Education and Training:

B.S., Mathematics, University of Washington, Seattle, WA (1984)

Ph.D., Biophysical Chemistry, Cornell University, Ithaca, NY (1992)

Postdoctoral Fellow, University of Colorado, Boulder, CO (1992-94)

Posdoctoral Fellow, University of Washington, Seattle, WA(1994-98)

Postdoctoral Fellow, Fred Hutchison Cancer Research Center, Seattle, WA (1998-99)

CURRENT RESEARCH INTERESTS

1.  Factor VIII interactions with membranes and von Willebrand factor
2.  Factor VIII interactions with inhibitory antibodies
3.  T-cellepitopes in factor VIII: mapping and modification
4.  Design of less immunogenic versions of factor VIII


RESEARCH DESCRIPTION

Background.
My laboratory is investigating the structure, function and pathologies due to defects in the blood coagulation cofactor known as factor VIII (FVIII). This large protein circulates in the plasma as a heterodimer, forming a noncovalent complex with von Willebrand factor (VWF), which protects it from degradation and delivers it to wound sites. VWF becomes anchored to collagen that is exposed upon injury, and it releases proteolytically activated FVIII (FVIIIa) as platelets aggregate to form a “platelet plug” to staunch blood loss. FVIIIa then associates with activated membrane surfaces, where it forms a complex with the serine protease factor IXa (FIXa). The substrate for FIXa is the zymogen form of the next serine protease in the coagulation cascade, factor X (FX), which is activated by cleavage of its propeptide. FVIIIa is not a protease; rather, it serves a cofactor function. By associating with FIXa at a negatively charged membrane surface, in the presence of calcium, it increases the catalytic efficiency of FIXa by approximately four orders of magnitude. This acceleratory activity constitutes a critical control point in hemostastasis. The importance of FVIII function is underscored by the fact that a severe deficiency in FVIII, which can be caused by gene rearrangements, deletions, or point mutations, results in the bleeding disorder hemophilia A.

Factor VIII interactions with membranes. 
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 FVIII C2 domain was expressed in the methylotrophic yeast Pichia pastoris, purified, and crystallized, and its three-dimensional structure was determined at 1.5 angstrom resolution (Pratt et al., 1999). This C-terminal domain is known to contain a membrane-binding site on the FVIII surface, and it is also known to be a region of association between FVIII and VWF. This domain forms an eight-stranded “beta sandwich”, and the loops formed by reverse turns at one end of the molecule have some unusual properties. Globular proteins tend to sequester amino acid side chains with a hydrophobic character in their interior, where they are not exposed to aqueous solvent. Exposure of large hydrophobic surfaces is usually energetically unfavorable, because a hydrophobic surface causes water molecules to order themselves into basket-like clathrate structures, decreasing the entropy of the system and therefore destabilizing the protein structure. Two of the 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, and the positively charged residues that cluster above the hydrophobic surface described above suggested a mode of membrane binding for this domain, specifically through 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 point mutagenesis of the putative membrane-contact residues in the C2 domain, resulting in a lower affinity for synthetic membrane surfaces. We have collaborated with Dr. Arthur Thompson’s group to test the contribution of the C1 domain to activated platelet surfaces using FACS analysis (Hsu et al., 2007). Ongoing projects in the lab are aimed at deducing the orientation of the C2 and C1 domains at the membrane surface (through evaluating point mutants of the C2 and C1C2 proteins and through 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. 
Up to a third of all patients with severe hemophilia A develop “inhibitors”, which are antibodies that neutralize the procoagulant function of FVIII. This very unfortunate phenomenon occurs because individuals with severe hemophilia A have little or no endogenous circulating FVIII, so when they are infused with FVIII concentrates their bodies mount an immune response to this “foreign” protein. Mild hemophilia A patients can also develop inhibitors, presumably because the infused wild-type FVIII differs from the endogenous, defective FVIII and therefore may provoke an immune response. Auto-antibodies to FVIII can also occur, and although this is rare, the resulting bleeding disorder is dangerous and can be extremely difficult and expensive to treat. Treatment of inhibitors involves various tolerance induction protocols. We are very interested in the mechanisms behind the immune response and also behind the establishment of tolerance to FVIII.

Production of antibodies requires helper T-cell involvement, and we are investigating the responses of CD4+ T cells to epitopes in the FVIII A2 and C2 domains (James et al., 2007; Ettinger et al., in preparation). We are also mapping B-cell epitopes on the C2 domain surface, using C2 proteins with specific amino acid substitutions. Several years ago, the crystal structure of the C2 domain bound to the Fab fragment of a patient-derived inhibitory monoclonal antibody was determined at high resolution (Spiegel et al, 2001) (Figure 1), and this structure showed that a major epitope is essentially equivalent to the C2 membrane-binding surface. Identification of additional B- and T-cell epitopes in FVIII will allow us to generate “rationally designed” FVIII proteins having point mutations that alter the immunological properties of this molecule. Our goal is to generate specific, minor modifications to the FVIII protein that will make it less immunogenic when used for cofactor replacement therapy.

Figure 1. The FVIII C2 domain (red) is shown in two orientations bound to the Fab fragment of the monoclonal antibody BO2C11 (yellow and green), which was derived from an inhibitor patient. The zoom-in on the right indicates FVIII residues that contact the Fab surface, defining this B-cell epitope.

SELECTED PUBLICATIONS

Hsu T-C, Pratt KP, Thompson AR.  The factor VIII C1 domain contributes to platelet binding. Blood 111:2399-407.

James EA, Kwok WW, Thompson AR, Pratt KP.  Analysis of CD4 T-cell responses to FVIII in a mild hemophilia A patient indicates early loss of tolerance to a C2 domain self peptide and sustained loss of tolerance to the wild-type peptide.  J. Thromb. Hemost. 5:2399-47, 2007.

Pratt KP, Zhukov O, Qu K, Thompson AR.  ELISA system for detection of immune responses to factor VIII: A study of 246 samples and correlation with the Bethesda assay. Hemophilia 13:317-22, 2007. 

Pratt KP, Lanelle A, Weiner H, Lillicrap D. Induction of immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain.  J. Thromb. Hemostas. 4:2172-79, 2006. 

Pratt KP, Qian J, Ellaban DK, Diethelm-Okita BM, Scott DW.  Immunodominant T-cell epitopes in the Factor VIII C2 domain are located within an inhibitory antibody binding site.  Thrombos. Haemost. 92:522-8, 2004.

Fuentes-Prior P, Fujikawa K, Pratt KP.  New insights into binding interfaces of coagulation factors V and VIII and their homologues – lessons from high-resolution crystal structures.  Current Protein and Peptide Science 3:313-9,  2002.

Spiegel PC, Jr., Jacquemin M, Saint-Remy J-MR, Stoddard BL, Pratt, KP.  Plenary paper:  Structure of a factor VIII C2-domain – Immunoglobulin G4 Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII.  Blood  98:13-19, 2001.

Liu, M-L, Shen BW, Nakaya S, Pratt KP, Takeshima K, Davie EW, Fujukawa K, Stoddard BL, Thompson AR.  Hemophilic factor VIII C1- and C2- domain missense mutations and their modeling to the 1.5-angstrom human C2-domain crystal structure.  Blood  96:979-87, 2000.

Pratt KP, Shen BW, Takeshima K, Davie EW, Fujikawa K, Stoddard BL.  Structure of the C2 domain of human factor VIII at 1.5 angstrom resolution.  Nature 402:439-42,  1999.

Pratt KP, HCF, Chung DW, Stenkamp RE, Davie EW.  The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal Gamma chain fragment complexed with the peptide Gly-Pro-Arg-Pro. Proc. Natl. Acad. Sci. USA 94:7176-84, 1997.