Towards More Effective Treatment for Blood Clotting Disorders: Pharmacogenetics of Von Willebrand Factor (VWF) and Related Proteins

Principal Investigator: Chava Kimchi-Sarfaty, PhD
Office / Division / Lab: OBRR / DH / LH

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Overview

Public Health Issue: Von Willebrand disease, the most common inherited blood disorder, is characterized by the inability to clot and serious bleeding risks. Thrombotic Thrombocytopenic Purpura (TTP) is a life threatening thrombotic condition resulting from the uncontrolled secretion of Ultra Large Von Willebrand Factor (ULVWF) multimers. Currently replacement therapy is the most effective treatment for severe VWD patients and for TTP - ADAMTS13 deficiency. However, as with all plasma-derived products, the risk of transmission of infectious agents, mainly prions, cannot be completely eliminated. A recombinant protein treatment is not yet available to patients. Therefore, expanding our knowledge of these possible recombinant proteins, and establishing assays to detect them is necessary in order to provide tools for evaluation of regulatory submissions on VWF and ADAMTS13 recombinant proteins received by OBRR.

Regulatory Contribution: (1) Establishing assays to determine the expression and function of Von Willebrand Factor (VWF) and its protease, ADAMTS13, for future applications to be submitted to FDA. (2) Studying different genetic polymorphic-or splicing forms of these recombinant proteins will reveal the highest expression and sequence of the functional synthetic proteins that may also have less immunogenicity, since it will be administered in lower concentrations. (3) Identifying other forms of ADAMTS13 and VWF will aid in determining the purity of future and currently used VWF plasma and recombinant proteins. Categorizing the various forms of the VWF proteins in mass spectrometry will help in regulation of new VWF products. Moreover, the splice forms may differ with respect to their half lives, pK values and pharmacokinetics. (4) Characterizing the cellular localization of ADAMTS13 to understand its extracellular and intracellular role, which will aid in determining the complete functionality of ADAMTS13 product applications. (5) Additional aberrant mRNA splicing forms might also be related to various types of VWD or TTP. Identifying mutations in VWD or TTP patients that were not classified earlier will help in regulation of new products and will assist in targeting treatment to the individual patient's disease. (6) Cyclosporine A and cisplatin are well known drugs that may cause side effects to VWF or ADAMTS13 expression and function.

Research Approach: We are developing sensitive assays to measure slight variations in expression or functional levels of VWF and ADAMTS13 using flow cytometry (FACS), confocal microscopy (in collaboration with Garfield from NCI, NIH), FACS, Western blots (in collaboration with Soeijima, Japan), FRETS, and ELISA. We have developed a flow cytometry assay and in addition, specific conformational sensitive antibodies to ADAMTS13 that are tested in flow cytometry in the presence of the VWF substrate (Sauna et al., submitted). We are focusing on finding new splicing sites in the ADAMTS13 and VWF genes (in collaboration with researchers from Dr. Chris Burge's lab at the M.I.T.), and we have found a new form in liver ADAMTS13 highly expressing cells (Shomron et al., in preparation). Moreover, we are evaluating the known polymorphic sites in these genes: Common synonymous and non-synonymous polymorphisms (Kimchi-Sarfaty et al., Science 2007) in these genes are generated in vectors using the site-directed mutagenesis method and then evaluated for gene expression and protein function.

Mission Relevance & Outcomes: The Division of Hematology is responsible for the evaluation of biologic products related to blood. Among these products are recombinant coagulation factors that are substitutes for their plasma-derived counterparts. A genetically designed recombinant protein, which does not necessarily include all the native exons, and is comprised of various polymorphisms, could convey improved function or expression as compared to the native wild-type protein. Such proteins, if administered at lower concentrations, may be less immunogenic. Our research will provide tools to determine the quality of these synthetic proteins.

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Publications

Mol Pharmacol 2008 Apr;73(4):1254-63
Modulation of Na+-Ca2+ exchanger expression by immunosuppressive drugs is isoform-specific.
Elbaz B, Alperovitch A, Gottesman MM, Kimchi-Sarfaty C, Rahamimoff H

Cancer Res 2007 Oct 15;67(20):9609-12
Silent polymorphisms speak: how they affect pharmacogenomics and the treatment of cancer.
Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM

Pharmacogenomics 2007 Jun;8(6):527-532
The sounds of silence: synonymous mutations affect function.
Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM

Ann N Y Acad Sci 2007 Mar;1099:204-14
Cyclosporin A-dependent downregulation of the Na+/Ca2+ exchanger expression.
Rahamimoff H, Elbaz B, Alperovich A, Kimchi-Sarfaty C, Gottesman MM, Lichtenstein Y, Eskin-Shwartz M, Kasir J

Science 2007 Jan 26;315(5811):525-8
A "Silent" Polymorphism in the MDR1 Gene Changes Substrate Specificity.
Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, Gottesman MM

Hum Gene Ther 2005 Sep;16(9):1110-5
Efficient Delivery of RNA Interference Effectors via In Vitro-Packaged SV40 Pseudovirions.
Kimchi-Sarfaty C, Brittain S, Garfield S, Caplen NJ, Tang Q, Gottesman MM.