Methods for testing Anti-thrombotic agents

Inactive Publication Date: 2009-08-13
THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0084]The invention is directed to understanding the effect that alterations in platelet size and shape have on the force-driven kinetics of the GPIb alpha-VWF-A1 tether bond. Platelets can bind to and translocate on surface-immobilized VWF under shear forces that preclude selectin-dependent adhesion of leukocytes to the vessel wall, due to their small discoid shape and not as a consequence of the unique kinetic properties of the GPIb alpha-VWF-A1 tether bond. The contribution of particle geometry in supporting the interactions between this receptor-ligand pair, we is performed by evaluating the interaction between vWF-A1 coated microspheres, ranging from 4 to 12 μm in diameter (platelet to leukocyte size), with surface-immobilized platelets under physiologic flow conditions. The utility of using polystyrene microspheres with a uniform shape and size to permit determination of the relationship between wall shear stress and the force acting on the GPIb alpha-vWF-A1 tether bond has been demonstrated. Moreover, it has been shown that the “sidedness” of the receptor or ligand does not alter the kinetic propertie

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.

Method used

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  • Methods for testing Anti-thrombotic agents
  • Methods for testing Anti-thrombotic agents
  • Methods for testing Anti-thrombotic agents

Examples

Experimental program
Comparison scheme
Effect test

example 1

VWF Characterization

[0206]VWF Microsphere Studies

[0207]The association and dissociation kinetics of the GPIb alpha-VWF-A1 bond and the impact of fluid shear and particle size on these parameters can be determined by measuring the frequency and duration of transient adhesive events, known as transient tethers, that represent the smallest unit of interaction observable in a parallel-platelet flow chamber.

[0208]Production of recombinant VWF-A1 protein and coating of microspheres. The generation of recombinant VWF-A1 protein (residues 1238 to 1472 of the mature, recombinant VWF) and its subsequent coupling to microspheres is performed as previously described (Doggett, T. A. et al. (2002) Biophys. J. 83, 194-205). Proper size, purity, and disulfide bonding of all proteins is assessed by Coomasie-blue staining of SDS-PAGE gels run under reducing and non-reducing conditions. Mass spectrometry is also employed to evaluate size and disulfide bonding pattern.

[0209]The resulting recombinant pr...

example 2

VWF-A1 Mutagenesis

[0230]Preliminary results indicate that minor differences may exist between murine and human VWF that would preclude one from studying human platelet behavior in a mouse model of thrombosis. However, our findings that the estimated off-rate values and structure of these domains are similar suggest that one can investigate the role of the biophysical properties of the GPIb alpha-VWF-A1 bond in regulating platelet-VWF interactions in vivo using a mouse model. However, neither a delineation of the binding region for GPIb alpha within the murine VWF-A1 domain nor determination of the impact of mutations on the kinetics of this interaction has been performed to date. Thus, both murine and human A1 crystal structures can be exploited to 1) identify candidate residues involved in the binding site for murine GPIb alpha and to determine their impact on the kinetic properties of this receptor-ligand pair, 2) identify residues that confer species specificity, and 3) ascertain...

example 3

Genetically Modified VWF-A1 Mice

[0251]Recent kinetic evaluation of mutations associated with type 2B and platelet-type vWD suggests that the intrinsic properties of the GPIb alpha-VWF-A1 tether bond contribute to the regulation of platelet interactions with VWF. This is also supported by our preliminary studies investigating the impact of botrocetin on the biophysical properties of this receptor-ligand pair. Thus, by using the information obtained in Example 2, mutations can be incorporated into the murine A1 domain of the VWF gene that increase or decrease the intrinsic on- and off-rates by varying degrees in order to truly understand the importance of these kinetic parameters in controlling platelet adhesion. Moreover, the role of the minor binding site, where the majority of type 2B mutations have been identified, can be further delineated by combining such mutations with those that significantly shorten the lifetime of interaction between GPIb alpha and VWF-A1. Results indicate ...

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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...

Claims

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Application Information

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IPC IPC(8): A61K51/00A61K49/00C12Q1/02C12N15/63C40B30/00C07K14/00C07H21/04A01K67/00
CPCA01K67/0275A01K2227/105C12N15/8509C07K14/755A01K2267/0381A01K67/0278A01K2207/15A01K2217/072A01K2267/0393G01N33/566G01N2333/70596G01N2500/02
Inventor DIACOVO, THOMASCHEN, JIANCHUN
Owner THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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