Project 4 - Abstract Novel approaches to enhance the biologic activity of FVIII and FIX are critical endeavors with potential for improving protein- and gene-based therapy for hemophilia. We have identified novel variants and new strategies that have a positive effect on the biological activity of FVIII and FIX. Promising preclinical studies on efficacy and safety in small and large animal models provide the basis for translational studies using these proteins; in particular the FIX-Padua (R338L) variant is already in early phase gene therapy clinical trials for hemophilia B. The main goal of this application is to understand the biochemistry of the variants and in the case of new variants, evaluate their potential in hemophilic models. We seek to use this approach to reveal mechanistic aspects of intrinsic Xase function and thus provide evidence-based insights into the potential of these variants in the treatment of human disease. The central tenets of our experimental strategies are based on the fact that even modest enhancements of procoagulant function, as judged from a biochemical perspective, can have a very significant impact on the success of therapeutic approaches.
In aim 1 we hypothesize that a FVIII derivative with a modified PACE/furin cleavage site results in its differential processing by thrombin and/or FXa yielding a more stable/active cofactor. A second class of variant is based on a molecule lacking the B-domain and acid region 3 with increased specific activity. These findings pose the question whether vWF engagement, while an important determinant of FVIII circulating half-life, limits activation by FXa, and if by relieving this constraint we can enhance available FVIIIa levels to promote clot formation.
In aim 2 we will characterize the FIX-Padua variant for which the molecular basis for its enhanced function is uncertain. We will use biochemical and biophysical approaches to identify the mechanism of the hyperfunctional molecule. Emerging structural information derived from FXa aptamers that prevent its binding to FVa and the known structure of snake FVa-FX implicate a region of Xa not previously considered central in Va binding. We hypothesize that the analogous interaction between FIXa and FVIIIa may be enhanced by modifications in the heparin-binding exosite. If successful, our goal is to combine FIX-Padua with modifications from the FIX-loop 90 to generate FIX variants with enhanced biologic properties.
In aim 3 we will pursue studies with variants of FIX that upon activation yield products with increasing zymogen-like character, are long-lived because of their resistance to protease inhibition but may be functionally rescued upon their assembly into the intrinsic Xase. Biochemical studies testing these ideas will be followed by approaches to assess if such variants, and those combined on the FIX-Padua background, have therapeutic value for hemophilia B. Together these approaches will provide new mechanistic insights into intrinsic Xase regulation and activity as well as provide a platform for the development of new therapeutic agents for bleeding disorders.

Public Health Relevance

The activation of coagulation by factor IXa (FIXa) and its cofactor, factor VIIIa, is critical for hemostasis. We have uncovered novel variants of FIX and FVIII with enhanced biological properties. We propose biochemical approaches to define the molecular bases of these variants, which have the potential to provide new insights into the biochemistry of coagulation and set the stage for the next generation of therapeutic modulation of hemophilia and other bleeding disorders.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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Warren, Ronald Q
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Children's Hospital of Philadelphia
United States
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