Puget Sound Blood Center | Research: Kathleen Pratt Laboratory - Studies with Recombinant FVIII Proteins and Fragments

Studies with Recombinant FVIII Proteins and Fragments
Large proteins, like many large problems, are best dealt with by breaking them down into smaller, more manageable pieces. Molecular biology makes this possible by providing techniques to express (produce) recombinant proteins in organisms such as E. coli and yeast. FVIII has a domain structure that can be represented as A1-A2-B-C3-C1-C2, where the A domains are homologous to the copper-binding protein ceruloplasmin and the C domains are members of the discoidin family (Fuentes-Prior, Fujikawa and Pratt, 2002). The three-dimensional structure of the FVIII C2 domain is known (Pratt et al., 1999, Figure 1).

FIGURE 1: Ribbon representation of the FVIII C2 domain, with side chains at the membrane-binding region shown explicitly. Hydrophobic side chains that insert into the phospholipid membrane are colored gold. The ring of basic residues colored blue is thought to interact favorably with negatively-charged phosphatidylserine head groups at the membrane surface (e.g. of activated platelets).

The C2 domain contains a membrane-binding site, and it is one of two regions on FVIII that bind to VWF. The C2 domain forms an eight-stranded “beta sandwich”, and two beta hairpin turns, with chain reversals at residues 2199-2000 and 2251-2252, expose the hydrophobic residues Met, Phe, Leu and Leu, respectively, to the solvent. FVIIIa associates with activated platelet membrane surfaces, which expose the negatively charged phosphatidylserine head group. The C domains are very basic, with a pI of approximately 8.9. A ring of positively charged residues that cluster above the hydrophobic surface described above suggested a mode of membrane binding, with insertion of the hydrophobic residues into the phospholipid bilayer, leading to favorable electrostatic interactions between the positively charged protein and the negatively charged phosphatidylserine surface.

This model of membrane binding has since been confirmed by several labs, including ours, through mutagenesis of putative membrane-contact residues. In collaboration with Dr. Arthur Thompson’s group, we tested the effect of point mutations on the binding of FVIII-C1C2 proteins to activated platelet surfaces (Hsu et al., Blood 111:200-208, 2008), and results confirmed the role of these residues in binding to physiological membrane surfaces. Ongoing projects in the lab are aimed at deducing the orientation of FVIIIa at the membrane surface (evaluating point mutants of recombinant FVIII proteins and fragments using various biophysical techniques), and mapping surfaces that bind to various partners of FVIII, in particular membranes, VWF and FVIII-neutralizing antibodies.

Immune Responses to FVIII
Approximately 1/4 of patients with hemophilia A develop “inhibitors”, which are antibodies that neutralize the pro-coagulant function of FVIII. This unfortunate phenomenon occurs because individuals with hemophilia A have either no endogenous circulating FVIII, or else a dysfunctional FVIII. Infused therapeutic FVIII may then be recognized by their immune systems as a “foreign” protein. Auto-antibodies to FVIII can also occur in people who do not have hemophilia, and although this is rare, the resulting bleeding disorders can be dangerous and extremely difficult and expensive to treat.

FIGURE 2: Drs. Eddie James (Benaroya Research Institute), Kate Pratt and Ruth Ettinger.

Production of anti-FVIII antibodies requires helper T-cell involvement, and we are therefore investigating the responses of CD4+ T cells to epitopes in FVIII (e.g. James EA et al., J. Thromb. Haemost. 5:2399-2407, 2007). We are particularly interested in studying the evolution of T-cell responses to FVIII over time. A recent study identified FVIII-specific Th17/Th1 cells in earlier but not later stages of an inhibitor response (Ettinger RA et al., Blood 114:1423-28, 2009). This study was the first report characterizing antigen-specific Th17/Th1 clones from human blood samples. Th17 cells play an important role in inflammation and are implicated in various autoimmune disorders; we are very interested in pursuing additional studies of their involvement in anti-FVIII immune responses.

We are very interested in understanding why some individuals develop immune tolerance to FVIII but others do not. Our studies are also shedding light on basic mechanisms of immune responses to foreign antigens, and on cellular signaling pathways leading to immune tolerance. The T-cell studies are carried out in collaboration with scientists at the Benaroya Research Institute, notably Drs. Eddie James and Bill Kwok. MHC Class II tetramers are utilized to label and isolate FVIII-specific T cells. Polyclonal and cloned cell lines are generated, allowing us to evaluate phenotypes of T cells at various stages of anti-FVIII immune responses, and also allowing us to test the effects of modifying epitopes. The latter experiments are aimed at rationally designing less immunogenic versions of FVIII.