Cleavage of Factor VIII Heavy Chain Is Required for the Functional Interaction of A2 Subunit with Factor IXa
2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês
10.1074/jbc.m009539200
ISSN1083-351X
AutoresPhilip J. Fay, Maria Mastri, Mary E. Koszelak, Hironao Wakabayashi,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoFactor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by ∼100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of ∼1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity. Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by ∼100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of ∼1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity. Factor VIII, the plasma protein deficient or defective in individuals with hemophilia A, is synthesized as a 300-kDa precursor (1Wood W.I. Capon D.J. Simonsen C.C. Eaton D.L. Gitschier J. Keyt B. Seeburg P.H. Smith D.H. Hollingshead P. Wion K.L. Delwart E. Tuddenham E.D.G. Vehar G.A. Lawn R.M. Nature. 1984; 312: 330-337Crossref PubMed Scopus (519) Google Scholar, 2Toole J.J. Knopf J.L. Wozney J.M. Sultzman L.A. Buecker J.L. Pittman D.D. Kaufman R.J. Brown E. Shoemaker C. Orr E.C. Amphlett G.W. Foster W.B. Coe M.L. Knutson G.J. Fass D.N. Hewick R.M. Nature. 1984; 312: 342-347Crossref PubMed Scopus (652) Google Scholar) with the domain structure A1-A2-B-A3-C1-C2 (3Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (652) Google Scholar). It is processed to a series of divalent metal ion-dependent heterodimers after cleavage at the B-A3 junction, generating a HC1 (A1-A2-B domains) and a LC (A3-C1-C2 domains). Additional cleavage sites within the B domain result in variably sized HCs minimally represented by contiguous A1-A2 domains. The two chains can be separated by chelating reagents (4Fass D.N. Knutson G.J. Katzmann J.A. Blood. 1982; 59: 594-600Crossref PubMed Google Scholar, 5Fay P.J. Anderson M.T. Chavin S.I. Marder V.J. Biochim. Biophys. Acta. 1986; 871: 268-278Crossref PubMed Scopus (123) Google Scholar) and isolated after ion exchange and/or immunoaffinity chromatography. Factor VIII activity can be reconstituted from the separated chains by combining them in the presence of a divalent metal ion (6Fay P.J. Arch. Biochem. Biophys. 1988; 262: 525-531Crossref PubMed Scopus (61) Google Scholar). Factor VIII functions in the intrinsic factor Xase complex as a cofactor for the serine protease, factor IXa in the surface-dependent conversion of factor X to Xa. This activity is dependent upon conversion of factor VIII to the active cofactor form, factor VIIIa, by thrombin or factor Xa. These enzymes cleave factor VIII HC at Arg-740, removing the B domain (or fragments) and at Arg-372, bisecting the HC into the A1 and the A2 subunits (7Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (392) Google Scholar). The proteases also cleave factor VIII LC at Arg-1689 (7Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (392) Google Scholar), liberating an acid-rich region and creating a new NH2 terminus. Thus, factor VIIIa is a heterotrimer of subunits designated as A1, A2, and A3-C1-C2 (8Lollar P. Parker C.G. Biochemistry. 1989; 28: 666-674Crossref PubMed Scopus (111) Google Scholar, 9Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar). The A1 and A3-C1-C2 subunits retain the divalent metal ion-dependent linkage, whereas the A2 subunit is weakly associated with the A1–A3-C1-C2 dimer by primarily electrostatic interactions (9Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar, 10Lollar P. Parker C.G. J. Biol. Chem. 1990; 265: 1688-1692Abstract Full Text PDF PubMed Google Scholar). Two regions of factor VIII have been identified as interactive sites for factor IXa. A high affinity site (K d∼15 nm (11Lenting P.J. Donath M.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: 7150-7155Abstract Full Text PDF PubMed Google Scholar)) was localized to the A3 domain of the LC in and around residues 1811–1818 (12Lenting P.J. van de Loo J.W. Donath M.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1996; 271: 1935-1940Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). A second, lower affinity site (K d∼300 nm (13Fay P.J. Koshibu K. J. Biol. Chem. 1998; 273: 19049-19054Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar)) was localized within the (isolated) A2 domain and comprises residues 558–565 (14Fay P.J. Beattie T. Huggins C.F. Regan L.M. J. Biol. Chem. 1994; 269: 20522-20527Abstract Full Text PDF PubMed Google Scholar). Recently, isolated A2 subunit was shown to stimulate thek cat for factor IXa-catalyzed conversion of factor X by ∼100-fold (13Fay P.J. Koshibu K. J. Biol. Chem. 1998; 273: 19049-19054Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). This property appeared unique to A2 and was not observed for either the isolated A1 or A3-C1-C2 subunit. However, the A1 subunit synergistically increased the cofactor activity of the isolated A2 subunit by ∼10-fold (15Fay P.J. Koshibu K. Mastri M. J. Biol. Chem. 1999; 274: 15401-15406Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Proteolysis of the LC during activation is responsible for the dissociation of factor VIIIa from its carrier protein, von Willebrand factor (16Hamer R.J. Koedam J.A. Beeser-Visser N.H. Sixma J.J. Eur. J. Biochem. 1987; 167: 253-259Crossref PubMed Scopus (43) Google Scholar). This cleavage also appears to increase the cofactor activity of factor VIIIa (17Donath M.S. 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, 18Regan L.M. Fay P.J. J. Biol. Chem. 1995; 270: 8546-8552Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The function of HC cleavage (at Arg-372) is not well understood. However, this step is essential for generating cofactor activity based upon mutations at this which result in severe hemophilia (19Kemball-Cook G. Tuddenham E.G.D. Nucleic Acids Res. 1997; 25: 128-132Crossref PubMed Scopus (50) Google Scholar). The isolated A2 subunit of factor VIIIa shows a several hundredfold weaker affinity for factor IXa and ∼1% of the cofactor activity compared with intact factor VIIIa (13Fay P.J. Koshibu K. J. Biol. Chem. 1998; 273: 19049-19054Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). It is unknown whether these low level activities are intrinsic to the HC or whether cleavage of the HC is necessary to manifest them. In this report we examine the capacity of isolated HC and derived subunits to modulate the catalytic activity of factor IXa in a purified system. Studies were performed in the absence of LC to preclude any interactions of this chain with factor IXa or factor X. Results show that intact HC possesses no detectable cofactor-like activity and fails to compete with isolated A2 subunit for interaction with factor IXa. However, its resultant cleavage by thrombin or (less efficiently) by factor Xa generates active subunits that show cofactor activity and modulation of factor IXa activity similar to that observed previously using the purified subunits. These results indicate that a primary role for HC cleavage during cofactor activation is the exposure of a functional factor IXa-interactive site. The reagents α-thrombin, factor IXaβ, factor X, factor Xa (Enzyme Research Laboratories), and Fl-FFR-factor IXaβ (Molecular Innovations) were purchased from the indicated vendors. Phospholipid vesicles composed of 20% PS, 40% PC, and 40% PE (Sigma) were prepared using octyl glucoside as described previously (20Mimms L.T. Zampighi G. Nozaki Y. Tanford C. Reynolds J.A. Biochemistry. 1981; 20: 833-840Crossref PubMed Scopus (554) Google Scholar). Tick anticoagulant peptide was a gift from Dr. S. Krishnaswamy. The anti-factor VIII monoclonal antibody, R8B12, which recognizes the COOH-terminal portion of the A2 domain (21Fay P.J. Smudzin T.M. Walker F.J. J. Biol. Chem. 1991; 266: 20139-20145Abstract Full Text PDF PubMed Google Scholar), was prepared as described (9Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar). Antibody 10104, an inhibitory monoclonal that binds the NH2-terminal region of factor VIII light chain (5Fay P.J. Anderson M.T. Chavin S.I. Marder V.J. Biochim. Biophys. Acta. 1986; 871: 268-278Crossref PubMed Scopus (123) Google Scholar), was obtained from QED BioScience. Monoclonal antibody 413 binds an epitope defined by residues 484–508 (22Healey J.F. Lubin I.M. Nakai H. Saenko E.L. Hoyer L.W. Scandella D. Lollar P. J. Biol. Chem. 1995; 270: 14505-14509Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and was a generous gift from Dr. Leon Hoyer. Recombinant factor VIII preparations were gifts from the Bayer Corporation and the Genetics Institute. Factor VIII HC was prepared as described previously (23Fay P.J. Smudzin T.M. J. Biol. Chem. 1989; 264: 14005-14010Abstract Full Text PDF PubMed Google Scholar) and is illustrated in Fig. 1. Potential trace levels of factor VIII LC present in the HC preparation were removed following chromatography using antibody 10104 coupled to Affi-Gel 10 as described previously (6Fay P.J. Arch. Biochem. Biophys. 1988; 262: 525-531Crossref PubMed Scopus (61) Google Scholar). The A2 subunit was prepared following fractionation of factor VIIIa using Mono S as described previously (9Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar). The rate of conversion of factor X to factor Xa was monitored in a purified system (24Lollar P. Fay P.J. Fass D.N. Methods Enzymol. 1993; 222: 128-143Crossref PubMed Scopus (93) Google Scholar). HC forms were reacted with factor IXa in 20 mm Hepes pH 7.2, 50 mm NaCl, 5 mm CaCl2, and 0.01% Tween 20 (buffer A) in the presence of 100 μg/ml bovine serum albumin and 10 μm phospholipid vesicles. Reactions were initiated with the addition of factor X (for reactant concentrations, see figure legends). Aliquots were removed at appropriate times to assess the initial rates of product formation and added to tubes containing EDTA (80 mm final concentration) to stop the reaction. Rates of factor Xa generation were determined by the addition of the chromogenic substrate, S-2765 (0.46 mm final concentration). Reactions were read at 405 nm using a V max microtiter plate reader (Molecular Devices). SDS-polyacrylamide gel electrophoresis was performed using the method of Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) with a Bio-Rad minigel system. Electrophoresis was performed at 200V for 1 h. Proteins were stained using Gel-Code Blue (Pierce). Alternatively, the proteins were transferred to a polyvinylidene difluoride membrane using a Bio-Rad mini-transblot apparatus at 0.5 amps for 30 min in a buffer containing 10 mm CAPS, pH 11, and 10% (v/v) methanol. Western blotting used the R8B12 monoclonal followed by goat anti-mouse horseradish peroxidase-conjugated secondary antibody. The secondary antibody signal was detected using the ECL system (Amersham Pharmacia Biotech) with luminol as substrate, and the blots were exposed to film for various times. Films were scanned, and band densities (obtained from a linear exposure range) were quantitated by using ImageQuant software (Molecular Devices). Rates of HC cleavage were calculated from the linear portion (initial time points) of the plotted data. Initial rates were estimated using best fit lines through points where ≤50% of the substrate had been converted to product. The concentration of HC remaining was calculated from band densities using the formula HC = (density of HC/(density of HC + density of A2 subunit)) × initial HC concentration. The concentration of A2 subunit formed following HC cleavage was calculated using the formula A2 subunit = (1− (density of HC/(density of HC + density of A2 subunit))) × initial HC concentration. Fluorescence anisotropy measurements were conducted using an Amico-Bowman series 2 spectrometer equipped with automatic polarizers arranged in an l-format. Reactions (0.2 ml) were run at room temperature in buffer A containing 90 nm Fl-FFR-IXa, 460 nm HC form, and 50 μm PSPCPE vesicles in the absence or presence of 500 nm factor X. Samples were excited at 495 nm, and the emission intensity was monitored at 520 nm (band pass = 4 nm) for 2 s at each polarizer position. Anisotropy values were calculated automatically after subtraction of blank readings. Data were averaged for at least five anisotropy measurements. Earlier we showed that the isolated A2 subunit of factor VIII stimulated the k cat for factor IXa-catalyzed conversion of factor X by ∼1% the level observed for factor VIIIa (13Fay P.J. Koshibu K. J. Biol. Chem. 1998; 273: 19049-19054Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The effect was enhanced further by 1 order of magnitude in the presence of saturating A1 subunit (15Fay P.J. Koshibu K. Mastri M. J. Biol. Chem. 1999; 274: 15401-15406Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Isolated factor VIII HC was assessed similarly for cofactor activity using a factor Xa generation assay and employing purified components. HC was obtained from EDTA-treated factor VIII as described under “Materials and Methods” and was essentially free from LC. Titration of intact HC in the factor Xa generation assay yielded virtually no activity increase under the reaction conditions described in Fig.2. However, prior cleavage of HC by thrombin to yield the HCIIa (free A1 and A2 subunits) resulted in a marked increase in the rate of factor Xa formed which was dose-dependent and saturable with respect to the cleaved HC. The concentration of substrate factor X used in these reactions (500 nm) represented near V maxlevels (data not shown). The extent of factor IXa stimulation observed at near saturating levels of HCIIa (1 μm;k cat ∼ 9 nm factor Xa generated/min/nmfactor IXa) was similar to that observed for factor IXa in the presence of an equivalent concentration of purified A2 plus A1 subunits (k cat ∼14 factor Xa generated/min/nm factor IXa) (15Fay P.J. Koshibu K. Mastri M. J. Biol. Chem. 1999; 274: 15401-15406Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). These results indicate that intact HC possesses no detectable factor IXa-stimulating activity and that this property is expressed only after cleavage of the HC to component subunits. To help exclude any contribution of trace factor VIII to these results, a series of experiments was performed in which HC was reacted first with one of three anti-factor VIII monoclonal antibodies prior to treatment with thrombin (Table I). Antibody 10104, a potent inhibitor that binds factor VIII LC (5Fay P.J. Anderson M.T. Chavin S.I. Marder V.J. Biochim. Biophys. Acta. 1986; 871: 268-278Crossref PubMed Scopus (123) Google Scholar), showed no effect in inhibiting HCIIa activity, consistent with the absence of functional factor VIII in the assay. R8B12, an anti-A2 domainal monoclonal with little inhibitory activity, also showed no effect. However, antibody 413, which binds an epitope defined by A2 domain residues 484–508 (22Healey J.F. Lubin I.M. Nakai H. Saenko E.L. Hoyer L.W. Scandella D. Lollar P. J. Biol. Chem. 1995; 270: 14505-14509Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and blocks the interaction between isolated A2 subunit and factor IXa (26Fay P.J. Scandella D. J. Biol. Chem. 1999; 274: 29826-29830Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), completely eliminated the cofactor activity of HCIIa. These results support the conclusion that the activity observed was attributed to HCIIa and was not the result of trace contamination of the HC preparation with functional factor VIII.Table IEffects of anti-factor VIII antibodies on the stimulation of factor IXa activity by thrombin-cleaved HCAntibody1-aPurified IgG (1 μm) was reacted with HC (300 nm) for 1 h prior to cleavage by thrombin and addition to the factor Xa generation assay containing 5 nm factor IXa, 10 μmPSPCPE vesicles, and 500 nm factor X in buffer A.Residual activity1-bFactor Xa generation was determined as described under “Materials and Methods” and is represented as percent of control (no antibody). Activity measured in the absence of antibody was 30 nm factor Xa generated/min.Epitope (LC/HC, residues)%None1001010498LC (1649–1689)R8B12102HC (563–740)413 5 μm. 2P. J. Fay, M. Mastri, and M. E. Koszelak, unpublished observation. Thus the phospholipid surface likely orients factor IXa such that collisions with A2 subunit are more productive. In this report, we show that a 10-fold molar excess of HC relative to A2 subunit marginally inhibited the A2-dependent stimulation of factor IXa activity, suggesting that the affinity of isolated HC for the enzyme is at least 10-fold weaker (>3 μm) than that of the derived A2 subunit. One model consistent with these observations is that cleavage of HC is required to expose the factor IXa-interactive site(s). Several lines of evidence demonstrate a conformational change in factor VIII HC following the conversion of factor VIII to factor VIIIa. For example, reaction of factor VIIIa with the zero-length cross-linker, EDC, resulted in formation of a covalent linkage between A1 and A2 subunits (32O'Brien L.M. Huggins C.F. Fay P.J. Blood. 1997; 90: 3943-3950Crossref PubMed Google Scholar), indicating the presence of a salt bridge at the site of cross-linking. However, treatment of factor VIII with EDC prior to cleavage by thrombin showed no linkage between the subunits, suggesting that the interdomain salt bridge was not present in the unactivated form. In addition, examination of binding of the apolar probe bisanilinonapthalsulfonic acid to isolated factor VIII and factor VIIIa subunits revealed two exposed hydrophobic sites on the isolated HC (33Sudhakar K. Fay P.J. J. Biol. Chem. 1996; 271: 23015-23021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) possessing affinities (K d values) of 0.21 and 1.4 μm. However, these sites contrast the single sites localized to the isolated A1 subunit (0.77 μm) and A2 subunit (0.11 μm), suggesting a change in conformation in and around these regions following thrombin cleavage. Finally, CD studies suggest an increase in β-sheet structure in factor VIIIa formed from factor VIII (34Curtis J.E. Helgerson S.L. Parker E.T. Lollar P. J. Biol. Chem. 1994; 269: 6246-6251Abstract Full Text PDF PubMed Google Scholar). Little evidence exists for a specific conformational change in and around the factor IXa-interactive site in A2 after cofactor activation. Suggestive evidence comes from observations of the inactivation of factor VIII and factor VIIIa by activated protein C. In collaboration with Walker, we showed that the bovine protease binds the cofactor near the COOH-terminal region of the A3 domain in the light chain (35Walker F.J. Scandella D. Fay P.J. J. Biol. Chem. 1990; 265: 1484-1489Abstract Full Text PDF PubMed Google Scholar) and preferentially attacks Arg-562 (21Fay P.J. Smudzin T.M. Walker F.J. J. Biol. Chem. 1991; 266: 20139-20145Abstract Full Text PDF PubMed Google Scholar) within the factor IXa-interactive site. Interestingly, the rate of cleavage at this site in factor VIIIa is ∼5-fold faster than its cleavage in factor VIII (21Fay P.J. Smudzin T.M. Walker F.J. J. Biol. Chem. 1991; 266: 20139-20145Abstract Full Text PDF PubMed Google Scholar). In factor VIIIa, this site is protected from cleavage by factor IXa (36Regan L.M. Lamphear B.J. Huggins C.F. Walker F.J. Fay P.J. J. Biol. Chem. 1994; 269: 9445-9452Abstract Full Text PDF PubMed Google Scholar). However, no factor IXa-dependent protection was observed using factor VIII (37O'Brien D.P. Johnson D. Byfield P. Tuddenham E.G. Biochemistry. 1992; 31: 2805-2812Crossref PubMed Scopus (41) Google Scholar). These results are compatible with differential exposure of the scissile bond at Arg-562 in the two substrates, with this region partially masked in the unactivated form. Taken together with the results of this study, these observations suggest that the factor IXa site localized to residues 558–565 in the A2 subunit is not fully formed in the contiguous A1-A2 domains of uncleaved HC. This lack of a functional factor IXa site in HC likely represents a primary requirement for cofactor activation at this domain junction and provides an explanation for the molecular basis of severe hemophilia attributed to cleavage-resistant mutations at Arg-372. We thank Debra Pittman of the Genetics Institute and James Brown and Lisa Regan of the Bayer Corporation for the gifts of recombinant factor VIII, Sriram Krishnaswamy for the tick anticoagulant peptide, and Leon Hoyer for the monoclonal antibody 413. factor VIII heavy chain factor VIII light chain factor IXa modified in its active site with fluorescein Phe-Phe-Arg chloromethyl ketone phosphatidylserine, PC, phosphatidylcholine phosphatidylethanolamine 3-(cyclohexylamino)propanesulfonic acid thrombin-cleaved HC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
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