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• W2013179005 abstract "SummaryBackground: Factor VIII (FVIII) is activated by thrombin to the labile FVIIIa, a heterotrimer of A1, A2 and A3C1C2 subunits, which serves as a cofactor for FIXa. A primary reason for the instability of FVIIIa is the tendency for the A2 subunit to dissociate from FVIIIa leading to an inactive cofactor and consequent loss of FXase activity. Objective: Based on our finding of low‐specific activity and a fast decay rate for a FVIII point mutation of Glu1829 to Ala (E1829A), we examined whether residue Glu1829 in the A3 subunit is important for A2 subunit retention. Results: The rate of activity decay of E1829A was ∼fourteenfold faster than wild‐type (wt) FVIIIa and this rate was reduced in the presence of added A2 subunit. Specific activity values for E1829A measured by one‐stage and two‐stage assays were ∼14% and ∼11%, respectively, compared with wt FVIII. Binding affinity for the A1 subunit to E1829A‐A3C1C2 was comparable to wt A3C1C2 (Kd = 20.1 ± 3.4 nm for E1829A, 15.3 ± 3.7 nm for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by ∼3.6‐fold as a result of the mutation (Kd = 526 ± 107 nm for E1829A, 144 ± 21 nm for wt). Conclusion: As modeling data suggest that Glu1829 is located at the A2‐A3 domain interface these results are consistent with Glu1829 contributing to the interactions involved with A2 subunit retention in FVIIIa. Background: Factor VIII (FVIII) is activated by thrombin to the labile FVIIIa, a heterotrimer of A1, A2 and A3C1C2 subunits, which serves as a cofactor for FIXa. A primary reason for the instability of FVIIIa is the tendency for the A2 subunit to dissociate from FVIIIa leading to an inactive cofactor and consequent loss of FXase activity. Objective: Based on our finding of low‐specific activity and a fast decay rate for a FVIII point mutation of Glu1829 to Ala (E1829A), we examined whether residue Glu1829 in the A3 subunit is important for A2 subunit retention. Results: The rate of activity decay of E1829A was ∼fourteenfold faster than wild‐type (wt) FVIIIa and this rate was reduced in the presence of added A2 subunit. Specific activity values for E1829A measured by one‐stage and two‐stage assays were ∼14% and ∼11%, respectively, compared with wt FVIII. Binding affinity for the A1 subunit to E1829A‐A3C1C2 was comparable to wt A3C1C2 (Kd = 20.1 ± 3.4 nm for E1829A, 15.3 ± 3.7 nm for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by ∼3.6‐fold as a result of the mutation (Kd = 526 ± 107 nm for E1829A, 144 ± 21 nm for wt). Conclusion: As modeling data suggest that Glu1829 is located at the A2‐A3 domain interface these results are consistent with Glu1829 contributing to the interactions involved with A2 subunit retention in FVIIIa. Factor VIII (FVIII), a plasma protein that participates in the blood coagulation cascade, is decreased or defective in individuals with hemophilia A. FVIII functions as a cofactor for the serine protease FIXa in the membrane surface‐dependent conversion of zymogen FX to the serine protease, FXa [1Davie E.W. Biochemical and molecular aspects of the coagulation cascade.Thromb Haemost. 1995; 74: 1-6Crossref PubMed Scopus (244) Google Scholar, 2Lollar P. Structure and function of Factor VIII.Adv Exp Med Biol. 1995; 386: 3-17Crossref PubMed Scopus (19) Google Scholar]. Deficiency of FVIII causes marked reductions in FIXa activity and in subsequent rates of FXa generation. FVIII is synthesized as an ∼300‐kDa single‐chain precursor protein [3Wood 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. Expression of active human factor VIII from recombinant DNA clones.Nature. 1984; 312: 330-7Crossref PubMed Scopus (521) Google Scholar, 4Toole 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. Molecular cloning of a cDNA encoding human antihaemophilic factor.Nature. 1984; 312: 342-7Crossref PubMed Scopus (660) Google Scholar] with a domain structure designated as A1‐A2‐B‐A3‐C1‐C2 [5Vehar 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: 337-42Crossref PubMed Scopus (658) Google Scholar]. FVIII is processed to a series of divalent metal ion‐dependent heterodimers [6Fass D.N. Knutson G.J. Katzmann J.A. Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein.Blood. 1982; 59: 594-600Crossref PubMed Google Scholar, 7Andersson L.O. Forsman N. Huang K. Larsen K. Lundin A. Pavlu B. Sandberg H. Sewerin K. Smart J. Isolation and characterization of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma.Proc Natl Acad Sci U S A. 1986; 83: 2979-83Crossref PubMed Scopus (104) Google Scholar, 8Fay P.J. Anderson M.T. Chavin S.I. Marder V.J. The size of human factor VIII heterodimers and the effects produced by thrombin.Biochim Biophys Acta. 1986; 871: 268-78Crossref PubMed Scopus (123) Google Scholar] by cleavage at the B‐A3 junction, generating a heavy chain (HC) minimally represented by the Al‐A2 domains; and a light chain (LC) consisting of the A3, C1 and C2 domains. Thrombin cleaves FVIII HC at Arg‐740, removing the B domain (or fragments) and at Arg‐372, bisecting the HC into the A1 and the A2 subunits [9Eaton 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: 505-12Crossref PubMed Scopus (397) Google Scholar]. The protease also cleaves FVIII LC at Arg‐1689 [9Eaton 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: 505-12Crossref PubMed Scopus (397) Google Scholar], liberating an acidic residue‐rich region and creating a new NH2 terminus. Thus, the FVIIIa heterotrimer is composed of A1, A2 and A3C1C2 subunits with the A2 subunit being weakly associated with the A1/A3C1C2 dimer by primarily electrostatic interactions [10Fay 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: 8957-62Abstract Full Text PDF PubMed Google Scholar, 11Lollar P. Parker C.G. pH‐dependent denaturation of thrombin‐activated porcine factor VIII.J Biol Chem. 1990; 265: 1688-92Abstract Full Text PDF PubMed Google Scholar]. This weak A2 subunit interaction accounts for the instability of FVIIIa activity. In fact, covalent association of A2 and A3 subunits by creating nascent disulfide bonds after mutagenesis to replace adjacent residues in the A2 and A3 domains with Cys residues [12Gale A.J. Pellequer J.L. An engineered interdomain disulfide bond stabilizes human blood coagulation factor VIIIa.J Thromb Haemost. 2003; 1: 1966-71Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar] or by deleting cleavage sites as well as a large portion of the B domain between the A2 and A3 domains [13Pipe S.W. Kaufman R.J. Characterization of a genetically engineered inactivation‐resistant coagulation factor VIIIa.Proc Natl Acad Sci U S A. 1997; 94: 11851-6Crossref PubMed Scopus (102) Google Scholar] generate recombinant FVIII molecules possessing more stable cofactor activity as a result of limited A2 subunit dissociation. Several FVIII point mutations at the A1‐A2 interface or A2‐A3 interface have been shown to facilitate A2 dissociation compared with wild‐type (wt) FVIII. These FVIII mutants include R531H, A284E, A284P and S289L at the modeled A1‐A2 domain interface [14Pipe S.W. Eickhorst A.N. McKinley S.H. Saenko E.L. Kaufman R.J. Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo.Blood. 1999; 93: 176-83Crossref PubMed Google Scholar, 15Pipe S.W. Saenko E.L. Eickhorst A.N. Kemball‐Cook G. Kaufman R.J. Hemophilia A mutations associated with 1‐stage/2‐stage activity discrepancy disrupt protein‐protein interactions within the triplicated A domains of thrombin‐activated factor VIIIa.Blood. 2001; 97: 685-91Crossref PubMed Scopus (84) Google Scholar] and N694I, R698L and R698W at the A2‐A3 domain interface [16Hakeos W.H. Miao H. Sirachainan N. Kemball‐Cook G. Saenko E.L. Kaufman R.J. Pipe S.W. Hemophilia A mutations within the factor VIII A2‐A3 subunit interface destabilize factor VIIIa and cause one‐stage/two‐stage activity discrepancy.Thromb Haemost. 2002; 88: 781-7Crossref PubMed Scopus (42) Google Scholar]. These molecules characteristically demonstrate a one‐stage/two‐stage assay discrepancy with as much as 50% reduced activity determined using a two‐stage assay compared with the one‐stage assay. This effect directly relates to enhanced rates of loss of A2 subunit from FVIIIa, which has a more pronounced impact on activity values determined by the two‐stage assay. Metal ions such as Ca2+ and Cu2+/+ play important roles in FVIII structure and activity [17Fay P.J. Smudzin T.M. Intersubunit fluorescence energy transfer in human factor VIII.J Biol Chem. 1989; 264: 14005-10Abstract Full Text PDF PubMed Google Scholar, 18Fay P.J. Reconstitution of human factor VIII from isolated subunits.Arch Biochem Biophys. 1988; 262: 525-31Crossref PubMed Scopus (61) Google Scholar, 19Nordfang O. Ezban M. Generation of active coagulation factor VIII from isolated subunits.J Biol Chem. 1988; 263: 1115-8Abstract Full Text PDF PubMed Google Scholar, 20Wakabayashi H. Koszelak M.E. Mastri M. Fay P.J. Metal ion‐independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter‐subunit affinity.Biochemistry. 2001; 40: 10293-300Crossref PubMed Scopus (55) Google Scholar]. Recent mutagenesis studies defined a Ca2+ binding site contained within a cluster of acidic residues in the A1 domain (residues 110–126) [21Wakabayashi H. Freas J. Zhou Q. Fay P.J. Residues 110‐126 in the A1 domain of factor VIII contain a Ca2+‐binding site required for cofactor activity.J Biol Chem. 2004; 279: 12677-84Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. In exploring other potential Ca2+ binding sites in FVIII, we prepared a number of point mutants in which single acidic residues were individually changed into Ala. Within one region of clustered acidic residues in the A3 domain, we observed atypical reduction in specific activity and an increase in the decay rate of FVIIIa activity for the E1829A mutant. In this study, we demonstrate that mutation of Glu1829 to Ala results in a significant decrease in the affinity of the A2 subunit for the A1/A3C1C2 dimer, whereas no effect on the interaction of A3C1C2 with A1 subunit was observed. This defective A2 interaction promotes a low‐specific activity and enhanced rate of FVIIIa decay. Recombinant FVIII (Kogenate™) was a gift from Dr Lisa Regan of Bayer Corporation (Berkeley, CA, USA). Phospholipid vesicles containing 20% phosphatidylcholine (PC), 40% phosphatidylethanolamine (PE) and 40% phosphatidylserine (PS) were prepared using octylglucoside as described previously [22Mimms L.T. Zampighi G. Nozaki Y. Tanford C. Reynolds J.A. Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside.Biochemistry. 1981; 20: 833-40Crossref PubMed Scopus (557) Google Scholar]. The reagents α‐thrombin, FIXaβ, FX and FXa (Enzyme Research Laboratories, South Bend, IN, USA), hirudin and phospholipids (DiaPharma, West Chester, OH, USA), the chromogenic Xa substrate, Pefachrome Xa (Pefa‐5523, CH3OCO‐D‐CHA‐Gly‐Arg‐pNA·AcOH; Pantapharm, Basel, Switzerland), and Phe‐Pro‐Arg‐chloromethylketone (PPACK; Calibiochem, La Jolla, CA, USA) were purchased from the indicated vendors. FVIII HC and LC subunits were isolated from FVIII as previously described [20Wakabayashi H. Koszelak M.E. Mastri M. Fay P.J. Metal ion‐independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter‐subunit affinity.Biochemistry. 2001; 40: 10293-300Crossref PubMed Scopus (55) Google Scholar]. FVIII A1 and A2 subunits were prepared from HC as follows. HC was digested with 20 nm thrombin in 20 mm HEPES, pH 7.2, 0.1 m NaCl, 0.02% Tween‐20, 2.5 mm CaCl2 for 30 min at 23°C. After quenching with 200 nm PPACK, the sample was applied to a HiTrap Heparin column (GE Healthcare, Piscataway, NJ, USA) using fast protein liquid chromatography (FPLC). The A2 subunit was eluted using a linear NaCl gradient (0.1–1 m). The flow‐through fraction containing A1 was then applied to MonoQ and the A1 subunit was eluted with a linear NaCl gradient (0.1–1 m). The A3C1C2 subunit was purified from LC after thrombin digestion [23Fay P.J. Mastri M. Koszelak M.E. Wakabayashi H. Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA.J Biol Chem. 2001; 276: 12434-9Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. Proteins were dialyzed into 10 mm 2‐[N‐morpholino]ethanesulfonic acid (MES), 0.3 m KCl, 0.01% Tween‐20, pH 6.5 (Buffer A), and stored at −80°C. The protein concentration was determined by the Bradford protein assay [24Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding.Anal Biochem. 1976; 72: 248-54Crossref PubMed Scopus (216428) Google Scholar] using bovine serum albumin (BSA) as a standard. Glu1829Ala and wt FVIII forms were constructed as a B‐domainless FVIII, stably expressed in BHK cells, and purified as described previously [21Wakabayashi H. Freas J. Zhou Q. Fay P.J. Residues 110‐126 in the A1 domain of factor VIII contain a Ca2+‐binding site required for cofactor activity.J Biol Chem. 2004; 279: 12677-84Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. Resultant FVIII forms were typically > 90% pure as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). FVIII concentration was measured by enzyme‐linked immunosorbent assay (ELISA) (see below), and FVIII activity was determined by one‐stage clotting assay and a two‐stage chromogenic FXa generation assay (see below). A sandwich ELISA was performed to measure the concentration of FVIII proteins as described before using purified commercial recombinant FVIII (Kogenate; Bayer Corporation) as a standard [25Wakabayashi H. Su Y.C. Ahmad S.S. Walsh P.N. Fay P.J. A Glu113Ala mutation within a factor VIII Ca(2 + )‐binding site enhances cofactor interactions in factor Xase.Biochemistry. 2005; 44: 10298-304Crossref PubMed Scopus (19) Google Scholar]. One‐stage clotting assays were performed using substrate plasma chemically depleted of FVIII and assayed in a Diagnostica Stago clotting instrument (Diagnostica Stago, Inc., Parsippany, NJ, USA) [26Over J. Methodology of the one‐stage assay of Factor VIII (VIII:C).Scand J Haematol Suppl. 1984; 41: 13-24PubMed Google Scholar]. Plasma was incubated with activated partial thromboplastin time (APTT) reagent (bioMerieux, Durham, NC, USA) for 6 min at 37°C at which time a dilution of FVIII was added to the cuvette. After 1 min the mixture was recalcified and time‐to‐clot formation determined and compared with a pooled normal plasma standard. The rate of conversion of FX to FXa was monitored in a purified system [27Lollar P. Fay P.J. Fass D.N. Factor VIII and factor VIIIa.Meth Enzymol. 1993; 222: 128-43Crossref PubMed Scopus (93) Google Scholar] according to methods previously described [20Wakabayashi H. Koszelak M.E. Mastri M. Fay P.J. Metal ion‐independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter‐subunit affinity.Biochemistry. 2001; 40: 10293-300Crossref PubMed Scopus (55) Google Scholar, 28Wakabayashi H. Schmidt K.M. Fay P.J. Ca(2 + ) binding to both the heavy and light chains of factor VIII is required for cofactor activity.Biochemistry. 2002; 41: 8485-92Crossref PubMed Scopus (25) Google Scholar]. Briefly, FVIII (1 nm) in buffer containing 20 mm N‐[2‐hydroxyethyl]piperazine‐N′‐[2‐ethanesulfonic acid] (HEPES), pH 7.2, 0.1 m NaCl, 0.01% Tween‐20, 0.01% BSA, 5 mm CaCl2, (Buffer B) and 10 μm PSPCPE vesicles was activated with 2 nmα‐thrombin for 1 min. The reaction was stopped by adding 5 U mL−1 hirudin and the resultant FVIIIa was reacted with FIXa (40 nm) for 1 min. FX (300 nm) was added to initiate the reaction and after 1 min, the reaction was quenched by addition of 50 mm ethylenediaminetetraacetic acid (EDTA). Amounts of FXa generated were determined after reaction with the chromogenic substrate Pefachrome Xa (0.46 mm final concentration). All reactions were run at 23°C. Activity is expressed as the amount of FXa generated (nm) per minute and converted to a value per nm FVIII subunit. FVIIIa activity decay was monitored using a modified FXa generation assay. FVIII (2 nm) in the presence of 40 nm FIXa in buffer B was mixed with 2 nmα‐thrombin for 1 min. The reaction was quenched by 5 U mL−1 hirudin, and aliquots were removed over a 30‐min time course and rates of FXa generation were determined. FVIIIa activity values as a function of time were fitted to a single exponential decay curve by non‐linear least square regression using the equation, 1 where A is residual FVIIIa activity (nm min−1 nm−1 HC), A0 is the initial activity, k is the apparent rate constant, and t is the time in minutes after thrombin activation was quenched. The binding of the A3C1C2 subunit purified from wt or E1829A FVIII with the A1 subunit was performed as follows. A3C1C2 subunits (10 nm) purified from wt and E1829A FVIII were reconstituted with 0–200 nm A1 subunit for 18 h in buffer A. Following subsequent incubation with the A2 subunit (400 nm) for 20 min, the mixture was diluted (1/20) in buffer B and reconstituted FVIIIa activity was measured by FXa generation assay. Data points were fitted to a quadratic equation by non‐linear least squares regression using the equation, 2 where A is reconstituted FVIIIa activity (nm min−1 nm−1 A3C1C2), [A3]0 is the concentration of added A3C1C2 (10 nm), [A1]0 is the concentration of added A1 (0–200 nm), Kd is the dissociation constant and Vmax is activity when A3C1C2 is saturated by A1. The binding of A1/A3C1C2 dimer with the A2 subunit was performed as follows. The A1/A3C1C2 dimer was reconstituted by mixing A1 (100 nm) and A3C1C2 (1 μm) purified from wt or E1829A FVIII in buffer A for 18 h. After dilution (1/10) in buffer B, 0–1 μm A2 was added and incubated for 20 min, and reconstituted FVIIIa activity was measured using the FXa generation assay. Data points were fitted to a quadratic equation by non‐linear least squares regression using the equation 2 after substitution of [A1]0 with the added A2 concentration and substitution of [A3]0 with the added A1 concentration. Non‐linear least‐squares regression analysis was performed by Kaleidagraph (Synergy, Reading, PA, USA). Comparison of average values was performed by the Student’s t‐test. In assessing a number of point mutations where acidic residues in the A3 domain were individually replaced with Ala and the proteins stably expressed and purified, we identified one such FVIII mutant, E1829A as showing a marked reduction in FVIII specific activity and an accelerated rate in activity decay after activation (see below). Specific activity values obtained for other purified point mutants in this region including D1828A, E1969A, E1970A, E1984A and E1987A were >60% the wt level of activity (data not shown) consistent with essentially benign effects of these mutations. The relative activity for the E1829A mutant was fractional to that obtained for the wt FVIII (P< 0.01, Table 1) as judged by both one‐ and two‐stage assays, with results from the latter assay representing < 80% of the activity value obtained by former assay (Table 1). Control experiments showed the mutant FVIII was activated by thrombin with the same kinetics as judged by peak activity generation and cleavages in the FVIII heavy and light chains (results not shown). Thus the observations that this mutation resulted in both a marked reduction in FVIII specific activity and FVIIIa decay compared with the wt protein, as well as a potential one‐stage/two‐stage assay discrepancy, suggested that the reduced activity value reflected an impaired interaction of the A2 subunit within FVIIIa.Table 1Relative activity obtained by one‐stage clotting and two‐stage factor Xa (FXa) generation assayswtE1829AOne‐stage clotting assay100 ± 2.014.9 ± 0.3*Two‐stage FXa generation assay100 ± 1.211.6 ± 0.8Activity values were measured as described in Materials and methods and are expressed as percent compared with the value of wild type (wt). *P< 0.01, compared with the activity determined by two‐stage FXa generation assay. Open table in a new tab Activity values were measured as described in Materials and methods and are expressed as percent compared with the value of wild type (wt). *P< 0.01, compared with the activity determined by two‐stage FXa generation assay. Attempts to accurately measure rates of activity decay for FVIII E1829A after activation by thrombin were hampered by the very low‐activity peak observed (∼10% that of wt) and its rapid decay (data not shown). Therefore, to better quantify these effects, thrombin activation experiments were performed in the presence of a twentyfold molar excess of FIXa and inclusion of phospholipid vesicles, based upon earlier observations that these conditions reduced FVIIIa lability [29Lollar P. Knutson G.J. Fass D.N. Stabilization of thrombin‐activated porcine factor VIII:C by factor IXa phospholipid.Blood. 1984; 63: 1303-8Crossref PubMed Google Scholar, 30Lamphear B.J. Fay P.J. Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3‐C1‐C2 dimer.J Biol Chem. 1992; 267: 3725-30Abstract Full Text PDF PubMed Google Scholar] by partially stabilizing the A2 subunit within FXase [31Fay P.J. Beattie T.L. Regan L.M. O’Brien L.M. Kaufman R.J. Model for the factor VIIIa‐dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa‐catalyzed proteolysis.J Biol Chem. 1996; 271: 6027-32Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar]. Using these conditions, the decay of the activity of FVIIIa E1829A could be quantified and the estimated apparent decay rate was ∼fourteenfold faster than that of wt (Fig. 1, Table 2). This result suggested the low steady‐state level of FVIIIa activity observed for the mutant in the absence of FIXa quickly reached this equilibrium as a result of a very fast rate of activity decay. Consistent with this observation, we noted that with the added A2 subunit (20 nm), the equilibrium activity of FVIIIa E1829A was ∼7‐fold higher (comparing the 8‐min time points with and without exogenous A2). Comparison of the slopes obtained from the initial portions of the time courses (≤ 5 min) showed the observed decay rate for FVIIIa E1829A was markedly decreased by the presence of excess A2. On the other hand, the activity of wt FVIIIa decayed at a much lower rate in the absence of the excess A2 subunit compared with the mutant and the effects observed in the presence of 20 nm exogenous A2 were more moderate, with an ∼1.4‐fold increase in the activity value at 30 min.Table 2Inter‐subunit binding and activity parameter valueswtE1829AApparent factor VIIIa activity decay rate (min−1)0.030 ± 0.0030.428 ± 0.026A1 and A3C1C2 interaction Kd (nm)15.3 ± 3.720.1 ± 3.4* Vmax (nm min−1 nm−1 A3C1C2)144 ± 891 ± 4A2 and A1/A3C1C2 interaction Kd (nm)144 ± 21526 ± 107† Vmax (nm min−1 nm−1 A1/A3C1C2)132 ± 5147 ± 13Apparent factor VIIIa (FVIIIa) activity decay rates were estimated by non‐linear least squares regression of the data shown in Fig. 1 as described in Materials and methods. The Kd and Vmax values for FVIII chain association were estimated by non‐linear least squares regression of the data shown in Fig. 2A (A1 and A3C1C2 interaction) and Fig. 2B (A2 and A1/A3C1C2 interaction) as described in Materials and methods. *P> 0.05, compared to wild‐type (wt). †P< 0.005, compared with wt. Open table in a new tab Apparent factor VIIIa (FVIIIa) activity decay rates were estimated by non‐linear least squares regression of the data shown in Fig. 1 as described in Materials and methods. The Kd and Vmax values for FVIII chain association were estimated by non‐linear least squares regression of the data shown in Fig. 2A (A1 and A3C1C2 interaction) and Fig. 2B (A2 and A1/A3C1C2 interaction) as described in Materials and methods. *P> 0.05, compared to wild‐type (wt). †P< 0.005, compared with wt. The above activity data were consistent with the E1829A mutation altering interaction of the A2 subunit with A1/A3C1C2 dimer in FVIIIa. To assess the separate interactions of the A1 subunit with the mutation‐containing A3C1C2 subunit and the A2 subunit with the A1/A3C1C2 dimer, we purified the A3C1C2 subunit from thrombin‐treated FVIII E1829A and from wt FVIII. These reagents were used in the presence of wt A1 and A2 subunits to reconstitute FVIIIa with titration of subunits performed to assess inter‐subunit affinity parameters. To evaluate the effects of the mutation on the interaction of A3C1C2 with the A1 subunit, variable levels of purified A1 subunit (0–200 nm) were reacted with either the mutant‐ or wt‐derived A3C1C2 (10 nm) as described in the Methods. After this reaction, an excess of the A2 subunit (400 nm) was added and the reconstituted FVIIIa was assessed for activity in a FXa generation assay. As only the intact heterotrimer demonstrates appreciable cofactor activity, the amount of FXa generated can be used as an indicator to approximate the extent of FVIIIa formed, which in this case represents a function of the concentration of the A1 subunit. Results shown in Fig. 2A indicate saturable increases in FXa generation relative to the A1 subunit concentration with ∼40% less activity observed for the reconstituted mutant protein. The reason for this reduced level of activity did not appear related to interaction of the A1 and A3C1C2 subunits as similar Kd values for this interaction were obtained independent of the mutation (Table 2). A similar evaluation of the interaction of the A2 subunit with mutant and wt A1/A3C1C2 dimers was undertaken. Dimers were preformed after reaction of the A1 subunit (100 nm) with either wt or mutant A3C1C2 (1 μm) as described in the Methods. The dimers were then reacted with varying levels of the purified A2 subunit and reconstituted FVIIIa was assayed by FXa generation. Results shown in Fig. 2B indicate that both FVIIIa forms yield a similar Vmax level for FXa generation when the A2 subunit is saturating (Table 2). However, we noted an ∼3‐ to 4‐fold reduced affinity of the A2 subunit for the mutant dimer compared with wt and suggest that this parameter is responsible for the reduced activity levels observed for the mutant in Fig. 2A when the A2 subunit is limiting. Activity assays for several FVIII mutants in which acidic amino acid residues in the A3 domain were individually replaced with alanine revealed one variant, E1829A, possessing reduced specific activity, rapid decay of FVIIIa activity and a lower value in a two‐stage assay compared with a one‐stage assay. These latter observations are suggestive of weakened affinity of the A2 subunit in FVIIIa [14Pipe S.W. Eickhorst A.N. McKinley S.H. Saenko E.L. Kaufman R.J. Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo.Blood. 1999; 93: 176-83Crossref PubMed Google Scholar, 15Pipe S.W. Saenko E.L. Eickhorst A.N. Kemball‐Cook G. Kaufman R.J. Hemophilia A mutations associated with 1‐stage/2‐stage activity discrepancy disrupt protein‐protein interactions within the triplicated A domains of thrombin‐activated factor VIIIa.Blood. 2001; 97: 685-91Crossref PubMed Scopus (84) Google Scholar, 16Hakeos W.H. Miao H. Sirachainan N. Kemball‐Cook G. Saenko E.L. Kaufman R.J. Pipe S.W. Hemophilia A mutations within the factor VIII A2‐A3 subunit interface destabilize factor VIIIa and cause one‐stage/two‐stage activity discrepancy.Thromb Haemost. 2002; 88: 781-7Crossref PubMed Scopus (42) Google Scholar] and this conclusion was confirmed after functional affinity determinations upon FVIIIa reconstitution from wt and mutant subunits. Our results demonstrated no apparent effect on association of the A1 subunit with the E1829A mutation‐containing A3C1C2 subunit, whereas we observed an ∼3‐ to 4‐fold reduced affinity of the A2 subunit for the mutant A1/A3C1C2 dimer and an ∼fourteenfold increased rate of A2 dissociation for this FVIIIa. Inasmuch as saturating levels of the A2 subunit yielded equivalent levels of cofactor activity for mutant and wt, we further conclude the low specific activity of the mutant derives from this defective inter‐subunit interaction. Mutation at this site to Ala and Gly has been reported in the Hemophilia A database and yield a mild phenotype, although the molecular basis for this defect has not been previously reported [32Kemball‐Cook G. Tuddenham E.G. Wacey A.I. The factor VIII structure and mutation resource site: HAMSTeRS version 4.Nucleic Acids Res. 1998; 26: 216-9Crossref PubMed Scopus (211) Google Scholar]. Several lines of evidence suggest interactions responsible for the A2 subunit retention in FVIIIa are largely the result of interaction with the A1 subunit. Fluorescence energy transfer data using the fluorophore‐labeled A1 subunit in the absence and presence of A3C1C2 showed that ∼90% of the binding energy for A2 in FVIIIa was derived from direct interaction with A1 [33Nogami K. Wakabayashi H. Schmidt K. Fay P.J. Altered interactions between the A1 and A2 subunits of factor VIIIa following cleavage of A1 subunit by factor Xa.J Biol Chem. 2003; 278: 1634-41Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. Furthermore, two large tryptic fragments of the A1 subunit comprising residues 37–121 and 221–336 inhibited the reconstitution of FVIIIa with Ki values similar to the Kd for the A2 subunit binding A1/A3C1C2, and effectively blocked the binding of the acrylodan‐labeled A2 subunit to the fluorescein‐labeled A1/A3C1C2 dimer as assessed by energy transfer [34Nogami K. Wakabayashi H. Ansong C. Fay P.J. Localization of a pH‐dependent, A2 subunit‐interactive surface within the factor VIIIa A1 subunit.Biochim Biophys Acta. 2004; 1701: 25-35Crossref PubMed Scopus (3) Google Scholar]. More recently, Parker et al. demonstrated that the slower rate of A2 subunit dissociation observed for porcine FVIIIa could be recapitulated in the human protein by replacing the human A1 domain with the porcine homolog [35Parker E.T. Doering C.B. Lollar P. A1 subunit‐mediated regulation of thrombin‐activated factor VIII A2 subunit dissociation.J Biol Chem. 2006; 281: 13922-30Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. Taken together, these studies suggest primary interactions of the A2 subunit occur over an extended interface largely comprised of A1 residues. While the role of A1 may make a dominant contribution to A2 retention, the contributions of the A3 domain to this interaction are less well defined. Several point mutations in A2 have been identified as yielding a one‐stage/two‐stage assay discrepancy consistent with enhanced A2 subunit dissociation and, from protein modeling these residues appear to localize near the A3 domain interface. Interestingly, several of the mutations that have been characterized as yielding increased rates for A2 subunit dissociation, N694I, R698L, R698W [16Hakeos W.H. Miao H. Sirachainan N. Kemball‐Cook G. Saenko E.L. Kaufman R.J. Pipe S.W. Hemophilia A mutations within the factor VIII A2‐A3 subunit interface destabilize factor VIIIa and cause one‐stage/two‐stage activity discrepancy.Thromb Haemost. 2002; 88: 781-7Crossref PubMed Scopus (42) Google Scholar], generate hydrophobic residues that may be disruptive to residue packing at the interface, inasmuch as the association of the A2 subunit is mediated primarily by electrostatic interactions [36Fay P.J. Smudzin T.M. Characterization of the interaction between the A2 subunit and A1/A3‐C1‐C2 dimer in human factor VIIIa.J Biol Chem. 1992; 267: 13246-50Abstract Full Text PDF PubMed Google Scholar]. Our results indicate the mutation E1829A results in an ∼fourteenfold increase in the dissociation rate for the A2 subunit. This value is somewhat greater than the ∼2‐ to 5‐fold rate increases observed for a number of other point mutations yielding one‐stage/two‐stage assay discrepancies (14–16) and may suggest an important role for Glu1829 in the interaction between the A1/A3C1C2 dimer and A2, leading to retention of the latter subunit in FVIIIa. Results from this study do not differentiate a direct or indirect contribution of this residue. However, a direct role for Glu1829 in interacting with a residue(s) in the A2 subunit may be possible based on evaluation of the modeled FVIII structure [37Pemberton S. Lindley P. Zaitsev V. Card G. Tuddenham E.G. Kemball‐Cook G. A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin.Blood. 1997; 89: 2413-21Crossref PubMed Google Scholar], which supports a possible hydrogen bond between a carboxyl hydrogen of Glu1829 with the carbonyl oxygen of Tyr664 consistent with the very short inter‐atomic distance (1.95 Å separation, Fig. 3). Because Glu1829 is conserved in all known species of FVIII [32Kemball‐Cook G. Tuddenham E.G. Wacey A.I. The factor VIII structure and mutation resource site: HAMSTeRS version 4.Nucleic Acids Res. 1998; 26: 216-9Crossref PubMed Scopus (211) Google Scholar], this hydrogen bond could contribute to the stability of the FVIIIa inter‐subunit structure. We thank Lisa M. Regan of Bayer Corporation for the gifts of recombinant human factor VIII. This work was supported by NIH grants HL38199 and HL76213. F. Varfaj acknowledges support from an American Heart Association Pre‐doctoral Fellowship. An account of this work was presented at the 48th annual meeting of the American Society of Hematology, Orlando, FL, December 9, 2006." @default.
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