Acidic Region Residues 1680–1684 in the A3 Domain of Factor VIII Contain a Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689

2021 ◽  
Author(s):  
Yuto Nakajima ◽  
Hiroaki Minami ◽  
Keiji Nogami
Keyword(s):  
Surface Plasmon Resonance ◽  
Factor Viii ◽  
Heavy Chain ◽  
Synthetic Peptides ◽  
C2 Domain ◽  
Wild Type ◽  
Cross Link ◽  
Domain Specific ◽  
Cofactor Activity ◽  
Acidic Region

AbstractFactor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg372, Arg740, and Arg1689. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg1689. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg1689. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr1664 and residues 1675–1685 with sulfated Try1680, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg1689. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg1689 in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg1689.

Contributions of Asn2198, Met2199, and Phe2200 in the factor VIII C2 domain to cofactor activity, phospholipid-binding, and von Willebrand factor-binding

2003 ◽  
Vol 89 (05) ◽  
pp. 795-802 ◽  
Author(s):  
Deborah Lewis ◽  
Mary Pound ◽  
Thomas Ortel
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Hydrophobic Surface ◽  
C2 Domain ◽  
Mutant Proteins ◽  
Phospholipid Binding ◽  
Cofactor Activity ◽  
The Individual ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe crystal structure of the factor VIII C2 domain consists of a β-sandwich core from which β-hairpins and loops extend to form a hydrophobic surface. The hydrophobic surface includes M2199 and F2200 at the tip of the 1st β-hairpin. To determine the individual contributions of residues N2198, M2199, and F2200 to phospholipid and von Willebrand factor (vWF) binding properties of factor VIII, we prepared mutant proteins with single alanine substitutions. We found that single mutations at N2198 and M2199 had relatively little impact on cofactor activity, or phospholipid and vWF binding. However the F2200A mutant had slightly lower cofactor activity at subsaturating phospholipid concentrations. Competitive ELISAs suggested that F2200 plays a more important role in both phospholipid-binding and vWF-binding than N2198 and M2199. All mutant proteins were still recognized by a monoclonal antibody and two factor VIII inhibitors that neutralized cofactor activity and blocked factor VIII binding to phospholipids.Presented in part at the XVIII Congress of the International Society on Thrombosis and Haemostasis, Paris, France, 6-12 July 2001, and the 43rd Annual Meeting of the American Society of Hematology, Orlando, Florida, 7-11 December 2001

The Specific Contribution of Amino Acids 334 and 335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1027-1027
Author(s):  
Melissa A. Blum ◽  
Tivadar Orban ◽  
Daniel O. Beck ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Factor Va ◽  
Cofactor Activity ◽  
Type Factor

Abstract The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.

B-Cell Epitope Mapping of Monoclonal Anti-Factor VIII C2 Domain Inhibitors: Identification of Amino Acids That Contribute Significant Antigen-Antibody Binding Affinity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1211-1211
Author(s):  
Jasper C. Lin ◽  
Jason T. Schuman ◽  
Shannon L. Meeks ◽  
John F. Healey ◽  
Arthur R. Thompson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Factor Viii ◽  
Dissociation Constants ◽  
Antibody Binding ◽  
Amino Acid Substitutions ◽  
C2 Domain ◽  
Wild Type ◽  
Mutant Proteins

Abstract The most troublesome clinical complication that can afflict hemophilia A patients who receive factor VIII (FVIII) infusions as replacement therapy is the development of an anti-FVIII immune response, in which antibodies bind to functionally important FVIII surfaces, thereby blocking the pro-coagulant function of this important plasma protein cofactor. These antibodies, commonly referred to as “FVIII inhibitors”, bind primarily to the FVIII A2 and C2 domains and to the C-terminal region of the C1 domain, and inhibitors mapping to other regions have also been seen. There are multiple epitopes on the FVIII C2 domain, reflecting both its immunogenicity/antigenicity and its diverse roles in mediating interactions between FVIII and other molecules. For example, the C2 domain is essential for binding of FVIII to its carrier protein von Willebrand factor (VWF). Proteolytic activation to FVIIIa causes its release from VWF and subsequent binding to negatively charged membrane surfaces, e.g. on activated platelets, whereupon a region that overlaps the VWF binding site contacts the membrane. The C2 domain also interacts with thrombin and factor Xa, which both can activate FVIII. To better understand the basis for FVIII inhibition, and to better delineate functionally important FVIII surfaces, a panel of 56 murine anti-C2 monoclonal antibodies was generated. Competition ELISAs and functional assays were used to classify the antibodies into five groups corresponding to distinct regions on the C2 surface, which comprised a larger number of distinct epitopes (Meeks et al., Blood110, 4234–42, 2007). The present study is a high-resolution mapping of the epitopes recognized by six representative antibodies (2-77, 2-117, 3D12, 3E6, I109 and I54) using surface plasmon resonance (SPR). Each antibody was immobilized covalently via amine coupling to a CM5 chip or was captured by a rat anti-mouse IgG attached covalently to a CM5 chip. Referring to the FVIII C2 domain crystal structure (Pratt et al., Nature402, 439–42, 1999), surface-exposed amino acids were selected for mutagenesis using the Stratagene Quik-Change system, and C2 constructs with single substitutions to alanine or amino acids that were structurally similar to the wild-type residues were generated. Forty-five of these proteins were expressed in E. coli and purified; their purity and structural integrity were confirmed by SDS-PAGE and Western blot analysis. The on- and off-rates for binding of these proteins to the six monoclonal antibodies were determined using a Biacore T100 instrument. Mutations that affected binding significantly were analyzed by measuring association and dissociation constants over a temperature gradient (10–40°C), yielding estimates of changes in antibody-binding energy (ΔΔGº) of these mutant proteins compared to wild-type C2. Van’t Hoff analysis was carried out to determine the relative contributions of enthalpy and entropy to the binding energies. Interestingly, C2 binding to each antibody was abrogated by 1–5 of the 45 amino acid substitutions tested. Each of these C2 mutants bound to other antibodies with affinities similar to that of wild-type C2, indicating that this was not an artifact due to protein misfolding. The following substitutions resulted in little or no binding, as evidenced by a completely abated signal (very low Rmax compared to the wild-type C2 protein): L2273A (2-77, 2-117), R2220A (3D12, I109), Q2231A (I54) and T2272A (I109). Additional mutant proteins with reduced binding to inhibitor(s) displayed markedly higher dissociation constants and sometimes less pronounced differences in association constants compared to wild-type C2. Although several FVIII residues contributed to more than one epitope, each antibody had a unique epitope map profile. Our results suggest that a limited number of amino acid substitutions could produce a modified FVIII protein capable of eluding immunodominant inhibitors. This approach could eventually find clinical application as a novel strategy to achieve hemostasis in patients with an established FVIII inhibitor.

Proteolytically Inactivatable Factor VIII is Less Immunogenic than Factor VIII in a Murine Hemophilia A Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 27-27
Author(s):  
Shannon Meeks ◽  
Ernest T Parker ◽  
Amy L. Dunn ◽  
John F Healey ◽  
Pete Lollar
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Factor Viii ◽  
Binding Site ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Foreign Protein ◽  
C2 Domain ◽  
Wild Type ◽  
Antibody Titers

Abstract Abstract 27 Patients with hemophilia A have a congenital deficiency of the factor VIII (fVIII) protein due to a mutation in the fVIII gene that frequently leads to absence of detectable expression of fVIII. Accordingly, the therapeutic replacement fVIII protein potentially is recognized as non-self by the immune system. Thirty percent of patients with severe hemophilia A develop detectable inhibitory anti-fVIII antibodies (inhibitors). Additionally, greater than 90 percent of hemophilia A mice treated with human fVIII develop inhibitors using dosing schedule that mimics use in humans. Because fVIII is an immunologically foreign protein, it might be expected that a hemophilia A patient would make a fVIII inhibitor. However, intravenous injection of soluble proteins in either humans or rodents usually results in tolerance rather than a humoral immune response. One major difference between fVIII and other proteins is that it is released from its large carrier protein von Willebrand factor (VWF) and is potentially exposed to the immune system at sites of active hemostasis and inflammation. Heat-inactivated, denatured fVIII, which maintains all T-cell epitopes but lacks several B-cell epitopes, is less immunogenic than native fVIII, suggesting that fVIII-dependent thrombin generation along the intrinsic pathway of blood coagulation may provide co-stimulatory signals necessary for the immune response (Skupsky BS, Zhang A, Scott DW Blood 2008; 112:1220a). We constructed a B domain-deleted human fVIII mutant, designated fVIIIi, which contains alanine substitutions at two critical thrombin cleavage sites, Arg372 and Arg1689, and purified it to homogeneity. FVIIIi does not develop procoagulant activity and is not released from VWF in response to thrombin. Therefore fVIIIi is less likely than wild-type fVIII to be exposed to the immune system at sites of active hemostasis and inflammation. Additionally, VWF binds to the immunodominant fVIII C2 domain and potentially hides part of fVIII from the immune system. FVIIIi was antigenically intact judging from intact binding to a panel of11 mouse anti-fVIII monoclonal antibodies whose epitope specificity was represented by all five domains of BDD fVIII. The immunogenicity of wild-type fVIII and fVIIIi was compared in a murine hemophilia A model in which groups of 25 mice received 8 weekly injections of physiologic doses of fVIII. Plasma was collected weekly for total anti-fVIII antibody titers by ELISA and one week following the last injection for total anti-fVIII antibody titers, inhibitor titers by Bethesda assay and for epitope mapping. Mice treated with fVIIIi had significantly lower levels of inhibitory as well as total anti-fVIII antibodies than mice treated with wild-type fVIII. Domain mapping using single human domain hybrid human/porcine molecules as ELISA antigens revealed that hemophilia A mice broadly recognized all fVIII domains in response to either wild-type or fVIIIi, although fVIIIi produced less anti-light chain antibodies. Mice in both the wild-type fVIII and fVIIIi groups produced antibodies that recognized the phospholipid-binding site of the C2 domain, even though this site overlaps the VWF binding site on fVIII. There was no difference in the isotype spectrum of the antibodies made to fVIII or fVIIIi. This study indicates that inactivatable fVIII is less immunogenic than native fVIII and suggests that the immunogenicity of fVIII is related either to its interaction with VWF or to events triggered by activation of the coagulation mechanism. Disclosures: No relevant conflicts of interest to declare.

The factor VIII heavy chain improves emicizumab-tenase assembly to enhance the factor VIII-mimicking cofactor activity

2018 ◽  
Vol 166 ◽  
pp. 77-79 ◽  
Author(s):  
Hiroaki Minami ◽  
Keiji Nogami ◽  
Tetsuhiro Soeda ◽  
Takehisa Kitazawa ◽  
Kunihiro Hattori ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Cofactor Activity

scholarly journals Inhibitors in German Hemophilia A Patients Treated with a Double Virus Inactivated Factor VIII Concentrate Bind to the C2 Domain of FVIII Light Chain

1999 ◽  
Vol 81 (01) ◽  
pp. 39-44 ◽  
Author(s):  
R. Laub ◽  
Di Giambattista ◽  
P. Fondu ◽  
H.-H. Brackmann ◽  
H. Lenk ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hepatitis A ◽  
Hemophilia A ◽  
Hepatitis A Virus ◽  
C2 Domain ◽  
Epitope Specificity ◽  
Previously Treated ◽  
Inhibitor Titer

SummaryTo reduce the risk of transmission of hepatitis A virus, an Octaphar-ma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

Discontinuous epitopes on the C2 domain of coagulation Factor VIII mapped by computer-designed synthetic peptides

2011 ◽  
Vol 155 (4) ◽  
pp. 487-497 ◽  
Author(s):  
Aurélien Lebreton ◽  
Violaine Moreau ◽  
Priscilla Lapalud ◽  
Christopher Cayzac ◽  
Sébastien André ◽  
...  
Keyword(s):  
Factor Viii ◽  
Synthetic Peptides ◽  
Coagulation Factor ◽  
Coagulation Factor Viii ◽  
C2 Domain ◽  
Discontinuous Epitopes

His2315/Gln2316 of the Factor VIII C2 Domain Interact with Phospholipid Membranes and Influence Activity of the Factor Xase Complex.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1727-1727
Author(s):  
Gary E. Gilbert ◽  
Randall J. Kaufman ◽  
Patricia Price ◽  
Hongzhi Mao ◽  
Steven W. Pipe
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Phospholipid Bilayer ◽  
Crystallographic Structure ◽  
Phospholipid Membranes ◽  
C2 Domain ◽  
Wild Type ◽  
Phospholipid Binding ◽  
Hydrophobic Amino Acids ◽  
Type Factor

Abstract A major phospholipid-binding motif of factor VIII is localized in the C2 domain. Two pairs of hydrophobic amino acids in the factor VIII C2 domain (Met2199/Phe2200, Leu2251/Leu2252) have previously been shown to interact with phospholipid membranes. However mutations of hydrophilic amino acids (Gln2213, Arg2215, Asn2217, Arg2220, Lys2249) that were predicted to interact with the membrane surface, based upon the crystallographic structure, have not altered phospholipid binding activity. The established hydrophobic interactions do not account for the demonstrated hydrophilic interactions of factor VIII with the phospholipid bilayer. We hypothesized that His2315 and/or Gln2316, interact with phospholipid membranes, based upon their contribution to the epitope for phospholipid-blocking mAb B02C11 and based upon phosphatidylserine binding by C2 domain peptides that include these residues. Factor VIII His2315Ala/Gln2316Ala (fVIII 2315/16) was prepared by PCR mutagenesis and expressed from COS-1 cells. Secreted factor VIII was purified by immunoaffinity chromatography and evaluated in ELISA assays, aPTT assays with factor VIII deficient plasma, and in defined assays with varying phospholipids. The specific activity of fVIII 2315/16 was 67 ± 9% of wild type factor in a commercial aPTT assay with a large excess of phospholipid. The apparent affinity for extruded phospholipid vesicles (0.1 μm diameter) of composition phosphatidylserine:phosphatidylethanolamine: phosphatidylcholine 4:20:76 was 32 ± 10% of wild type factor VIII in a factor Xase assay with varying phospholipid. When the phospholipid concentration was saturating, the Vmax was 55% of factor Xase with wild type factor VIII. We conclude that His2315 and/or Gln2316 constitute the first identified hydrophilic amino acids of factor VIII that interact with a phospholipid membrane. His2315/Gln2316, combined with the two pairs of phospholipid-interactive hydrophobic amino acids constitute three spatially separated phospholipid-interactive points that define a phospholipid-interactive plane on the factor VIII C2 domain. This is consistent with the hypothesis that the phospholipid-binding plane of the C2 domain overlaps substantially with the epitope for BO2C11 and is nearly coincident with the phospholipid-binding surface predicted from the crystallographic structure.

Identification of a Plasmin-Interactive Site within the A2 Domain of the Factor VIII Heavy Chain.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1018-1018 ◽  
Author(s):  
Keiji Nogami ◽  
Midori Shima ◽  
Katsumi Nishiya ◽  
Evgueni L. Saenko ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
Rate Constant ◽  
Heavy Chain ◽  
Active Site ◽  
Inactivation Rate ◽  
Wild Type ◽  
Inactivation Rate Constant ◽  
Factor Ixa ◽  
A2 Domain

Abstract Factor VIII (FVIII) is inactivated by limited proteolytic cleavage by plasmin immediately after the activation. However, the plasmin-interactive region(s) in FVIII remain to be determined. Recently, we reported that the A2 domain may interact with plasmin during FVIII inactivation by this protease (Abst #1991, BLOOD102, 2002). In the current study, several approaches were employed to examine the localization and role of plasmin-interactive region(s). Activation and inactivation rate constants of plasmin-catalyzed FVIII and FVIIIa by the addition of isolated A2 subunit were reduced by ~4 and ~13-folds, respectively, in dose-dependent manners using one-stage clotting assay. The addition of Glu-Gly-Arg active-site modified factor IXa, interacts with the A2 domain, also reduced the rate constant of FVIIIa inactivation by ~4-fold. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody 413, recognizing residues 484–509 in factor IXa-interactive site, blocked the plasmin-catalyzed cleavages at Arg336, Arg372, and Arg740 in the heavy chain. Surface plasmon resonance-based assay using anhydro-plasmin, catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that FVIII and isolated A2 subunit bound to anhydro-plasmin with Kd values of 4 and 21 nM, respectively. The binding assay using ELISA with immobilized anhydro-plasmin also showed the similar binding affinities. Monoclonal antibody 413 blocked the A2 subunit binding to anhydro-plasmin by ~80% (IC50: 151 nM). Furthermore, synthetic peptide with sequences 479–504 inhibited this binding by ~55% (Ki: 3 microM), however, peptide with sequences 489–514 had a very weak inhibition (by <20%). To investigate the responsible residues in A2 domain for plasmin binding, the mutant forms of the A2 domain were expressed in baculovirus system and purified. Compared with wild type (23 nM), the affinity of R484A mutant was dramatically decreased by ~250-fold, and the affinities of K377A, K466A, R471A, and K523A mutants also were decreased by 10~40-folds, respectively. Especially, the addition of R484A mutant was reduced inactivation rate constant of plasmin-catalyzed FVIIIa by only ~40% of that of wild type. These findings demonstrate that Arg484 in the A2 domain contains plasmin-binding site responsible for plasmin-catalyzed FVIII(a) inactivation.

Six Amino Acid Residues in a 1200 Å2 Interface Mediate Binding of Factor VIII to An IgG4 Inhibitory Antibody.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3380-3380 ◽  
Author(s):  
Jasper C. Lin ◽  
Jason T. Schuman ◽  
Shelley M. Nakaya ◽  
Vasudha Kaushik ◽  
Marc Jacquemin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
High Morbidity ◽  
Side Chains ◽  
C2 Domain ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Solvent Exposure ◽  
Fab Fragment ◽  
Antigen Antibody

Abstract Approximately one quarter of severe hemophilia A patients who receive Factor VIII (FVIII) injections develop antibodies, clinically referred to as “inhibitors”, which interfere with FVIII procoagulant activity. The effects of these antibody inhibitors can be difficult and quite expensive to manage. Inhibitors are associated with high morbidity and mortality and impaired quality of life; therefore, there is a compelling need to develop new therapeutic options. One approach is to design recombinant versions of FVIII that are less immunogenic (less likely to stimulate T cells) or less antigenic (containing fewer B-cell epitopes, i.e. surfaces that bind to anti-FVIII IgG). Proteins with reduced antigenicity will by definition bind to inhibitory IgG with lower affinity and therefore could be useful in attempting to achieve hemostasis in patients with an established inhibitor response. To design such FVIII proteins, common inhibitor epitopes must be characterized by determining which amino acid residues are essential to form high-affinity antigen-antibody complexes. A crystal structure of the FVIII C2 domain bound to an Fab fragment from a patient-derived inhibitory IgG4 antibody, BO2C11, provides the most detailed characterization to date of a human inhibitor epitope (Spiegel et al., Blood38, 13–19, 2001). Although this structure clearly shows which FVIII residues interact with the antibody surface, the contributions of particular residues to the overall affinity must be determined experimentally. In this study, we systematically modified each of the C2 side chains at the C2-Fab interface, which buries 1200 Å2 of each protein surface, then used surface plasmon resonance (SPR) to measure the contributions of individual residues to the kon and koff rates and to the overall affinity. The experiments were carried out on a Biacore T100 instrument, which allowed us to analyze several samples in parallel and to carry out SPR runs at different temperatures. Substitutions at only six sites decreased the affinity significantly relative to that of wild-type C2. R2220A and R2220Q completely abrogated binding to BO2C11, while F2196A, N2198A, M2199A, L2200A and R2215A displayed markedly higher off-rate kinetic constants compared to wild-type C2 but retained some binding affinity. SPR runs were carried out for the latter five proteins using a temperature gradient (10–40°C), and thermodynamic values derived from van’t Hoff analysis were used to roughly quantitate the energetic consequences of these mutations compared to wild-type C2 binding. Although a relative order of energetic contributions was established (F2200 > F2196 = R2215 > N2198 > M2199) the ΔΔGº values were similar (approx. 11 ± 5 kJ/mol). Furthermore, the data suggest that the loss of binding energy was mostly an entropic, not enthalpic, effect, as the ΔH values were remarkably stable for the set of C2 mutants. In other words, the mutations increased the ordering of the system consisting of BO2C11 bound to C2 plus solvent, or else they increased the disorder of the uncomplexed system, e.g. by allowing greater flexibility of protein side chains or backbone, or by changing the solvent exposure of hydrophobic residues, thereby affecting ordering of water molecules. Interestingly, only one of two beta-hairpin turns that comprise part of this epitope contributes appreciably to the binding of the C2 domain to BO2C11. Substitutions at L2251 and L2252 in the second hairpin turn had surprisingly little effect on the off-rate and overall affinity, despite their extensive contact with the antibody that shielded this hydrophobic region from solvent. IgG4 antibodies are common in anti-FVIII immune responses, as is inhibition of FVIII binding to activated membranes and von Willebrand factor. BO2C11 is a human-derived IgG4 that inhibits these binding interactions. Our results for this prototypical inhibitor suggest that a limited number of amino acid substitutions could produce modified FVIII proteins capable of eluding inhibitors that bind to similar epitopes, even in the case of antibodies that form an extensive antigen-antibody interface.

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