Methods and kits for measuring von willebrand factor
Inventors
Assignees
Versiti Blood Research Institute Foundation Inc • Medical College of Wisconsin • Blood Center Research Foundation Inc
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Publication Number
US-9046535-B2
Publication Date
2015-06-02
Expiration Date
Abstract
Methods and kits for measuring levels of von Willebrand factor function in a sample without using a platelet aggregation agonist, such as ristocetin, comprising recombinant glycoprotein Ibα having at least two of a G233V, D235Y and M239V mutations and an agent to detect a complex between the recombinant glycoprotein Ibα and von Willebrand factor.
Core Innovation
The invention describes ristocetinless von Willebrand factor function assays and corresponding kits for measuring von Willebrand factor function. The assays use a recombinant gain-of-function platelet glycoprotein Ibα (GPIbα) with at least two mutations relative to residues 1-290 of SEQ ID NO:11, including D235Y. The recombinant GPIbα is capable of forming a complex with von Willebrand factor (VWF), enabling detection of a VWF function readout without dependence on ristocetin/botrocetin.
The VWF function measurement is demonstrated as VWF:IbCo signaling in an agonistless or ristocetinless format. Complex formation occurs with immobilized or heterologously expressed mutant GPIbα on cells, supporting spontaneous VWF binding and detection of VWF:IbCo without reliance on ristocetin/botrocetin. Detection approaches include use of anti-VWF antibodies and anti-GPIbα antibodies with assay formats including flow cytometry (FC)/FACS and enzyme-linked immunosorbent assay (ELISA).
The invention also provides ELISA solid-phase implementations using immobilized mutant GPIbα constructs. The document describes that VWF measurements can be obtained in formats where ristocetin is added or omitted, and it discusses solid-phase and GPIbα construct requirements for obtaining comparable measurements. In patient-plasma testing, the assay performance is reported across von Willebrand disease (VWD) subtypes and apparent phenotypes, including type 2M, type 2B, type 3, and SNP-related cases, with an alignment to in vivo function described in the document.
Claims Coverage
The independent claim coverage centers on an isolated GPIbα polypeptide or functional fragment engineered to form a complex with VWF, with at least two specified mutations including D235Y, and with the sequence constrained by high identity to residues 1-290 of SEQ ID NO:11. One inventive feature is identified in the independent claim coverage, with additional dependent claims refining mutation combinations and assay-related constraints.
Mutant GPIbα capable of forming a complex with VWF
An isolated GPIbα polypeptide or functional fragment comprising an amino acid sequence with at least 90% sequence identity to residues 1-290 of SEQ ID NO:11, including at least two mutations relative to residues 1-290 of SEQ ID NO:11, wherein one of the mutations is D235Y, and wherein the isolated GPIbα polypeptide or fragment is capable of forming a complex with von Willebrand factor (VWF).
Overall claim coverage is directed to engineered isolated GPIbα or functional fragments defined by high sequence identity to residues 1-290 of SEQ ID NO:11 and inclusion of at least two mutations including D235Y, where the construct forms a complex with VWF, enabling downstream ristocetinless VWF function readouts described in the document.
Stated Advantages
Enables VWF function measurement without dependence on ristocetin/botrocetin.
Provides VWF:IbCo signal in an agonistless or ristocetinless format through complex formation between mutant GPIbα and VWF.
Shows patient-plasma performance across VWD subtypes and apparent phenotypes, including type 2M, type 2B, type 3, and SNP-related cases.
Demonstrates improved alignment with in vivo function as described in the document.
Documented Applications
Ristocetinless VWF function assays performed in FC/FACS formats using immobilized or heterologously expressed mutant GPIbα on cells with detection of VWF complexes.
Ristocetinless VWF function assays performed in ELISA formats using solid-phase immobilized mutant GPIbα for measuring VWF binding/function readouts.
Patient-plasma testing for VWD subtypes and apparent phenotypes, including type 2M, type 2B, type 3, and SNP-related cases.
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