Methods of testing effectiveness of a treatment for thromobotic thrombocytopenic purpura by measuring ADAMTS13-mediated in vivo cleavage of von Willebrand factor
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. An in vivo method for testing effectiveness of a treatment for thrombotic thrombocytopenic purpura (TTP) in a subject comprising measuring a level of von Willebrand Factor (VWF) or recombinant VWF cleavage fragments in a blood sample from the subject before and after the treatment, wherein a decrease in the level of ultra-large multimer forms of VWF with a reduced triplet structure or an increase in the level of recombinant VWF fragment with satellite bands after the treatment indicates that the treatment is effective in increasing a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity or concentration in vivo in the subject thereby indicating effectiveness of the treatment for TTP.
2. The method of claim 1 , wherein measuring the level of ultra-large multimer forms of VWF with a reduced triplet structure or the level of recombinant VWF cleavage fragments with satellite bands comprises performing Western blot analysis with a VWF antibody to visualize the level of the ultra-large multimer forms of VWF or the level of recombinant VWF 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 the level of ultra-large multimer forms of VWF or the level of recombinant VWF fragments is visualized through use of a VWF antibody conjugated to a marker.
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.
8. The method of claim 1 , wherein the level of ultra-large multimer forms of VWF or the level of recombinant VWF fragments is visualized by using high resolution horizontal SDS-agarose gel electrophoresis followed by immunostaining with a polyclonal rabbit anti-human VWF antibody.
9. The method of claim 1 , wherein the level of recombinant VWF cleavage fragments is detected at a sensitivity level of about 0.025 to about 0.05 Ag U/mL VWF.
10. The method of claim 1 , wherein the blood sample is plasma or serum.
11. The method of claim 10 , wherein the blood sample is plasma.
12. The method of claim 1 , wherein the subject is a mammal.
13. The method of claim 12 , wherein the mammal is a human.
14. The method of claim 1 , wherein the increase in recombinant VWF cleavage fragment level is detected by measuring the level of one or more of a 140 kDa VWF fragment or a 176 kDa VWF fragment.
15. The method of claim 14 , wherein the recombinant VWF fragment is a 176 kDa VWF fragment.
16. The method of claim 1 , comprising measuring the level of ultra-large multimer forms of VWF wherein a decrease in the level of ultra-large multimer forms of VWF with a reduced triplet structure after the treatment indicates that the treatment is effective in increasing ADAMTS13 activity or concentration in the subject.
17. The method of claim 1 , comprising measuring the level of recombinant VWF cleavage fragments wherein an increase in the level of recombinant VWF fragments with satellite bands after the treatment indicates that the treatment is effective in increasing ADAMTS13 activity or concentration in the subject.