2003; baxter - Supplements - Haematologica

12P. Lollaragainst a limited number of immunodominantepitopes. For example, the immune response tothe Mr 76,000 influenza A hemagglutinin heterodimeris directed against four immunodominantB-cell epitopes 5 that cover only a fraction ofthe protein surface.The mechanisms that produce this restrictionof the immune response are not known. Oneview is that T-cells orchestrate the process andthe structure of the antigen itself is relativelyunimportant. However, it is possible that thepotential antibody repertoire is biased to recognizeendemic pathogens and to avoid self-reactivespecificities. 6 When there is a loss of tolerance,intrinsic structural properties of the antigen mayinfluence immunodominance. For example, theautoantibody response to cytochrome c found insystemic lupus erythematosus (SLE) and otherdisorders is directed to a limited number of siteswhich are similar to those recognized by mice thatare immunized with human cytochrome C. 7Analysis of the polyclonal response within asingle immunodominant epitope reveals considerableheterogeneity when the component monoclonalantibodies are studied. An exhaustiveanalysis of the number of anti-hemagglutininmurine B-cell hybridomas produced in responseto infection with an influenza A strain producedan estimate of 1,500 different antibodies in therepertoire directed against the four immunodominantepitopes. 8 Thus, an immunodominantepitope could be viewed as an area underneathan antibody footprint, or set of overlappingfootprints, in which there is considerable variationat the clonal level in the atomic contacts thatdetermine the strength of the interaction.Structural analysis of antigen-antibody complexesby X-ray crystallography has demonstratedthat typically 20-25 amino acid residues of eachcomponent are buried. 9 However, most of thestructural epitope does not contribute significantlyto the binding energy because most of theresidues in contact with antibody can be mutatedwithout loss of binding energy. 10 However,mutation of one of a few key residues typicallyresults in a major decrease in affinity. The set ofthese residues has been defined as the functionalepitope. 2,10FVIII structure and functionWith this background, we can discuss factorVIII (FVIII) as a potential target of the immunesystem. FVIII is a large, complex plasma glycoprotein.It contains 2,332 amino acids and ispost-translationally glycosylated and sulfated. Themolecular weight of the mature protein is approximately300,000. FVIII circulates bound noncovalentlyto von Willebrand factor (vWf). vWfis a huge, multimeric protein that weights severalmillion Daltons and has a hydrodynamicradius in the submicron range. 11 Thus, the FVIIIvWfcomplex may look something like a bacteriumor virus to the immune system. Disruption ofthis interaction, for example in severe von Willebrand’sdisease or naturally occurring vWf mutations,results in rapid clearance of FVIII and a secondarydeficiency of FVIII.FVIII contains a sequence of domains designatedA1-A2-B-ap-A3-C1-C2, where ap is an activationpeptide. FVIII is cleaved intracellularly withinthe B domain by a protease known asPACE/furin. 12 This results in fragmentation ofthe B domain and production of a heterodimer,which still requires proteolytic activation in thecoagulation cascade. During the activation ofFVIII by thrombin, cleavages occur between A1and A2 at Arg 372 , between A2 and B at Arg 740 , andbetween ap and A3 at Arg 1689 . Consequently, theB domain fragments and the acidic 41-residue appeptide are released, producing an A1/A2/A3-C1-C2 activated FVIII (FVIIIa) heterotrimer. 13 FVIIIalso can be activated by factor Xa, 14 which producesa more complex cleavage pattern. 15The only known function of FVIII is to participate,in activated form, as a cofactor for factor IXaduring intrinsic pathway factor X activation. Theenzymatic complex consists of factor IXa, FVIIIaand factor X on a phospholipid membrane surface.FVIII is assayed in coagulation or chromogenicassays under conditions in which it islimiting relative to factor IXa, factor X and phospholipid.Inhibitory antibodies are most commonlymeasured using the Bethesda assay 16 or theNijmegen modification of this assay. 17 Theseassays measure the loss of FVIII activity in a coagulationassay and return a single number — thedilution of antibody sample that produces 50%inhibition — as a global measurement of inhibitoryantibody activity.FVIII inhibitorsThe FVIII molecule represents the most commonlytargeted plasma protein for the developmentof inhibitory antibodies. Hemophilia A isthe most common severe hereditary bleeding disorderand produces a clinical setting in whichpatients are infused with a potentially immunogenicprotein. Most patients with severe hemophiliahave gene deletions, insertions, inversionsor nonsense mutations, all of which could possiblylead to complete lack of circulating FVIII. Inthis setting FVIII would represent a foreign proteinand would be fair game for the immune system.Alloantibodies arise in approximately 25%of patients with hemophilia A 18 and it is perhapssurprising that the incidence is not higher.Patients with severe hemophilia usually do nothave detectable levels of FVIII antigen. However,it is possible that in some of these patients, thereis a small amount of antigen that is sufficient toproduce tolerance.It is not possible to predict whether a previouslyuntreated patient with hemophilia A will developan inhibitor. However, patients with missensemutations and circulating levels of FVIII antigenare less likely to develop inhibitors, either becauseof a state of immune tolerance or because themissense mutation does not produce severehaematologica vol. 88(supplement n. 12):september 2003