Methods for testing Anti-thrombotic agents

Abstract

The invention provides a transgenic non-human animal expressing von Willebrand Factor A1 protein containing at least one mutation selected from the group consisting of: 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, and any combination thereof.

Description

[0001]This application is a continuation-in-part of International Application No. PCT / US2007 / 015043 filed on Jun. 28, 2007, which claims the benefit of priority of U.S. Ser. No. 60 / 817,600 filed on Jun. 29, 2006, the contents of which are hereby incorporated in their entirety.GOVERNMENT INTERESTS[0002]This invention was made with support from the U.S. Federal Government under Grant No. 5RO1HL63244-7 awarded by the National Heart, Lung, and Blood Institute (NHLBI). As such, the United States government has certain rights in this invention.[0003]Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.[0004]This patent disclosure contains material that is subject to copyright protection. The copyright owner has no o...

AI Technical Summary

Benefits of technology

[0073]The invention provides for where the mutation in the A1 domain of mouse VWF comprises 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>1,1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, and any combination thereof, wherein the mutation corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6. In addition, the mouse VWF A1 domain can be fully or partially replaced with the human VWF A1 domain. In one embodiment, the mutation in mouse VWF-A1 is 1326R>H. In another embodiment, the mouse model comprises a device within a vessel, such as a stent or a graft, or mechanical, chemical, or heat-induced disruption of vascular endothelium in vivo. This model system is useful for testing compounds in an in vivo environment. The compounds can be tested for an effect on the interaction between human platelets and human-like (the mutant VWF-A1, 1326R>H, for example), or the actual human VWF-A1 domain. For example, the animal model can be used for pre-clinical testing of drugs in order to determine whether 1) there is a desired effect on hemostasis and / or thrombus formation or anti-thrombotic effect by the test drug or 2) there is an undesired effect on hemostasis and / or thrombus formation or anti-thrombotic effect by a test drug not specifically designed to alter hemostasis and / or thrombus formation. In the latter case, many drugs are only identified as having an effect on clotting or bleeding once they are in human clinical trials, this animal model will fill an unmet need, which is to test such effects prior to clinical trials. The invention also permits testing of compounds targeted to the VWF-A1 domain that can correct the bleeding phenotype associated with a loss-of-function mutations (1326R>H, for example) by altering the kinetics of the interaction between GPIbα and VWF-A1 (for example, enhancing the on-rate and / or prolonging the bond lifetime as shown for the snake venom protein botrocetin.

[0082]The invention provides a method for rapidly detecting an internal vascular injury site in a subject. The method comprises: administering to a subject a targeted molecular imaging agent, wherein the molecule circulates for an effective period of time in order to bind to the injury site within the subject; tracking a deposition of the labeled targeted molecular imaging agent in the subject; and identifying the site of a thrombus formation in the subject by imaging the targeted molecular imaging agent, thereby the deposition of the targeted molecular imaging agent at the internal vascular injury site is indicative of internal bleeding within a subject. In one embodiment, the targeted molecular imaging agent is administered by subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; by oral, nasal, or topical delivery; or a combination of the routes listed. In another embodiment, the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination of the agents listed. In a further embodiment, the nanoparticle comprises a perfluorocarbon, while in particular embodiments, the nanoparticle is coupled to an antibody, a small molecule, a peptide, or a receptor trap. In some embodiments, the targeted molecular imaging agent specifically binds to a platelet receptor, or a VWF protein, or a portion thereof. In other embodiments, the targeted molecular imaging agent has a T1 / 2 of at least 30 minutes. In further embodiments, imaging comprises a PET scan, MRI, IR scan, ultrasound, nuclear imaging, or a combination of the methods listed. In a particular embodiment, the subject is further administered a pro-thrombotic compound. In a further embodiment, the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold.

Problems solved by technology

Inappropriate deposition of these hemostatic cells in arterial blood vessels due to pathological disease processes such as atherosclerosis can result in lack of blood flow to vital organs such as the heart and brain.

Thus a delicate balance exists between providing adequate hemostasis without causing blockage of blood vessels by excessive platelet deposition (a.k.a. thrombus formation).

One major hurdle hindering drug development in this field is the lack of an appropriate small animal model of thrombosis to test promising therapies.

Moreover, this issue cannot be overcome by simply transfusing mice with human platelets as we have observed that mouse VWF does not support significant interactions with human cells (see below).

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