWTand FIXaloop3 displayed normal binding properties (Table I) when compared with plasma-derived FIXa (FIXaN). Addition of FVIIIa and FX to the binding reaction was not sufficient to correct the binding deficiencies of FIXaloop1 and FIXaloop2(Table I).Table IK d and B max values in the absence and presence of FVIIIaFVIIIa and FXAbsentPresentFIXaK dB maxK dB maxnmsites × platelet−1nmsites × platelet−1Normal2.9 ± 0.45610 ± 600.55 ± 0.06585 ± 52Wild type3.5 ± 0.5600 ± 750.80 ± 0.08640 ± 80Loop 127 ± 5.0310 ± 6519 ± 4.0380 ± 70Loop 265 ± 12.0250 ± 5035 ± 9.0290 ± 44Loop 35.0 ± 0.90580 ± 591.1 ± 0.09555 ± 60Values are mean ± S.E.M (standard error of the mean) of independent measurements taken on different days using blood from different donors. K d values are in nm,B max values are in sites per platelet. Plasma-derived FIXa (n), FIXaWT (wild type), FIXaloop1 (loop 1), FIXaloop2 (loop 2), FIXaloop3 (loop 3). Open table in a new tab Values are mean ± S.E.M (standard error of the mean) of independent measurements taken on different days using blood from different donors. K d values are in nm,B max values are in sites per platelet. Plasma-derived FIXa (n), FIXaWT (wild type), FIXaloop1 (loop 1), FIXaloop2 (loop 2), FIXaloop3 (loop 3). The effects of the chimeric substitutions and point mutations on FX activation using platelets as a reaction surface were examined. Surface occupancy of platelet binding sites by FIXa was found to have a high correlation with rate of FX activation (8Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 20012-20016Abstract Full Text PDF PubMed Google Scholar). It was of interest to determine whether the FIXaloop1 and FIXaloop2proteins were deficient in FX activation in conjunction with their binding deficiencies. Titration of FIXa proteins into the reaction was used to determine a K d (app) value (FIXa concentration resulting in half-maximal rate of FX activation) as well as a V max9 value (maximum rate of FX activation in the presence of saturating FIXa concentration).K d (app) andV max9 values were determined from the isotherms shown in Fig. 2 and are reported in Table II. In addition to the proteins studied above, we also examined the FIXaR94D, FIXaFVIIEGF2, and FI" @default.
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• W2091215711 date "2002-02-01" @default.
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• W2091215711 title "The Factor IXa Second Epidermal Growth Factor (EGF2) Domain Mediates Platelet Binding and Assembly of the Factor X Activating Complex" @default.
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• W2091215711 doi "https://doi.org/10.1074/jbc.m107753200" @default.
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