Methods of measuring ADAMTS13-mediated in vivo cleavage of von Willebrand factor and uses thereof
View Patent ↗The invention generally relates to methods of measuring cleaved von Willebrand factor (VWF) fragments. More specifically, the invention relates to methods of measuring the ability of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) to cleave VWF in vivo. The invention also relates to methods of using various animal models which demonstrate ADAMTS13 activity similar to that of a human. The invention further relates to methods of measuring the cleavage products of rVWF in mammals, particularly in humans and in human plasma.
1. A method for determining aberrant in vivo a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity in a test subject comprising the step of measuring recombinant von Willebrand Factor (rVWF) cleavage fragments in a blood sample from the test subject after
(a) administering rVWF to the test subject, or
(b) adding rVWF to the blood sample from the test subject and subjecting the blood sample to shear stress,
wherein a change in rVWF cleavage fragment levels in the blood sample of the test subject compared to rVWF cleavage fragment levels in a blood sample from a control subject known to have normal ADAMTS13 activity indicates aberrant in vivo ADAMTS13 activity in the test subject.
2. The method of claim 1 wherein the step of measuring of rVWF cleavage fragment level in the blood sample comprises performing Western blot analysis with a VWF antibody to visualize rVWF cleavage fragments.
3. The method of claim 2 wherein the Western blot analysis is carried out under non-reducing conditions.
4. The method of claim 2 wherein rVWF fragments are visualized by detecting a marker conjugated to an antibody that binds rVWF fragments in the blood sample.
5. The method of claim 4 wherein the marker is alkaline phosphatase (ALP) or horseradish peroxidase (HRP).
6. The method of claim 4 wherein the marker is detected with enhanced chemiluminescence (ECL).
7. The method of claim 2 wherein the VWF antibody is monoclonal or polyclonal.
8. The method of claim 1 wherein rVWF cleavage fragments are detected at a sensitivity level of about 0.025 to about 0.05 Ag U/mL VWF.
9. The method of claim 1 wherein the blood sample is plasma or serum.
10. The method of claim 9 wherein the blood sample is plasma.
11. The method of claim 1 wherein the subject is a mammal.
12. The method of claim 11 wherein the mammalian subject is human, rabbit, monkey, dog, rat, mouse, or pig.
13. The method of claim 12 wherein the subject is human, rabbit, monkey, or dog.
14. The method of claim 13 wherein the subject is human.
15. The method of claim 1 wherein rVWF cleavage fragment level in the blood sample of the test subject is increased compared to rVWF cleavage fragment level in the blood sample from the control subject indicating increased in vivo ADAMTS13 activity in the test subject compared to the control subject.
16. The method of claim 1 wherein rVWF cleavage fragment level in the blood sample of the test subject is decreased compared to rVWF cleavage fragment level in the blood sample from the control subject indicating decreased in vivo ADAMTS13 activity in the test subject compared to the control subject.
17. The method of claim 1 wherein the change in rVWF cleavage fragment level is detected by measuring the level of one or more of a 140 kDa VWF cleavage fragment or a 176 kDa VWF cleavage fragment.
18. The method of claim 17 wherein the rVWF fragment is a 176 kDa VWF fragment.