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• W2040257513 abstract "The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys1967 and Lys1968 of region Thr1964-Tyr1971 are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys1967-Lys1968 within region Thr1964-Tyr1971 has an opposite contribution to the stability of FVIIIa. The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys1967 and Lys1968 of region Thr1964-Tyr1971 are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys1967-Lys1968 within region Thr1964-Tyr1971 has an opposite contribution to the stability of FVIIIa. Factor VIII (FVIII) 2The abbreviations used are: FVIIIfactor VIIIFVIIIaactivated FVIIICIDcollision-induced dissociationdis-FVIIIadissociated FVIIIaFIXaactivated factor IXFXfactor XFXaactivated FXHCDhigher energy CIDRUresponse unitsTMTtandem mass tag. serves its role in the coagulation cascade as a cofactor for activated factor IX (FIXa) during the proteolytic conversion of factor X (FX) into activated FX (1.Fay P.J. Factor VIII structure and function.Int. J. Hematol. 2006; 83: 103-108Crossref PubMed Scopus (78) Google Scholar). To perform this role, FVIII comprises multiple domains that are grouped in a heavy chain (domains A1-a1-A2-a2-B) and a light chain (domains a3-A3-C1-C2) (2.Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Structure of human factor VIII.Nature. 1984; 312: 337-342Crossref PubMed Scopus (658) Google Scholar). The A domains are homologous to the A domains of ceruloplasmin, the C domains to those of discoidin, and the B domain is unique to FVIII. a1, a2, and a3 are spacer regions that are rich in acidic amino acid residues (3.Church W.R. Jernigan R.L. Toole J. Hewick R.M. Knopf J. Knutson G.J. Nesheim M.E. Mann K.G. Fass D.N. Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins.Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 6934-6937Crossref PubMed Scopus (134) Google Scholar). Because of limited proteolysis of the B domain, FVIII circulates in plasma as a heterogeneous protein of which the light chain is noncovalently linked to the heavy chain (1.Fay P.J. Factor VIII structure and function.Int. J. Hematol. 2006; 83: 103-108Crossref PubMed Scopus (78) Google Scholar). factor VIII activated FVIII collision-induced dissociation dissociated FVIIIa activated factor IX factor X activated FX higher energy CID response units tandem mass tag. FVIII is found in plasma in a tight complex with its carrier protein von Willebrand factor (4.Lollar P. Hill-Eubanks D.C. Parker C.G. Association of the factor VIII light chain with von Willebrand factor.J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar). The role of von Willebrand factor is to protect FVIII from rapid clearance from the circulation and to prevent premature binding of FVIII to its ligands (5.Lillicrap D. Extending half-life in coagulation factors: where do we stand?.Thromb. Res. 2008; 122: S2-S8Abstract Full Text PDF PubMed Scopus (24) Google Scholar, 6.Lenting P.J. Donath M.J. van Mourik J.A. Mertens K. Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.J. Biol. Chem. 1994; 269: 7150-7155Abstract Full Text PDF PubMed Google Scholar). Proteolytic cleavage between a3 and the A3 domain by thrombin results in the dissociation of the FVIII-von Willebrand factor complex (4.Lollar P. Hill-Eubanks D.C. Parker C.G. Association of the factor VIII light chain with von Willebrand factor.J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar). Additional cleavages by thrombin between a1 and the A2 domain, and between a2 and the B domain, are required to convert FVIII into the activated heterotrimeric protein (FVIIIa) (7.Duffy E.J. Parker E.T. Mutucumarana V.P. Johnson A.E. Lollar P. Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.J. Biol. Chem. 1992; 267: 17006-17011Abstract Full Text PDF PubMed Google Scholar, 8.Camire R.M. Bos M.H. The molecular basis of factor V and VIII procofactor activation.J. Thromb. Haemost. 2009; 7: 1951-1961Crossref PubMed Scopus (66) Google Scholar). FVIIIa subsequently binds via its C1 domain and C2 domain to phosphatidylserine-containing procoagulant surfaces thereby forming a platform for high affinity interaction with FIXa (1.Fay P.J. Factor VIII structure and function.Int. J. Hematol. 2006; 83: 103-108Crossref PubMed Scopus (78) Google Scholar, 9.Lü J. Pipe S.W. Miao H. Jacquemin M. Gilbert G.E. A membrane-interactive surface on the factor VIII C1 domain cooperates with the C2 domain for cofactor function.Blood. 2011; 117: 3181-3189Crossref PubMed Scopus (41) Google Scholar, 10.Meems H. van den Biggelaar M. Rondaij M. van der Zwaan C. Mertens K. Meijer A.B. C1 domain residues Lys2092 and Phe2093 are of major importance for the endocytic uptake of coagulation factor VIII.Int. J. Biochem. Cell Biol. 2011; 43: 1114-1121Crossref PubMed Scopus (33) Google Scholar). FVIIIa is rapidly inactivated to prevent the unlimited production of activated FX (FXa). Several mechanisms have been proposed that may drive this inactivation. One mechanism involves proteolytic cleavage of FVIIIa by activated protein C. It has been proposed that activated protein C inactivates FVIIIa through proteolytic cleavages in the A1 and A2 domain (11.Fay P.J. Smudzin T.M. Walker F.J. Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa: identification of cleavage sites and correlation of proteolysis with cofactor activity.J. Biol. Chem. 1991; 266: 20139-20145Abstract Full Text PDF PubMed Google Scholar, 12.Fay P.J. Haidaris P.J. Huggins C.F. Role of the COOH-terminal acidic region of A1 subunit in A2 subunit retention in human factor VIIIa.J. Biol. Chem. 1993; 268: 17861-17866Abstract Full Text PDF PubMed Google Scholar, 13.Eaton D. Rodriguez H. Vehar G.A. Proteolytic processing of human factor VIII: correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity.Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (397) Google Scholar). It has also been shown that FIXa, FXa, and plasmin are able to cleave FVIIIa inducing inactivation of the cofactor (13.Eaton D. Rodriguez H. Vehar G.A. Proteolytic processing of human factor VIII: correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity.Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (397) Google Scholar, 14.Lamphear B.J. Fay P.J. Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa.Blood. 1992; 80: 3120-3126Crossref PubMed Google Scholar, 15.O'Brien D.P. Johnson D. Byfield P. Tuddenham E.G. Inactivation of factor VIII by factor IXa.Biochemistry. 1992; 31: 2805-2812Crossref PubMed Scopus (41) Google Scholar, 16.Nogami K. Shima M. Matsumoto T. Nishiya K. Tanaka I. Yoshioka A. Mechanisms of plasmin-catalyzed inactivation of factor VIII: a crucial role for proteolytic cleavage at Arg336 responsible for plasmin-catalyzed factor VIII inactivation.J. Biol. Chem. 2007; 282: 5287-5295Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Apart from protease-assisted inactivation of the cofactor, spontaneous dissociation of the A2-a2 domain from the A1-a1/A3-C1-C2 dimer also leads to dampening of the activity of the FXa-generating complex (17.Lollar P. Parker E.T. Structural basis for the decreased procoagulant activity of human factor VIII compared with the porcine homolog.J. Biol. Chem. 1991; 266: 12481-12486Abstract Full Text PDF PubMed Google Scholar, 18.Lollar P. Parker C.G. pH-dependent denaturation of thrombin-activated porcine factor VIII.J. Biol. Chem. 1990; 265: 1688-1692Abstract Full Text PDF PubMed Google Scholar, 19.Fay P.J. Haidaris P.J. Smudzin T.M. Human factor VIIIa subunit structure: reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit.J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar). It has been demonstrated that residues that control the dissociation rate of the A2-a2 domain from A1-a1/A3-C1-C2 dimer, and as such affect the stability of FVIIIa, are of critical importance for cofactor function (20.Gale A.J. Radtke K.P. Cunningham M.A. Chamberlain D. Pellequer J.L. Griffin J.H. Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants.J. Thromb. Haemost. 2006; 4: 1315-1322Crossref PubMed Scopus (37) Google Scholar, 21.Wakabayashi H. Fay P.J. Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa.J. Biol. Chem. 2008; 283: 11645-11651Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). For a number of hemophilia A variants carrying single amino acid substitutions, it has been suggested that a decreased stability of FVIIIa is the cause for the bleeding disorder (21.Wakabayashi H. Fay P.J. Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa.J. Biol. Chem. 2008; 283: 11645-11651Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 22.Kemball-Cook G. Tuddenham E.G. Wacey A.I. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4.Nucleic Acids Res. 1998; 26: 216-219Crossref PubMed Scopus (211) Google Scholar). Employing molecular modeling studies on the available FVIII structures in combination with site-directed mutagenesis, Fay and co-workers have now successfully identified several amino acid residues that enhance or decrease the stability of FVIII (21.Wakabayashi H. Fay P.J. Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa.J. Biol. Chem. 2008; 283: 11645-11651Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 23.Wakabayashi H. Griffiths A.E. Fay P.J. Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations.J. Thromb. Haemost. 2009; 7: 438-444Crossref PubMed Scopus (22) Google Scholar, 24.Wakabayashi H. Varfaj F. Deangelis J. Fay P.J. Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface.Blood. 2008; 112: 2761-2769Crossref PubMed Scopus (36) Google Scholar, 25.Wakabayashi H. Griffiths A.E. Fay P.J. Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability.J. Biol. Chem. 2011; 286: 25748-25755Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The recently solved crystal structures of FVIII have also assisted the biochemical studies addressing FVIIIa stability (26.Ngo J.C. Huang M. Roth D.A. Furie B.C. Furie B. Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex.Structure. 2008; 16: 597-606Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 27.Shen B.W. Spiegel P.C. Chang C.H. Huh J.W. Lee J.S. Kim J. Kim Y.H. Stoddard B.L. The tertiary structure and domain organization of coagulation factor VIII.Blood. 2008; 111: 1240-1247Crossref PubMed Scopus (201) Google Scholar). They have, for instance, served as a basis for molecular dynamics studies from which conclusions have been drawn about the critical contacts between the individual FVIII domains (28.Venkateswarlu D. Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study.BMC Struct. Biol. 2010; 10: 7Crossref PubMed Scopus (16) Google Scholar). Yet, a limitation of the crystal structures is their relatively low resolution. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to FVIIIa stability. We hypothesized that those lysine residues, which are buried in FVIII and not in dissociated FVIIIa (dis-FVIIIa), may contribute to the interaction between the A2-a2 domain and the A1-a1/A3-C1-C2 dimer. To identify these residues, we took advantage of the notion that buried lysine residues are less prone to chemical modification than those that are exposed to the protein surface (29.Shkriabai N. Datta S.A. Zhao Z. Hess S. Rein A. Kvaratskhelia M. Interactions of HIV-1 Gag with assembly cofactors.Biochemistry. 2006; 45: 4077-4083Crossref PubMed Scopus (121) Google Scholar). The lysines with the largest change in surface exposure upon FVIIIa dissociation were therefore evaluated for their contribution to the stability of FVIIIa. HEPES was from SERVA (Heidelberg, Germany), NaCl was from FAGRON (Rotterdam, The Netherlands) and Tris-HCl from Invitrogen. All other fine chemicals were from Merck. Monoclonal antibodies EL14, KM33, and CLB-CAg 9 have been described previously (30.Meems H. Meijer A.B. Cullinan D.B. Mertens K. Gilbert G.E. Factor VIII C1 domain residues Lys2092 and Phe2093 contribute to membrane binding and cofactor activity.Blood. 2009; 114: 3938-3946Crossref PubMed Scopus (55) Google Scholar, 31.van Helden P.M. van den Berg H.M. Gouw S.C. Kaijen P.H. Zuurveld M.G. Mauser-Bunschoten E.P. Aalberse R.C. Vidarsson G. Voorberg J. IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A.Br. J. Haematol. 2008; 142: 644-652Crossref PubMed Scopus (80) Google Scholar, 32.Stel, H. V., (1984) Monoclonal antibodies against factor VIII-von Willebrand factor. Ph.D. thesis, University of Amsterdam, 1984,.Google Scholar). B domain-deleted FVIII and the FVIII variants thereof have been constructed and purified as described (10.Meems H. van den Biggelaar M. Rondaij M. van der Zwaan C. Mertens K. Meijer A.B. C1 domain residues Lys2092 and Phe2093 are of major importance for the endocytic uptake of coagulation factor VIII.Int. J. Biochem. Cell Biol. 2011; 43: 1114-1121Crossref PubMed Scopus (33) Google Scholar, 33.van den Biggelaar M. Meijer A.B. Voorberg J. Mertens K. Intracellular cotrafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles.Blood. 2009; 113: 3102-3109Crossref PubMed Scopus (30) Google Scholar) with the exception that FVIII was stored in 50 mm HEPES (pH 7.4), 0.8 m NaCl, 5 mm CaCl2, and 50% glycerol. Plasma-derived FVIII was purified from Aafact (Sanquin, Amsterdam) according to the same procedure. To this end, Aafact was taken up in 50 mm imidazole (pH 6.7), 50 mm CaCl2, and 0.8 m NaCl prior to purification. The purification of FX, FIXa, and thrombin is described by Mertens and Bertina (34.Mertens K. Bertina R.M. Pathways in the activation of human coagulation factor X.Biochem. J. 1980; 185: 647-658Crossref PubMed Scopus (51) Google Scholar). FXa was from Enzyme Research (South Bend, IN). 70 nm plasma-derived FVIII was incubated with 2 nm thrombin in 50 mm HEPES (pH 7.4), 150 mm NaCl, and 5 mm CaCl2 for 2 h at 37 °C to ensure dissociation. Thrombin activation was terminated by the addition of 1.6 units/ml hirudin. FVIII was chemically modified by tandem mass tag (TMT)-127 for 2 h at 37 °C (Thermo Fisher Scientific) according to instructions of the manufacturer for labeling whole proteins. 100 mm lysine dissolved in 140 mm Tris-HCl was employed to stop the reaction. To label nonactivated FVIII with TMT-126, FVIII was first incubated with hirudin and then with thrombin prior to the addition of TMT-126. The TMT-labeled proteins were pooled in a one-to-one molar ratio, and the free cysteines were alkylated employing 2.5 mm dithiothreitol for 15 min at 50 °C followed by a 30-min incubation at 37 °C with 15 mm iodoacetamide in a buffer containing 50 mm ammonium bicarbonate and 2 mm CaCl2. Finally, the sample was cleaved by the addition of 0.1 μg chymotrypsin for an overnight incubation at 37 °C. Obtained peptides were concentrated and washed employing a C18 Ziptip (Millipore Corporation) according to the instructions of the manufacturer. FVIII peptides were separated by reverse-phase chromatography and sprayed in a LTQ OrbitrapXL mass spectrometer (Thermo Fisher Scientific) essentially as described (35.van Haren S.D. Herczenik E. ten Brinke A. Mertens K. Voorberg J. Meijer A.B. HLA-DR-presented peptide repertoires derived from human monocyte-derived dendritic cells pulsed with blood coagulation factor VIII.Mol. Cell Proteomics. 2011; 10 (M110.002246)Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). During reverse-phase chromatography, we employed a 40-min gradient from 0 to 35% (v/v) acetonitrile with 0.05% (v/v) acetic acid. Collision-induced dissociation (CID) spectra and higher energy collision-induced dissociation (HCD) spectra were acquired as described by Dayon et al. (36.Dayon L. Pasquarello C. Hoogland C. Sanchez J.C. Scherl A. Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags.J. Proteomics. 2010; 73: 769-777Crossref PubMed Scopus (96) Google Scholar). The three most intense precursor ions in the full scan (300–2000 m/z, resolving power 30,000) with a charge state of 2+ or higher were selected for CID using an isolation width of 2 Da, a 35% normalized collision energy, and an activation time of 30 ms. The same precursor ions were subjected to HCD with a normalized collision energy of 60%, which allows for the identification of the reporter group from the TMT label. The identity of the peptides, including TMT labeled lysine residues, and the TMT-127/TMT-126 ratio thereof were assessed employing Proteome discoverer 1.1 software (Thermo Fisher Scientific). The SEQUEST search algorithm and protein data base 25.H_sapiens.fasta were used to identify the peptides. The presence of the TMT label on peptides generates additional ion fragments in the tandem MS spectra that negatively influence the SEQUEST Xcorr score (37.Thingholm T.E. Palmisano G. Kjeldsen F. Larsen M.R. Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency.J. Proteome Res. 2010; 9: 4045-4052Crossref PubMed Scopus (100) Google Scholar). We therefore employed the following peptide selection criteria: (i) the peptide is identified in three of four independent experiments; (ii) the peptide is identified from both the CID and the HCD spectrum; (iii) lysine residues within the peptide are all modified by a TMT label (+225.1558 Da) and the cysteines are alkylated; (iv) peptides that included (or are close to) a thrombin cleavage site were excluded from the analysis. TMT ratios were normalized to the average ratio obtained for all peptides within one experiment. Factor Xase activity measurement and the preparation of phospholipid vesicles were performed as described before (30.Meems H. Meijer A.B. Cullinan D.B. Mertens K. Gilbert G.E. Factor VIII C1 domain residues Lys2092 and Phe2093 contribute to membrane binding and cofactor activity.Blood. 2009; 114: 3938-3946Crossref PubMed Scopus (55) Google Scholar). Briefly, 25 μm sonicated phospholipid vesicles comprising 15% phosphatidylserine, 20% phosphatidylethanolamine, and 65% phosphatidylcholine were mixed with 0.3 nm FVIII, 200 nm FX, and 0–16 nm FIXa in a buffer containing 40 mm Tris-HCl (pH 7.8), 150 mm NaCl, 0.2% (w/v) bovine serum albumin (BSA) (Merck). The reaction was started with the addition of 1.5 mm CaCl2 and 1 nm thrombin. The amount of FXa generated per minute was subsequently assessed as described (30.Meems H. Meijer A.B. Cullinan D.B. Mertens K. Gilbert G.E. Factor VIII C1 domain residues Lys2092 and Phe2093 contribute to membrane binding and cofactor activity.Blood. 2009; 114: 3938-3946Crossref PubMed Scopus (55) Google Scholar). For the FVIIIa stability analysis, the FVIII variants were first incubated at varying time intervals with 2 nm thrombin and 1.5 mm CaCl2 in the presence and absence of increasing concentrations of FIXa (0–16 nm) at 25 °C in 40 mm Tris-HCl (pH 7.8), 150 mm NaCl, 0.2% (w/v) BSA, and 25 μm phospholipid vesicles. The generated amount of FXa was assessed as described (30.Meems H. Meijer A.B. Cullinan D.B. Mertens K. Gilbert G.E. Factor VIII C1 domain residues Lys2092 and Phe2093 contribute to membrane binding and cofactor activity.Blood. 2009; 114: 3938-3946Crossref PubMed Scopus (55) Google Scholar). Residual FVIII activity was derived from the initial rate of FXa formation as a function of the FIXa concentration. This residual FVIIIa activity was assessed 2, 5, 7.5, 12.5, 20, and 40 min after thrombin activation of at least three independent experiments. The inactivation rate constant, kinact, was estimated by fitting the mean data to a one-phase exponential decay equation (24.Wakabayashi H. Varfaj F. Deangelis J. Fay P.J. Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface.Blood. 2008; 112: 2761-2769Crossref PubMed Scopus (36) Google Scholar) by nonlinear least square regression analysis using Prism 5 (GraphPad Software). Surface plasmon resonance analysis was performed on a BIAcoreTM3000 system (Uppsala, Sweden) essentially as described (10.Meems H. van den Biggelaar M. Rondaij M. van der Zwaan C. Mertens K. Meijer A.B. C1 domain residues Lys2092 and Phe2093 are of major importance for the endocytic uptake of coagulation factor VIII.Int. J. Biochem. Cell Biol. 2011; 43: 1114-1121Crossref PubMed Scopus (33) Google Scholar, 33.van den Biggelaar M. Meijer A.B. Voorberg J. Mertens K. Intracellular cotrafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles.Blood. 2009; 113: 3102-3109Crossref PubMed Scopus (30) Google Scholar). EL14 was first coupled to a CM5 chip (GE Healthcare) to a density of 5000 response units (RU) employing the amide coupling kit as prescribed by the manufacturer. FVIII WT, K1967A, and K1968A were bound to immobilized EL14 by passing the proteins over EL14 at a flow rate of 20 μl/min 20 mm HEPES (pH 7.4), 150 mm NaCl, 5 mm CaCl2 and 0.005% Tween 20. After thrombin activation on the chip, the remaining FVIII light chain and/or A2 domain was assessed by passing either 100 nm KM33 or 100 nm CLB-CAg 9 over the immobilized proteins at a flow rate of 20 μl/min in the same buffer as described above. To identify FVIII lysine residues that are less prone to chemical modification in FVIII than in dis-FVIIIa, we labeled the lysine residues in plasma-derived FVIII with TMT-126 and those in dis-FVIIIa with TMT-127. Labeled FVIII and dis-FVIIIa were subsequently pooled and cleaved into peptides by chymotrypsin. A unique property of the TMT labels is that the same peptide ions carrying a TMT-126 or a TMT-127 exhibit an identical mass. However, fragmentation of these ions employing HCD generates a reporter ion from the TMT label as well as a mass spectrum of the peptide. The intensity of the signals of the reporter ions within each mass spectrum allows for calculating the TMT-127/TMT-126 ratio of the lysine-containing peptides (36.Dayon L. Pasquarello C. Hoogland C. Sanchez J.C. Scherl A. Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags.J. Proteomics. 2010; 73: 769-777Crossref PubMed Scopus (96) Google Scholar, 38.Thompson A. Schäfer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Johnstone R. Mohammed A.K. Hamon C. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 75: 1895-1904Crossref PubMed Scopus (1717) Google Scholar). As such, it can be assessed whether a lysine residue is more readily labeled in dis-FVIIIa or in FVIII (supplemental Fig. S1). To facilitate peptide identification, we also performed CID of the peptide ions. The above-mentioned approach was employed on plasma-derived FVIII. Fig. 1A shows the identified peptides that comprise TMT-modified lysine residues. Peptide identification scores are shown in supplemental Table S1. The average TMT-127/TMT-126 ratio obtained from peptides comprising the same TMT-modified lysine residues are displayed in Fig. 1B. The result reveals that most of the identified peptides exhibit an increased TMT-127/TMT-126 ratio. However, region Thr1964-Tyr1971 comprising the residues Lys1967 and Lys1968 was most prone to chemical modification in dis-FVIIIa (supplemental Fig. S2). This finding implies that Lys1967 and Lys1968 are buried within the protein core in FVIII and not in dis-FVIIIa. The mass spectrometry results suggest that the lysine residues 1967 and 1968 may contribute to the stability of FVIIIa. Intriguingly, replacement of Lys1967 with an isoleucine has been associated with mild hemophilia A, implying that this lysine may also directly contribute to cofactor activity (22.Kemball-Cook G. Tuddenham E.G. Wacey A.I. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4.Nucleic Acids Res. 1998; 26: 216-219Crossref PubMed Scopus (211) Google Scholar). Replacement of Lys1967 by an alanine in FVIII, however, did not result in a marked change in the specific one-stage clotting activity and the specific two-stage chromogenic activity. In addition, the relative specific activities of a K1967I FVIII variant were still 70% of FVIII WT. Surprisingly, a K1968A variant revealed a moderate (if any) increase in the specific chromogenic activity and a 2-fold increase in specific clotting activity (supplemental Table S2). To assess FVIII activity under more defined experimental conditions, we next evaluated the FVIII variants for their ability to enhance the activity of the FXa-generating complex employing purified proteins. To this end, FXa generation was assessed at increasing concentrations of FIXa in the presence of the activated FVIII variants (Fig. 2). The results showed that there was only a small difference in the efficiency of FXa generation for K1967I and K1967A compared with that of FVIII WT. However, K1968A displayed a markedly more effective FXa generation relative to FVIII WT. This was reflected by an almost 2-fold decrease in the apparent affinity constant (KD) for FIXa binding (KD = 2 nm for FVIII WT and 1.2 nm for K1968A) and an increased maximum rate of FXa generation. The finding that the hemophilic K1967I variant displays almost normal cofactor function suggests that Lys1967 may indeed contribute to the stability of FVIIIa. Because the A2 domain dissociation from FVIIIa inactivates the cofactor, we addressed whether or not mutation of the residues 1967 and 1968 affects the activity of FVIIIa over time. To this end, we activated FVIII WT and variants thereof with thrombin, and we measured the time-dependent decrease in cofactor activity at increasing FIXa concentrations. As expected, the results showed that there was a decline in cofactor activity over time for FVIII WT (Fig. 3). However, the rate of inactivation was about 3-fold increased for K1967I and 2-fold for K1967A. Strikingly, K1968A showed an about 3-fold decrease in the rate of inactivation relative to that of FVIIIa WT (Fig. 3E). This finding suggests that there is a decreased" @default.
• W2040257513 created "2016-06-24" @default.
• W2040257513 creator A5026414692 @default.
• W2040257513 creator A5026910493 @default.
• W2040257513 creator A5042863699 @default.
• W2040257513 creator A5056038004 @default.
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• W2040257513 date "2012-02-01" @default.
• W2040257513 modified "2023-10-18" @default.
• W2040257513 title "Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII" @default.
• W2040257513 cites W1497014448 @default.
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