Methods for measuring ADAMTS13 activity and protein on platelets and in plasma
Provided are assays to detect ADAMTS13 activity using peptide substrates. These assays can be used for diagnostic applications and to evaluate treatment of thrombotic thrombocytopenic purpura (TTP). Also provided is a novel form of ADAMTS13 found on platelets.
1. A method for measuring the presence of platelet ADAMTS13 comprising:
a) incubating a sample comprising ADAMTS13 with a substrate, wherein the sample is selected from the group consisting of platelet rich plasma (PRP), pooled normal plasma (PNP) and washed platelets, wherein the substrate comprises a peptide moiety and a chromogenic or fluorogenic moiety, wherein the peptide moiety comprises X-Val-Tyr, X-Leu-Tyr or X-Ile-Tyr, wherein X is any amino acid, and wherein the chromogenic moiety is not para-nitroanaline (pNA); and
b) measuring optical density or fluorescence of the sample;
thereby measuring the presence of platelet ADAMTS13.
2. The method of claim 1 , wherein X is Leu.
3. The method of claim 1 , wherein the peptide moiety is Leu-Val-Tyr.
4. The method of claim 1 , wherein the peptide moiety is Leu-Leu-Val-Tyr (SEQ ID NO:2).
5. The method of claim 1 , wherein the peptide moiety is Suc-Leu-Leu-Val-Tyr (Suc-SEQ ID NO:2).
6. The method of claim 1 , wherein the chromogenic moiety is selected from the group consisting of s-benzyl, 5-amino-2-nitrobenzoic acid and 6-amino-1-naphthalenesulfonamides.
7. The method of claim 1 , wherein the fluorogenic moiety is selected from the group consisting of coumarins, fluoresceins, rhodamines, resorufins and dimethylacridinones.
8. The method of claim 7 , wherein the fluorogenic moiety is a coumarin.
9. The method of claim 8 , wherein the coumarin is 7-amino-4-methylcoumarin (AMC).
10. A method for measuring the presence of platelet ADAMTS13 comprising:
a) incubating a sample comprising ADAMTS13 with a substrate, wherein the sample is selected from the group consisting of PRP, PNP and washed platelets, wherein the substrate comprises X-peptide moiety-Z, wherein the peptide moiety comprises Val-Tyr-Met, Leu-Tyr-Met or Ile-Tyr-Met, wherein X is a donor moiety and Z is an acceptor moiety, and wherein the donor and acceptor moieties mediate fluorescence resonance energy transfer; and
b) measuring fluorescence of the sample;
thereby measuring the presence of platelet ADAMTS13.
11. The method of claim 10 , wherein the peptide moiety is Asn-Leu-Val-Tyr-Met-Val-Thr-Gly-Asp (SEQ ID NO:3).
12. The method of claim 10 , wherein the donor moiety is EDANS.
13. The method of claim 12 , wherein the acceptor moiety is DABCYL.
14. The method of claim 10 , wherein the substrate is NH 2 -Arg-Lys-(DABCYL)-Asn-Leu-Val-Tyr-Met-Val-Thr-Gly-Asp-(EDANS)-Arg-COOH-(NH 2 -Arg-Lys-(DABCYL)-SEQ ID NO:3-(EDANS)-Arg-COOH)—.
15. A method for identifying an inhibitor of platelet ADAMTS13 comprising:
a) incubating a sample comprising platelet ADAMTS13 with a candidate inhibitor of ADAMTS13, wherein the sample is selected from the group consisting of PRP, PNP and washed platelets; and
b) incubating the sample with a substrate comprising a peptide moiety and a chromogenic or fluorogenic moiety, wherein the peptide moiety comprises X-Val-Tyr, X-Leu-Tyr or X-Ile-Tyr, wherein X is any amino acid and wherein the chromogenic moiety is not pNA; and
c) measuring the optical density or fluorescence of the sample;
wherein the inhibitor is identified by reduced optical density or fluorescence of the sample compared with optical density or fluorescence of a control sample comprising platelet ADAMTS13, wherein the control sample is selected from the group consisting of PRP, PNP and washed platelets, and wherein the control sample was not incubated with the candidate inhibitor.
16. The method of claim 15 , wherein X is Leu.
17. The method of claim 15 , wherein the peptide moiety is Leu-Val-Tyr.
18. The method of claim 15 , wherein the peptide moiety is Leu-Leu-Val-Tyr (SEQ ID NO:2).
19. The method of claim 15 , wherein the peptide moiety is Suc-Leu-Leu-Val-Tyr (Suc-SEQ ID NO:2).
20. The method of claim 15 , wherein the chromogenic moiety is selected from the group consisting of s-benzyl, 5-amino-2-nitrobenzoic acid and 6-amino-1-naphthalenesulfonamides.
21. The method of claim 15 , wherein the fluorogenic moiety is selected from the group consisting of coumarins, fluoresceins, rhodamines, resorufins and dimethylacridinones.
22. The method of claim 21 , wherein the fluorogenic moiety is a coumarin.
23. The method of claim 22 , wherein the coumarin is 7-amino-4-methylcoumarin (AMC).
24. The method of claim 15 , wherein the candidate inhibitor is from a chemical compound library, a phage display library, a natural chemical library or a combinatorial chemistry library.
25. The method of claim 15 , wherein the inhibitor is an antibody.
26. A method for identifying an inhibitor of platelet ADAMTS13 comprising:
a) incubating a sample comprising platelet ADAMTS13 with a candidate inhibitor of ADAMTS13, wherein the sample is selected from the group consisting of PRP, PNP and washed platelets; and
b) incubating the sample with a substrate comprising a peptide moiety and a chromogenic or fluorogenic moiety, wherein the peptide moiety comprises X-Val-Tyr, X-Leu-Tyr or X-Ile-Tyr, wherein X is any amino acid and wherein the chromogenic moiety is not pNA; and
c) measuring the optical density or fluorescence of the sample;
wherein the inhibitor is identified by reduced optical density or fluorescence of the sample compared with optical density or fluorescence of a control sample comprising platelet ADAMTS13, wherein the control sample is selected from the group consisting of PRP, PNP and washed platelets, and wherein the control sample was not incubated with the candidate inhibitor.
27. The method of claim 26 , wherein the peptide moiety is Asn-Leu-Val-Tyr-Met-Val-Thr-Gly-Asp (SEQ ID NO:3).
28. The method of claim 26 , wherein the donor moiety is EDANS.
29. The method of claim 28 , wherein the acceptor moiety is DABCYL.
30. The method of claim 26 , wherein the substrate is NH 2 -Arg-Lys-(DABCYL)-Asn-Leu-Val-Tyr-Met-Val-Thr-Gly-Asp-(EDANS)-Arg-COOH (NH 2 -Arg-Lys-(DABCYL)-SEQ ID NO:3-(EDANS)-Arg-COOH)—.
31. The method of claim 26 , wherein the candidate inhibitor is from a chemical compound library, a phage display library, a natural chemical library or a combinatorial chemistry library.
32. The method of claim 26 , wherein the inhibitor is an antibody.
33. A method for diagnosing TTP in a subject comprising:
a) measuring the amount of platelet ADAMTS13 in a test sample from the subject according to the method of claim 1 , wherein the test sample is selected from the group consisting of PRP and washed platelets; and
b) comparing the amount of platelet ADAMTS13 in the test sample to the amount of platelet ADAMTS13 in a control sample having a normal amount of platelet ADAMTS13 wherein the control sample is selected from the group consisting of PRP, PNP and washed platelets;
wherein TTP is diagnosed by reduced amount of platelet ADAMTS13 in the test sample compared with the control sample.
34. The method of claim 33 , wherein X is Leu.
35. The method of claim 33 , wherein the peptide moiety is Leu-Val-Tyr.
36. The method of claim 33 , wherein the peptide moiety is Leu-Leu-Val-Tyr (SEQ ID NO:2).
37. The method of claim 33 , wherein the peptide moiety is Suc-Leu-Leu-Val-Tyr (Suc-SEQ ID NO:2).
38. The method of claim 33 , wherein the chromogenic moiety is selected from the group consisting of s-benzyl, 5-amino-2-nitrobenzoic acid and 6-amino-1-naphthalenesulfonamides.
39. The method of claim 33 , wherein the fluorogenic moiety is selected from the group consisting of coumarins, fluoresceins, rhodamines, resorufins and dimethylacridinones.
40. The method of claim 39 , wherein the fluorogenic moiety is a coumarin.
41. The method of claim 40 , wherein the coumarin is 7-amino-4-methylcoumarin (AMC).
42. The method of claim 33 , wherein the TTP is congenital.
43. The method of claim 33 , wherein the TTP is acquired.
44. A method for diagnosing TTP in a subject comprising:
a) measuring the amount of platelet ADAMTS13 in a test sample from the subject according to the method of claim 10 , wherein the test sample is selected from the group consisting of PRP and washed platelets; and
b) comparing the amount of platelet ADAMTS13 in the test sample to the amount of platelet ADAMTS13 in a control sample having a normal amount of platelet ADAMTS13, wherein the control sample is selected from the group consisting of PRP, PNP and washed platelets;
wherein TTP is diagnosed by reduced amount of platelet ADAMTS13 activity in the test sample compared with the control sample.
45. The method of claim 44 , wherein the peptide moiety is Asn-Leu-Val-Tyr-Met-Val-Thr-Gly-Asp (SEQ ID NO:3).
46. The method of claim 44 , wherein the donor moiety is EDANS.
47. The method of claim 46 , wherein the acceptor moiety is DABCYL.
48. The method of claim 44 , wherein the substrate is NH 2 -Arg-Lys-(DABCYL)-Asn-Leu-Val-Tyr-Met-Val-Thr-Gly-Asp-(EDANS)-Arg-COOH (NH 2 -Arg-Lys-(DABCYL)-SEQ ID NO:3-(EDANS)-Arg-COOH)—.
49. The method of claim 44 , wherein the TTP is congenital.
50. The method of claim 44 , wherein the TTP is acquired.
51. A method for diagnosing acquired TTP in a subject comprising:
a) incubating a sample comprising platelet ADAMTS13 with plasma from the subject, wherein the sample is selected from the group consisting of PRP, PNP and washed platelets; and
b) measuring the amount of platelet ADAMTS13 in the sample according to the method of claim 1 ;
wherein acquired TTP is diagnosed by reduced amount of platelet ADAMTS13 activity in the sample compared with the amount of platelet ADAMTS13 in a control sample having a normal amount of platelet ADAMTS13.
52. The method of claim 51 , wherein X is Leu.
53. The method of claim 51 , wherein the peptide moiety is Leu-Val-Tyr.
54. The method of claim 51 , wherein the peptide moiety is Leu-Leu-Val-Tyr (SEQ ID NO:2).
55. The method of claim 51 , wherein the peptide moiety is Suc-Leu-Leu-Val-Tyr (Suc-SEQ ID NO:2).
56. The method of claim 51 , wherein the chromogenic moiety is selected from the group consisting of s-benzyl, 5-amino-2-nitrobenzoic acid and 6-amino-1-naphthalenesulfonamides.
57. The method of claim 51 , wherein the fluorogenic moiety is selected from the group consisting of coumarins, fluoresceins, rhodamines, resorufins and dimethylacridinones.
58. The method of claim 57 , wherein the fluorogenic moiety is a coumarin.
59. The method of claim 58 , wherein the coumarin is 7-amino-4-methylcoumarin (AMC).
60. A method for diagnosing acquired TTP in a subject comprising:
a) incubating a sample comprising platelet ADAMTS13 with plasma from the subject, wherein the sample is selected from the group consisting of PRP, PNP and washed platelets; and
b) measuring the amount of platelet ADAMTS13 in the sample according to the method of claim 12 ;
wherein acquired TTP is diagnosed by reduced amount of platelet ADAMTS13 in the sample compared with the amount of platelet ADAMTS13 in a control sample having a normal amount of platelet ADAMTS 13.
61. The method of claim 60 , wherein the peptide moiety is Asn-Leu-Val-Tyr-Met-Val-Thr-Gly-Asp (SEQ ID NO:3).
62. The method of claim 60 , wherein the donor moiety is EDANS.
63. The method of claim 62 , wherein the acceptor moiety is DABCYL.
64. The method of claim 60 , wherein the substrate is NH 2 -Arg-Lys-(DABCYL)-Asn-Leu-Val-Tyr-Met-Val-Thr-Gly-Asp-(EDANS)-Arg-COOH (NH 2 -Arg-Lys-(DABCYL)-SEQ ID NO:3(EDANS)-Arg-COOH)—.