Methods and kits for measuring von Willebrand factor
View Patent ↗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.
1. A nucleic acid vector encoding a GPIbα polypeptide or functional fragment thereof, comprising an amino acid sequence that has at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 and that includes at least two mutations relative to residues 1-290 of SEQ ID NO:11, wherein one of the mutations is D235Y.
2. The vector of claim 1 , wherein the GPIbα polypeptide further comprises a C65A mutation.
3. The vector of claim 2 , wherein the GPIbα polypeptide has a mutation that is selected from the group consisting of G233V and M239V.
4. The vector of claim 3 , wherein the GPIbα polypeptide lacks a transmembrane region.
5. An isolated cell transformed with the vector of claim 1 .
6. An isolated cell comprising a nucleic acid vector encoding a GPIbα polypeptide or functional fragment thereof, comprising an amino acid sequence that has at least 90% sequence identity to residues 1-290 of SEQ ID NO:11, that has at least one mutation that is D235Y, and that lacks a transmembrane region, wherein the polypeptide is expressed in the isolated cell.
7. The isolated cell of claim 6 , wherein the GPIbα polypeptide has a C65A mutation.
8. The isolated cell of claim 6 , wherein the GPIbα polypeptide has a second mutation that is selected from the group consisting of G233V and M239V.
9. An isolated cell comprising a nucleic acid vector encoding a GPIbα polypeptide or functional fragment thereof, comprising an amino acid sequence that has at least 90% sequence identity to residues 1-290 of SEQ ID NO:11, that has at least one mutation that is D235Y, and that is modified to prevent dimerization with a second GPIbα polypeptide, wherein the polypeptide is expressed in the isolated cell.
10. The isolated cell of claim 9 , wherein the GPIbα polypeptide lacks a transmembrane region.
11. The isolated cell of claim 9 , wherein the GPIbα polypeptide has a second mutation that is selected from the group consisting of G233V and M239V.
12. An isolated cell comprising a nucleic acid vector encoding a GPIbα polypeptide or functional fragment thereof, comprising an amino acid sequence that has at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 and that has at least one mutation that is D235Y, wherein the polypeptide is expressed in the isolated cell.