Methods and kits for determining von Willebrand factor activity in the absence of ristocetin and for determining the activity of ADAMTS-13 protease
AssigneeSiemens Healthcare Diagnostics Products GmbH
InventorsHarald Althaus, Tobias Obser, Juergen Patzke, Reinhard Schneppenheim
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Abstract
Described herein are method(s), kit(s), reagent(s) and the like for determining von Willebrand factor (VWF) activity in a sample in the absence of ristocetin.
Claims12
A method for determining the von Willebrand factor (VWF)-cleaving activity of a disintegrin metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) protease in a sample, comprising the steps of:
- a) mixing a sample which is suspected of containing the ADAMTS-13 protease with isolated VWF as a substrate to provide a reaction mix, wherein the VWF substrate is a high molecular weight, multimeric VWF which includes VWF monomers having at least one amino acid sequence variation resulting in said VWF substrate being broken down by ADAMTS-13 in an accelerated manner compared to a VWF substrate that is high molecular weight, multimeric VWF composed of wild-type VWF monomers;
- b) incubating the reaction mix;
- and
- c) determining residual VWF activity in the reaction mix by mixing the reaction mix or an aliquot thereof with isolated GPIbα protein comprising an amino acid sequence which, when compared to the wild-type sequence of human GPIbα protein (SEQ ID NO:
- 1), includes at least the amino acid residues 1-268 of SEQ ID NO:
- 1 except that amino acid substitutions are found in position 233 and 239;
- and by adding neither ristocetin nor botrocetin.
A method for determining the von Willebrand factor (VWF)-cleaving activity of a disintegrin metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) protease in a sample, comprising the steps of:
- a) mixing a sample which is suspected of containing the ADAMTS-13 protease with (i) isolated VWF and (ii) recombinant or synthetic GPIbα protein to provide a reaction mix;
- wherein the VWF substrate is a high molecular weight, multimeric VWF which includes VWF monomers having at least one amino acid sequence variation resulting in said VWF substrate being broken down by ADAMTS-13 in an accelerated manner compared to a VWF substrate that is high molecular weight, multimeric VWF composed of wild-type VWF monomers;
- wherein the recombinant or synthetic GPIbα protein comprises an amino acid sequence which, when compared to the wild-type sequence of human GPIbα protein (SEQ ID NO:
- 1), includes at least the amino acid residues 1-268 of SEQ ID NO:
- 1 except that amino acid substitutions are found in positions 233 and 239;
- wherein the amino acid substitution in position 233 and/or position 239 of the GPIbα protein comprises a valine or a serine residue;
- and
- wherein the GPIbα protein comprises at least one of SEQ ID NO:
- 4, SEQ ID NO:
- 5, and SEQ ID NO:
- 6;
- b) incubating the reaction mix;
- and
- c) determining residual VWF activity in the reaction mix.
Dependencies are inferred from the claim text.