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• W2136078026 abstract "The transition of the factor IX zymogen into the enzyme factor IXaβ was investigated. For this purpose, the activation intermediate factors IXα and IXaα were purified after cleavage of the Arg145-Ala146 and Arg180-Val181 bonds, respectively. These intermediates were compared for a number of functional properties with factor IXaβ, which is cleaved at both positions. Factor IXaα was equal to factor IXaβ in hydrolyzing the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide (kcat/Km≈ 120 s-1M-1) but was less efficient in factor X activation. Factor IXα was incapable of generating factor Xa but displayed reactivity toward p-nitrophenol p-guanidinobenzoate and the peptide substrate. The catalytic efficiency, however, was 4-fold lower compared with factor IXaα and factor IXaβ. Factor IXα and factor IXaβ had similar affinity for the inhibitor benzamidine (Ki≈ 2.5 mM), and amidolytic activity of both species was inhibited by Glu-Gly-Arg-chloromethyl ketone and antithrombin III. Unlike factor IXaβ, factor IXα was unable to form SDS stable complexes with antithrombin III. Moreover, inhibition of factor IXaβ and factor IXα by Glu-Gly-Arg-chloromethyl ketone followed distinct pathways, because factor IXα was inhibited in a nonirreversible manner and displayed only minor incorporation of the dansylated inhibitor into its catalytic site. These data demonstrate that the catalytic site of factor IXα differs from that of the fully activated factor IXaβ. Factor IX and its derivatives were also compared with regard to complex assembly with factor VIII in direct binding studies employing the immobilized factor VIII light chain. Factor IXα and factor IXaβ displayed a 30-fold higher affinity for the factor VIII light chain (Kd≈ 12 nM) than the factor IX zymogen. Factor IXaα showed lower affinity (Kd≈ 50 nM) than factor IXα and factor IXaβ, which may explain the lower efficiency of factor X activation by factor IXaα. Collectively, our data indicate that cleavage of the Arg180-Val181 bond develops full amidolytic activity but results in suboptimal binding to the factor VIII light chain. With regard to cleavage of the Arg145-Ala146 bond, we have demonstrated that this results in the transition of the factor IX zymogen into an enzyme that lacks proteolytic activity. Moreover, the same cleavage fully exposes the binding site for the factor VIII light chain, suggesting that cleavage of the Arg145-Ala146 bond serves a previously unrecognized role in the assembly of the factor IX-factor VIII complex. The transition of the factor IX zymogen into the enzyme factor IXaβ was investigated. For this purpose, the activation intermediate factors IXα and IXaα were purified after cleavage of the Arg145-Ala146 and Arg180-Val181 bonds, respectively. These intermediates were compared for a number of functional properties with factor IXaβ, which is cleaved at both positions. Factor IXaα was equal to factor IXaβ in hydrolyzing the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide (kcat/Km≈ 120 s-1M-1) but was less efficient in factor X activation. Factor IXα was incapable of generating factor Xa but displayed reactivity toward p-nitrophenol p-guanidinobenzoate and the peptide substrate. The catalytic efficiency, however, was 4-fold lower compared with factor IXaα and factor IXaβ. Factor IXα and factor IXaβ had similar affinity for the inhibitor benzamidine (Ki≈ 2.5 mM), and amidolytic activity of both species was inhibited by Glu-Gly-Arg-chloromethyl ketone and antithrombin III. Unlike factor IXaβ, factor IXα was unable to form SDS stable complexes with antithrombin III. Moreover, inhibition of factor IXaβ and factor IXα by Glu-Gly-Arg-chloromethyl ketone followed distinct pathways, because factor IXα was inhibited in a nonirreversible manner and displayed only minor incorporation of the dansylated inhibitor into its catalytic site. These data demonstrate that the catalytic site of factor IXα differs from that of the fully activated factor IXaβ. Factor IX and its derivatives were also compared with regard to complex assembly with factor VIII in direct binding studies employing the immobilized factor VIII light chain. Factor IXα and factor IXaβ displayed a 30-fold higher affinity for the factor VIII light chain (Kd≈ 12 nM) than the factor IX zymogen. Factor IXaα showed lower affinity (Kd≈ 50 nM) than factor IXα and factor IXaβ, which may explain the lower efficiency of factor X activation by factor IXaα. Collectively, our data indicate that cleavage of the Arg180-Val181 bond develops full amidolytic activity but results in suboptimal binding to the factor VIII light chain. With regard to cleavage of the Arg145-Ala146 bond, we have demonstrated that this results in the transition of the factor IX zymogen into an enzyme that lacks proteolytic activity. Moreover, the same cleavage fully exposes the binding site for the factor VIII light chain, suggesting that cleavage of the Arg145-Ala146 bond serves a previously unrecognized role in the assembly of the factor IX-factor VIII complex. The blood coagulation pathway comprises a cascade of sequential steps in which proenzymes are converted into active serine proteases (1). The serine protease precursor factor IX (FIX)1( 1The abbreviations used are: FIXfactor IXATIIIantithrombin IIIDEGR-CKdansyl-L-glutamyl-L-glycyl-L-arginine chloromethyl ketoneEGR-CKL-glutamyl-L-glycyl-L-arginine chloromethyl ketoneHSAhuman serum albuminFVIIIfactor VIIIFVIIIafactor VIIIaFIXαfactor IXαFIXaαfactor IXaαFIXaβfactor IXaβFXfactor XFXafactor XaFXIafactor XIaCH3SO2-LGR-pNACH3SO2-D-leucyl-L-glycyl-L-arginine-p-nitroanilideNPGBp-nitrophenol p-guanidinobenzoatePAGEpolyacrylamide gel electrophoresisEGFepidermal growth factor.) circulates in plasma as a single-chain polypeptide (Mr = 57,000) (2DiScipio R.G. Hermodson M.A. Yates S.G. Davie E.W. Biochemistry. 1977; 16: 698-706Crossref PubMed Scopus (415) Google Scholar) that comprises a number of discrete domains(3Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (989) Google Scholar). At the amino-terminal site of the molecule the so-called “Gla domain” is located. This domain contains several glutamic acid residues that have been carboxylated to yield Gla(4DiScipio R.G. Davie E.W. Biochemistry. 1979; 18: 899-904Crossref PubMed Scopus (170) Google Scholar). The presence of Gla residues allows this region to bind metal ions (5Sperling R. Furie B.C. Blumenstein M. Keyt B. Furie B. J. Biol. Chem. 1978; 253: 3898-3906Abstract Full Text PDF PubMed Google Scholar) and is essential for surface binding at platelets and endothelial cells(6Cheung W.-F. Hamaguchi N. Smith K.J. Stafford D.W. J. Biol. Chem. 1992; 267: 20529-20531Abstract Full Text PDF PubMed Google Scholar, 7Rawala-Sheikh R. Ahmad S.S. Monroe D.M. Roberts H.R. Walsh P.N. Blood. 1992; 79: 398-405Crossref PubMed Google Scholar). Adjacent to the Gla domain a region is located that shares homology with the epidermal growth factor (EGF) (8). This region consists of two distinct EGF-like domains, which are important for FIX function. The first EGF-like domain contains a single calcium binding site(8Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar). The second EGF-like domain is connected to the activation peptide, a segment that is liberated during zymogen activation(9DiScipio R.G. Kurachi K. Davie E.W. J. Clin. Invest. 1978; 61: 1528-1538Crossref PubMed Scopus (177) Google Scholar). Finally, the carboxyl-terminal portion comprises the trypsin-like serine protease domain, which contains a single metal ion binding site (10Bajaj S.P. Sabharwal A.K. Gorka J. Birktoft J.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 152-156Crossref PubMed Scopus (70) Google Scholar) and the catalytic centre of FIX(11Kurachi K. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6461-6464Crossref PubMed Scopus (295) Google Scholar). factor IX antithrombin III dansyl-L-glutamyl-L-glycyl-L-arginine chloromethyl ketone L-glutamyl-L-glycyl-L-arginine chloromethyl ketone human serum albumin factor VIII factor VIIIa factor IXα factor IXaα factor IXaβ factor X factor Xa factor XIa CH3SO2-D-leucyl-L-glycyl-L-arginine-p-nitroanilide p-nitrophenol p-guanidinobenzoate polyacrylamide gel electrophoresis epidermal growth factor. In contrast to most serine proteases, FIX requires two cleavages to yield full enzymatic activity(2DiScipio R.G. Hermodson M.A. Yates S.G. Davie E.W. Biochemistry. 1977; 16: 698-706Crossref PubMed Scopus (415) Google Scholar, 12Lindquist P.A. Fujikawa K. Davie E.W. J. Biol. Chem. 1978; 253: 1902-1909Abstract Full Text PDF PubMed Google Scholar, 13Walsh P.N. Bradford H. Sinha D. Piperno J. Tuszynski G.P. J. Clin. Invest. 1982; 73: 1392-1399Crossref Scopus (37) Google Scholar, 14Bajaj S.P. Rapaport S.I. Russell W.A. Biochemistry. 1983; 22: 4047-4053Crossref PubMed Scopus (44) Google Scholar, 15Fujikawa K. Coan M.H. Legaz M.E. Davie E.W. Biochemistry. 1974; 13: 4508-4516Crossref PubMed Scopus (129) Google Scholar). Dependent on the sequence of cleavage, FIX activation can follow two distinct pathways. Physiological activators first cleave the Arg145-Ala146 bond, resulting in the transient activation intermediate FIXα. So far, FIXα has been characterized as being enzymatically inactive(16Lawson J.H. Mann K.G. J. Biol. Chem. 1991; 266: 11317-11327Abstract Full Text PDF PubMed Google Scholar), which is underscored by the notion that FIXα lacks clotting activity(12Lindquist P.A. Fujikawa K. Davie E.W. J. Biol. Chem. 1978; 253: 1902-1909Abstract Full Text PDF PubMed Google Scholar, 15Fujikawa K. Coan M.H. Legaz M.E. Davie E.W. Biochemistry. 1974; 13: 4508-4516Crossref PubMed Scopus (129) Google Scholar). Subsequent cleavage at the amino terminus of the protease domain (i.e. position Arg180) then results in the active enzyme FIXaβ. The nonphysiological activator isolated from Russell's viper venom first cleaves at position Arg180(12Lindquist P.A. Fujikawa K. Davie E.W. J. Biol. Chem. 1978; 253: 1902-1909Abstract Full Text PDF PubMed Google Scholar). The resulting intermediate FIXaα displays proteolytic activity, although its clotting activity is just 20-50% of that of the enzyme FIXaβ(12Lindquist P.A. Fujikawa K. Davie E.W. J. Biol. Chem. 1978; 253: 1902-1909Abstract Full Text PDF PubMed Google Scholar, 17Griffith M.J. Breitkreutz L. Trapp H. Brit E. Noyes C.M. Lundblad R.L. Roberts H.R. J. Clin. Invest. 1985; 75: 4-10Crossref PubMed Scopus (36) Google Scholar). Subsequent cleavage of the Arg145-Ala146 bond then converts FIXaα into the fully active FIXaβ. Within the blood coagulation cascade, FIXaβ activates the zymogen factor X (FX) in a process that requires the presence of phospholipids, calcium ions, and factor VIIIa (FVIIIa)(18van Dieijen G. Tans G. Rosing J. Hemker H.C. J. Biol. Chem. 1981; 256: 3433-3442Abstract Full Text PDF PubMed Google Scholar, 19Mertens K. Bertina R.M. Thromb. Haemostasis. 1985; 54: 654-660Crossref PubMed Scopus (58) Google Scholar). During FX activation, FIXaβ is in complex with its protein cofactor FVIIIa(20Lamphear B. Fay P.J. J. Biol. Chem. 1992; 267: 3725-3730Abstract Full Text PDF PubMed Google Scholar, 21Duffy E.J. Parker E.T. Mutucumarana V.P. Johnson A.E. Lollar P. J. Biol. Chem. 1992; 267: 17006-17011Abstract Full Text PDF PubMed Google Scholar). This interaction involves the FVIII light chain, which contains a high affinity binding site for FIXaβ(22Lenting P.J. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: 7150-7155Abstract Full Text PDF PubMed Google Scholar). Complex formation with FVIIIa results in structural changes within the active site of FIXaβ(20Lamphear B. Fay P.J. J. Biol. Chem. 1992; 267: 3725-3730Abstract Full Text PDF PubMed Google Scholar). Maximal response requires the presence of the carboxyl-terminal portion of the FVIII heavy chain(20Lamphear B. Fay P.J. J. Biol. Chem. 1992; 267: 3725-3730Abstract Full Text PDF PubMed Google Scholar), indicating that the FVIII heavy chain is involved in complex formation with FIXaβ as well. Optimal FX activation requires the two-step cleavage of FIX into FIXaβ, because point mutations at Arg145 or Arg180 are both associated with the bleeding disorder hemophilia B(23Roberts H.R. Thromb. Haemostasis. 1993; 70: 1-9Crossref PubMed Scopus (51) Google Scholar). Whereas cleavage of the Arg180-Val181 bond corresponds with the zymogen-activating site that FIX is sharing with many other serine protease precursors(24Kraut J. Annu. Rev. Biochem. 1977; 46: 331-358Crossref PubMed Scopus (1075) Google Scholar), cleavage of the Arg150-Ala146 bond is unique for FIX. The aim of the present study was to elucidate the role of cleavage of the Arg145-Ala153 bond in the activation of the FIX zymogen. For this purpose FIXα was compared with other FIX activation products with regard to a number of parameters that are associated with FIXa enzyme function. These included reactivity toward synthetic and natural substrates and inhibitors and interaction with the cofactor FVIII. This approach allowed us to establish that cleavage at Arg145 plays a major role in the assembly of the FIX-FVIII complex. Protein A-Sepharose CL4B and CNBr-Sepharose CL4B were from Pharmacia LKB Biotechnology AB (Uppsala, Sweden). Microtiter plates (Immulon) were from Dynatech (Plockingen, Germany) unless stated otherwise. Glu-Gly-Arg-chloromethyl ketone (EGR-CK) and dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CK) were from Calbiochem. CH3SO2-D-Leu-Gly-Arg-p-nitroanilide (CH3SO2-LGR-pNA), product name CBS 31.39, was from Diagnostica Stago (Asnières, France). Heparin (grade 1-A) was obtained from Sigma. p-Nitrophenol p-guanidinobenzoate (NPGB) was from BDH Chemicals Ltd. (Poole, United Kingdom). The anti-FVIII antibodies CLB-CAg 12 and CLB-CAg 69 have been described previously(25Stel, H. V., 1984, Monoclonal Antibodies against Factor VIII-von Willebrand Factor. Ph.D. thesis, pp. 51-72, University of Amsterdam.Google Scholar, 26Leyte A. Mertens K. Distel B. Evers R.F. de Keyzer-Nellen M.J.M. Groenen-van Dooren M.M.C.L. de Bruin J. Pannekoek H. van Mourik J.A. Biochem. J. 1989; 263: 187-194Crossref PubMed Scopus (51) Google Scholar). The murine anti-FIX antibodies CLB-FIX 10 and CLB-FIX 11 were obtained as outlined previously(22Lenting P.J. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: 7150-7155Abstract Full Text PDF PubMed Google Scholar), employing a screening strategy based on binding to immobilized FIX in the presence or absence of calcium ions. Binding of CLB-FIX 10 to FIX was calcium-independent, whereas binding of CLB-FIX 11 was markedly enhanced in the presence of calcium ions. Both antibodies CLB-FIX 10 and CLB-FIX 11 strongly inhibit FIX activity (results not shown). The murine anti-FIX antibody CLB-FIX D4 has been described elsewhere(27Mertens, K., van Mourik, J. A., (March 17, 1994) International Patent Application, Patent Cooperation Treaty, Publication WO 94/05692.Google Scholar). This antibody is directed against the FIX sequence Asn136-Asp154, which comprises the Arg145-Ala146 activation site. By virtue of the location of its epitope, antibody CLB-FIX D4 distinguishes between intact FIX and cleaved FIX (i.e. FIXα and FIXaβ)(27Mertens, K., van Mourik, J. A., (March 17, 1994) International Patent Application, Patent Cooperation Treaty, Publication WO 94/05692.Google Scholar). All FIX antibodies used were from the IgG1k isotype. Monoclonal antibodies were purified from culture medium employing protein A-Sepharose as recommended by the manufacturer. Polyclonal antibodies against human FIX were obtained as described previously (22). Antibodies were conjugated with horseradish peroxidase as described(28Nakane P.K. Kawaoi A. J. Histochem. Cytochem. 1974; 22: 1084-1091Crossref PubMed Scopus (1880) Google Scholar). The human FVIII light chain was purified as described(22Lenting P.J. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: 7150-7155Abstract Full Text PDF PubMed Google Scholar). FX was prepared as described(29Mertens K. Bertina R.M. Biochem. J. 1980; 185: 647-658Crossref PubMed Scopus (51) Google Scholar). The factor X-activating protein from Russell's viper venom was purified as described (30Kisiel W. Hermodson M.A. Davie E.W. Biochemistry. 1976; 15: 4901-4906Crossref PubMed Scopus (134) Google Scholar) and coupled to CNBr-Sepharose (2 mg/ml) according to the manufacturer's instructions. Purified factor XIa (FXIa) was obtained from Enzyme Research Laboratories. Purified Antithrombin III (ATIII), C1-inhibitor, and human serum albumin (HSA) were obtained from the Division of Products of our institute. Bovine serum albumin was from Miles Inc. Purified α1-antitrypsin and α2-antiplasmin were gifts from Dr. W. Wuillemin, Department of Autoimmune Diseases of our institute. All proteins used, including FIX and its activation products (see below) were homogeneous as assessed by SDS-polyacrylamide gel electrophoresis (PAGE) (see Figs. 3 and 5). Human FIX was purified from a concentrate of prothrombin, FIX, and FX (31Mertens K. Brit E. Giles A.R. Thromb. Haemostasis. 1990; 64: 138-144Crossref PubMed Scopus (17) Google Scholar) obtained from the Division of Products of our institute. NaCl, benzamidine, and sodium citrate (pH 7.4) were added to the concentrate to final concentrations of 0.15 M, 0.01 M, and 0.02 M, respectively, and the mixture was subjected to immunoaffinity chromatography employing the anti-FIX antibody CLB-FIX D4 (5 mg/ml CNBr-Sepharose). After extensive washing with 0.15 M NaCl, 0.01 M benzamidine, 0.02 M sodium citrate (pH 7.4), FIX was eluted in a linear gradient (0-2 M KSCN). FIX-containing fractions were pooled and stored at −20°C in 0.1 M NaCl, 0.05 M Tris (pH 7.4). The specific activity of the FIX preparations ranged between 300 and 350 units/mg. FIXaβ was prepared by incubating purified FIX (4 μM) with human FXIa (0.23 μM) for 2 h at 37°C in 0.1 M NaCl, 2 mM CaCl2, 0.05 M Tris (pH 7.4). After the reaction was terminated by the addition of EDTA (0.01 M final concentration), residual FIX and FIXα were removed from the incubation mixture by rechromatography on the CLB-FIX D4 affinity column. In this immunoaffinity step, FIXaβ and FXIa did not bind to the column, whereas FIX and FIXα remained bound (27). Finally, FIXaβ and FXIa were separated employing anion exchange chromatography as described previously(32Mertens K. Bertina R.M. Thromb. Haemostasis. 1982; 47: 96-100Crossref PubMed Scopus (22) Google Scholar). FIXaβ was stored at −20°C in 50% glycerol, 0.1 M NaCl, 0.05 M Tris (pH 7.4). The FIXaβ preparations were more than 90% active as determined by active site titrations employing NPGB(33Byrne R. Link R.P. Castellino F.J. J. Biol. Chem. 1980; 255: 5336-5341Abstract Full Text PDF PubMed Google Scholar). FIXα was prepared by incubating purified FIX (4 μM) with human FXIa (16 nM) in the presence of 6.8 mM MnCl2 in 0.1 M NaCl, 0.05 M Tris (pH 7.4). After incubation for 2 h at 37°C, the reaction was terminated by the addition of EDTA and benzamidine (0.01 M final concentrations). Under these conditions, approximately 90% of FIX was converted into FIXα as judged by SDS-PAGE. FIXα was then separated from FIX and FXIa employing CLB-FIX D4 affinity chromatography. In this step FXIa and possible traces of FIXaβ passed through the column, while FIXα and residual FIX were bound. After extensive washing, FIX and FIXα were eluted separately in a linear gradient (0-3 M KSCN)(27Mertens, K., van Mourik, J. A., (March 17, 1994) International Patent Application, Patent Cooperation Treaty, Publication WO 94/05692.Google Scholar). The FIXα-containing fractions were pooled and stored at −20°C in 0.1 M NaCl, 0.05 M Tris (pH 7.4). The position of cleavage in FIXα was assessed by NH2-terminal amino acid sequence analysis employing automated equipment (Applied Biosystems, Warrington, UK; Eurosequence, Groningen, the Netherlands). The resulting sequence, Ala-Glu-Thr-Val-Phe, corresponds with the five NH2-terminal amino acids of the FIXα activation peptide region, demonstrating that indeed cleavage had occurred at Arg145-Ala146 (11). The FIXα preparations were more than 95% active as determined by NPGB titration(33Byrne R. Link R.P. Castellino F.J. J. Biol. Chem. 1980; 255: 5336-5341Abstract Full Text PDF PubMed Google Scholar). FIXaα was prepared from purified FIX essentially as described (17Griffith M.J. Breitkreutz L. Trapp H. Brit E. Noyes C.M. Lundblad R.L. Roberts H.R. J. Clin. Invest. 1985; 75: 4-10Crossref PubMed Scopus (36) Google Scholar) but modified in that the purified FX-activating enzyme from Russell's viper venom instead of the crude snake venom was immobilized on CNBr-Sepharose. Protein was measured by the method of Bradford using HSA as a standard(34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215990) Google Scholar). FIX activity was measured employing a commercially available chromogenic method (Baxter-DADE, Düdingen, Switzerland). Antigen concentrations of FIX or its derivatives were quantified by an immunological assay as described previously (22Lenting P.J. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: 7150-7155Abstract Full Text PDF PubMed Google Scholar) but modified in that immunopurified polyclonal anti-FIX antibodies were immobilized (0.2 μg/well) instead of monoclonal antibody CLB-FIX 2. Dose-response curves were transformed by plotting logit absorbance versus log concentration and were linear between 0.07 and 7 nM. Within this range the coefficient of variation was approximately 5%. Antigen values were converted into molar concentrations using the purified FIX derivatives as standards. Molar concentrations were calculated from protein concentrations employing Mr = 57,000 for FIX, FIXα, and FIXaα and Mr = 45,000 for FIXaβ (2). In experiments in which FIXaβ or FIX was used as competitor for the binding of FIXα to the FVIII light chain, nonbound FIXα was assayed by a FIXα-specific assay employing the antibody CLB-FIX D4. Samples containing FIXα and FIX or FIXaβ were incubated with the immobilized antibody (0.5 μg/well) in 3 M NaCl, 0.1% (v/v) Tween-20, 1% (w/v) HSA, 0.05 M Tris (pH 7.2). After washing with 0.15 M NaCl, 0.1% (v/v) Tween-20, 0.05 M Tris (pH 7.2), bound FIXα was detected employing peroxidase-conjugated polyclonal anti-FIX IgG in the washing buffer. Under these conditions FIXα but not FIX or FIXaβ binds to the immobilized antibody CLB-FIX D4. Dose-response curves were transformed by plotting logit absorbance versus log concentration and were linear between 0.1 and 10 nM. The ability of FIX or its cleaved derivatives to activate FX was assayed essentially as described previously (35Donath M.J.S.H. Lenting P.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1995; 270: 3648-3655Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) employing acetylated FX to prevent cleavage of the Arg145-Ala146 bond by the product FXa(16Lawson J.H. Mann K.G. J. Biol. Chem. 1991; 266: 11317-11327Abstract Full Text PDF PubMed Google Scholar). FX was acetylated according to Neuenschwander and Jesty(36Neuenschwander P. Jesty J. Anal. Biochem. 1990; 184: 347-352Crossref PubMed Scopus (9) Google Scholar). The modified FX zymogen had lost more than 95% of its biological activity, whereas amidolytic activity was fully maintained. Cleavage of CH3SO2-LGR-pNA by FIX or its derivatives was assayed in 0.2% (w/v) HSA, 0.1 M NaCl, 0.01 M CaCl2, 0.05 M Tris (pH 8.4). Substrate hydrolysis was initiated by the addition of 50 μl of a 2.5 mM solution of CH3SO2-LGR-pNA to a 50-μl sample in a microtiter plate (Costar, type flat bottom). Initial rates of substrate hydrolysis were measured at 37°C by monitoring absorbance at 405 nm in time. Kinetic parameters of substrate hydrolysis by FIX cleavage products were determined employing substrate concentrations between 0 and 15 mM at two different enzyme concentrations. Absorbance values were converted into molar concentrations using a molar extinction coefficient of 9.65 ´ 103M-1 cm-1 for p-nitroanilide and a pathlength of 0.35 cm for a 100-μl volume. The experimental data were fitted in the Michaelis-Menten equation using EnzFitter software (Elsevier, Amsterdam, the Netherlands) to obtain Km and kcat values. The binding of FIX or its cleaved derivatives to the immobilized FVIII light chain and calculation of binding parameters were performed as described(22Lenting P.J. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: 7150-7155Abstract Full Text PDF PubMed Google Scholar). The role of the individual cleavages at Arg145 and Arg180 in human FIX was investigated with respect to the development of enzymatic activity. FIX and its cleaved derivatives were compared for their ability to activate FX in the presence of calcium ions, phospholipids, and FVIIIa. As expected, the enzyme FIXaβ efficiently activated FX under these conditions (Fig. 1A). FXa was also generated by the intermediate FIXaα, although at a lower rate than by FIXaβ. In contrast, both the intermediate FIXα and the FIX zymogen were incapable of activating FX. These data demonstrate that cleavage at Arg180 converts FIX into an active protease but that the additional cleavage at Arg145 develops full proteolytic activity. To investigate whether limited proteolysis of FIX had a similar effect on amidolytic activity, the reactivity of the zymogen FIX and its cleaved derivatives toward the synthetic substrate CH3SO2-LGR-pNA was tested. No substrate cleavage occurred in the presence of the FIX zymogen (Fig. 1B). In contrast, all cleaved forms of FIX, including the intermediate FIXα, were capable of hydrolyzing this synthetic substrate. The kinetic parameters for the hydrolysis of CH3SO2-LGR-pNA by FIXaβ, FIXaα, and FIXα were determined. As listed in Table I, FIXaβ and FIXaα display similar catalytic efficiency. The catalytic efficiency of FIXα, however, appears to be 4-fold lower, which is mainly due to a decreased kcat. The possibility was considered that the lower catalytic efficiency could be due to FIXα being only partially active. However, active site titrations employing the active site titrant NPGB indicated that the extent of the p-nitrophenol burst corresponded to 90-95% of the protein concentrations of both the FIXα and FIXaβ preparations employed (see “Experimental Procedures”). It was noted that titration of FIXaβ requires about 4 min to reach completion(33Byrne R. Link R.P. Castellino F.J. J. Biol. Chem. 1980; 255: 5336-5341Abstract Full Text PDF PubMed Google Scholar), whereas the p-nitrophenol burst lasts 7-8 min for FIXα (results not shown). This slight difference in reactivity toward NPGB was not further elaborated. Collectively, these results confirm that the zymogen FIX is an inactive species, whereas the enzyme FIXaβ displays activity toward FX, CH3SO2-LGR-pNA, and NPGB. With regard to the activation intermediates, FIXaα equals the enzyme FIXaβ in synthetic substrate hydrolysis but is less efficient in FX activation. FIXα, however, is extremely inefficient in generating FXa but at the same time hydrolyzes CH3SO2-LGR-pNA and reacts with the active site titrant NPGB. This demonstrates that cleavage at Arg145 converts the FIX zymogen into an enzymatic form that lacks proteolytic activity.Table I:Kinetic parameters for the hydrolysis of CH3SO2-LGR-pNA by FIXα, FIXaα, or FIXaβ Open table in a new tab Although the concentration of active sites was in good agreement with the protein concentrations of purified FIXaβ and FIXα, these experiments do not fully exclude the possibility that traces of other serine proteases could contribute to the observed CH3SO2-LGR-pNA hydrolysis by FIXα or FIXaβ. Therefore, the effect of a number of serine protease inhibitors was tested. Amidolytic activity of FIXα or FIXaβ appeared to be unaffected by the presence of serine protease inhibitors including hirudin, soybean trypsin inhibitor, α1-antitrypsin, α2-antiplasmin, and C1-inhibitor (data not shown). This demonstrates that the amidolytic activity is not likely to be associated with the presence of a variety of potential contaminants. In contrast, CH3SO2-LGR-pNA hydrolysis was effectively inhibited in the presence of the monoclonal anti-FIX antibody CLB-FIX 10 (see Fig. 5). Control experiments demonstrated that this antibody does not inhibit CH3SO2-LGR-pNA hydrolysis by thrombin, FXa, or FXIa. This strongly suggests that the observed CH3SO2-LGR-pNA hydrolysis originates from the enzyme FIXaβ or the activation intermediate FIXα. Additional experiments were performed to determine the affinity of FIXα and FIXaβ for the inhibitor benzamidine. This reversible inhibitor is known to inhibit the various coagulation enzymes with Ki values ranging from 0.04 to 11 mM(37Pedersen A.H. Lund-Hansen T. Bisgaard-Frantzen H. Olsen F. Petersen L.C. Biochemistry. 1989; 28: 9331-9336Crossref PubMed Scopus (80) Google Scholar, 38Geratz J.D. Stevens F.M. Polakoski K.L. Parrish R.F. Tidwell R.R. Arch. Biochem. Biophys. 1979; 197: 551-559Crossref PubMed Scopus (45) Google Scholar, 39Tans G. Janssen-Claessen T. Rosing J. Griffin J.H. Eur. J. Biochem. 1987; 164: 637-642Crossref PubMed Scopus (43) Google Scholar) and as such could contribute to the identification of the active species in FIXα. Under the same experimental conditions as in Fig. 1B, FIXα inhibition proved to be similar to that of FIXaβ, with Ki values of 2.9 ± 0.3 and 2.0 ± 0.2 mM, respectively. These data demonstrate that FIXα and FIXaβ share similar inhibition characteristics by benzamidine. The enzymatic properties of FIXaβ and FIXα were examined in more detail using the synthetic serine protease inhibitor EGR-CK. Binding of EGR-CK to FIXα or FIXaβ was determined by continuously monitoring CH3SO2-LGR-pNA hydrolysis by FIXα or FIXaβ in the presence of various concentrations of EGR-CK. As expected, substrate hydrolysis by FIXaβ displayed progress curves that are" @default.
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• W2136078026 title "Cleavage at Arginine 145 in Human Blood Coagulation Factor IX Converts the Zymogen into a Factor VIII Binding Enzyme" @default.
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