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,
Postdoctoral Fellow, Fred Hutchison Cancer Research Center, Seattle, WA (1998-99)
|Kathleen P. Pratt, Ph.D.
Research Assistant Professor of Medicine
Division of Hematology
University of Washington School of Medicine
Puget Sound Blood Center
Puget Sound Blood Center Research Institute
1551 Eastlake Ave E
Seattle, WA 98102
Phone: (206) 568-2232
Fax: (206) 587-6056
Research lab website:
CURRENT RESEARCH INTERESTS
Factor VIII (FVIII) structure and function, immune responses to FVIII,
immune tolerance and design of less immunogenetic therapeutic
Principal Investigator: Dr. Pratt is a Research Assistant Professor in the Department of Medicine (Division of Hematology) and an Assistant Member at the Puget Sound Blood Center Research Institute in Seattle. Dr. Pratt's research program focuses on Factor VIII (FVIII structure and function, immune responses to FVIII, immune tolerance and design of less immunogenic therapeutic proteins.
The Pratt 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 associates 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 hemostasis. 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 .
A is treated by infusing patients with FVIII. Unfortunately, approximately one quarter of the patients who receive these infusions develop antibodies that bind to FVIII and interfere with its pro-coagulant function. These immune responses seem to occur more frequently in African American and Hispanic populations, and we are trying to better understand the basis of these observed differences between populations. Current projects in my laboratory are focused on characterizing these immune responses and designing less immunogenic versions of FVIII.
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 C2 domain contains a
membrane-binding site, and it is one of two regions on FVIII that bind
to VWF. 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 been confirmed by several labs,
including ours, through mutagenesis of putative membrane-contact
residues. Ongoing projects in the lab are aimed at mapping surfaces in
FVIII that bind to membranes, to VWF and to FVIII-neutralizing
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. Production of anti-FVIII
antibodies requires helper T-cell involvement, and we are
investigating the responses of CD4+ T cells to epitopes in FVIII. 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 also are
investigating why some individuals develop immune tolerance to FVIII
but others do not. Our studies are 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. In addition to the
T-cell studies, we are mapping B-cell epitopes, i.e. the surface
regions of FVIII that bind to anti-FVIII antibodies, using surface
plasmon resonance. The binding sites for both human and murine
monoclonal antibodies are being characterized. A central goal of our
immunological studies is to identify additional B- and T-cell epitopes
that will allow us to generate “rationally designed” FVIII
proteins having reduced immunogenicity.
Pratt KP. Inhibitory antibodies in hemophilia A. Current Opinion in Hematology, in
James EA, van Haren SD, Ettinger RA, Fjnvandraat K, Liberman J, Kwok WW, Voorberg J, Pratt KP. T-cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site. J Thromb Haemostas, 9:689-99, 2010.
Ettinger RA, James EA, Kwok WW, Thompson AR, Pratt KP. HLA-restricted T-cell responses to FVIII epitopes in a mild haemophilia A family with missense mutation FVIII-A2201P. Haemophilia 16:44-55, 2010.
Ettinger RA, James EA, Kwok WW, Thompson AR, Pratt KP.
of human T-cell clones, including TH17/TH1
cells, isolated at different stages of anti-factor VIII immune
responses. Blood 114: 1423-8, 2009.
Pratt KP, Thompson AR. B-Cell and T-Cell Epitopes in
Anti-factor VIII Immune Responses. Clin Rev Allergy Immunol 37:
EA, Kwok WW, Thompson AR, Pratt
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.
Qian J, Ellaban E, Okita,
DK, Diethelm-Okita BM, Conti-Fine, 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.
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.
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.