Plasma prekallikrein activation and kallikrein production assay
A activator for prekallikrein includes isolated, purified, or substantially purified heat shock protein 90, a phosphate ion, and mixtures thereof. The prekallikrein activator is part of an in vitro system for activating prekallikrein in the absence of FXIIa. The system includes the activator, high-molecular weight kininogen, and zinc ions. A method for activating prekallikrein to produce kallikrein in the absence of FXIIa includes mixing prekallikrein with the activator and a composition comprising high molecular weight kininogen and zinc ions. A method for evaluating the activity of a test drug for the production of kallikrein comprises activating prekallikrein and determining the kallikrein produced. The activity (promoting or inhibiting ability) of the tested drug against the production of kallikrein in the absence of FXIIa can be easily measured. The test drug may be an analgesic, anti-allergy, anti-inflammatory drug.
1. A method for evaluating the activity of a test drug towards the production of kallikrein comprising activating prekallikrein by mixing prekallikrein with high molecular weight kininogen, zinc ions and isolated or purified heat-shock protein 90 with said drug, and determining the degree of production of kallikrein.
2. The method for evaluating the activity of a test drug towards the production of kallikrein of claim 1 , wherein the test drug is selected from the group consisting of analgesic, anti-allergy and anti-inflammatory drugs.
3. The method for evaluating the activity of a test drug towards the production of kallikrein of claim 1 , wherein the produced plasma kallikrein is quantitatively determined by using a substrate to the plasma kallikrein.
4. The method for evaluating the activity of a test drug towards the production of kallikrein of claim 3 , wherein said substrate to the plasma kallikrein is selected from the group consisting of a natural substrate, a coloring synthetic substrate and a fluorescent synthetic substrate.
5. The method for evaluating the activity of a test drug of claim 1 wherein the degree of the prekallikrein activation is measured by determination of bradykinin production.
6. The method for evaluating the activity of a test drug of claim 1 wherein the prekallikrein is activated to produce kallikrein in the absence of FXIIa.
7. The method for evaluating the activity of a test drug of claim 1 , wherein the heat-shock protein 90 is purified.
8. A method for activating prekallikrein comprising mixing prekallikrein with high molecular weight kininogen, zinc ions and isolated or purified heat-shock protein 90 to form an activated mixture.
9. The method for activating prekallikrein of claim 8 , wherein said mixture further comprises phosphate.
10. The method for activating prekallikrein of claim 8 wherein the prekallikrein is activated to produce kallikrein in the absence of FXIIa.
11. The method for activating prekallikrein of claim 8 , wherein the heat-shock protein 90 is purified.
12. The method for activating prekallikrein of claim 8 , wherein the prekallikrein is separated or purified.
13. The method for activating prekallikrein of claim 8 , wherein the prekallikrein is purified.
14. The method for activating prekallikrein of claim 8 , wherein the high molecular weight kininogen is separated or purified.
15. The method for activating prekallikrein of claim 8 , wherein the high molecular weight kininogen is purified.
16. The method for activating prekallikrein of claim 8 , wherein said mixing is carried out in vitro.
17. A composition for evaluating the activity of a test drug towards the production of kallikrein comprising prekallikrein, high molecular weight kininogen, zinc ions and isolated or purified heat-shock protein 90.
18. The composition of claim 17 further comprising phosphate ions.
19. The composition of claim 17 , wherein the heat-shock protein 90 is purified.
20. The composition of claim 17 , wherein the prekallikrein is separated or purified.
21. The composition of claim 17 , wherein the prekallikrein is purified.
22. The composition of claim 17 , wherein the high molecular weight kininogen is separated or purified.
23. The composition of claim 17 , wherein the high molecular weight kininogen is purified.