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-9778272-B2
Publication Date
2017-10-03
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 relates to ristocetinless/agonistless methods and kits for measuring von Willebrand factor (VWF) function. The approach uses recombinant platelet glycoprotein Ibb1 (GPIbb1) gain-of-function mutants to form a VWFGPIbb1 complex that is detectable using a labeled anti-VWF antibody.
The described mutants include GPIbb1 polypeptide variants having at least two mutations selected from G233V, D235Y, and M239V, with an option for a triple mutant. The mutants enable spontaneous binding in the absence of ristocetin/botrocetin, supporting VWF:IbCo type measurements that are described as better reflecting VWF function and avoiding assay variability associated with VWF:RCo.
Assay formats described include flow cytometry/FACS and solid-phase ELISA, with VWFGPIbb1 interaction measured using patient samples. The document further describes comparative measurement and correlation of assay readouts with clinical VWF:RCo and VWF antigen (VWF:Ag) in patient cohorts, and includes ELISA discussion addressing surface effects and measurement comparability.
Claims Coverage
The independent claims are directed to engineered nucleic acid vectors and isolated cells expressing GPIbb1 variants with specified sequence identity and mutation constraints, including D235Y, and additional constraints related to dimerization and structural features such as absence of a transmembrane region. Across the independent claims, the inventive features center on four engineered GPIbb1-based claim groups.
Vector encoding GPIbb1 with defined identity and D235Y plus additional mutation
A nucleic acid vector encoding a GPIbb1 polypeptide (or functional fragment) comprising an amino acid sequence having at least 90% sequence identity to residues 1390 of SEQ ID NO:11 and including at least two mutations relative to residues 1390 of SEQ ID NO:11, wherein one of the mutations is D235Y.
Isolated cell expressing D235Y GPIbb1 lacking a transmembrane region
An isolated cell comprising a nucleic acid vector encoding a GPIbb1 polypeptide (or functional fragment) with an amino acid sequence having at least 90% sequence identity to residues 1390 of SEQ ID NO:11, at least one mutation being D235Y, and lacking a transmembrane region, wherein the polypeptide is expressed in the isolated cell.
Isolated cell expressing D235Y GPIbb1 preventing dimerization
An isolated cell comprising a nucleic acid vector encoding a GPIbb1 polypeptide (or functional fragment) comprising an amino acid sequence having at least 90% sequence identity to residues 1390 of SEQ ID NO:11, including at least one mutation that is D235Y, and that is modified to prevent dimerization with a second GPIbb1 polypeptide, wherein the polypeptide is expressed in the isolated cell.
Isolated cell expressing GPIbb1 with D235Y mutation
An isolated cell comprising a nucleic acid vector encoding a GPIbb1 polypeptide (or functional fragment) comprising an amino acid sequence having at least 90% sequence identity to residues 1390 of SEQ ID NO:11 and that has at least one mutation that is D235Y, wherein the polypeptide is expressed in the isolated cell.
The claim set covers engineered nucleic acid vectors and isolated cells expressing GPIbb1 polypeptides having at least 90% identity to SEQ ID NO:11 residues 1390, including the D235Y mutation, and includes further inventive structural and functional constraints in specific independents, such as lacking a transmembrane region and being modified to prevent dimerization with a second GPIbb1 polypeptide.
Stated Advantages
Avoids ristocetin dependence and assay variability associated with VWF:RCo.
Enables VWF:IbCo measurements that better reflect VWF function.
Documented Applications
Measuring von Willebrand factor (VWF) function using ristocetinless/agonistless methods and kits based on formation of a VWFGPIbb1 complex detectable with labeled anti-VWF, including flow cytometry/FACS and solid-phase ELISA formats, including patient-sample testing.
Correlating VWFGPIbb1-based assay results with clinical VWF:RCo and VWF antigen (VWF:Ag) in patient cohorts.
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