Compositions and methods for in vitro assays of fibrin activity
The present invention provides compositions exhibiting in vivo activity of fibrin in an in vitro setting, in vitro assays comprising such compositions, methods of producing such compositions, and methods of using such compositions and assays. The compositions of the invention include molecules with the biochemical properties of 1) high affinity binding to fibrin receptors and 2) activation of cell-signaling systems comparable to that observed in vivo by fibrin. The fibrin compositions of the invention are compatible both in biochemical assays and cell-based assays, and thus useful for in vitro assays for screening of test agents that modulate cell activation and/or signaling pathways mediated by fibrin or associated with fibrin activity.
1. An assay method for identification of test agents that modulate CD11b/CD18 signaling through fibrin, comprising the steps of:
immobilizing a fibrin composition to a solid support surface, wherein the fibrin composition displays an exposed, pro-inflammatory, cryptic fibrin epitope capable of binding to CD11b/CD18 integrin;
introducing the test agent to the support surface;
measuring the binding affinity of the test agent to the fibrin composition immobilized on the solid support surface; and
identifying agents that exhibit a significant increased binding affinity to the fibrin composition in vitro compared to a control lacking the fibrin composition.
2. A method for identification of a binding partner for modulation of a biological process involving CD11b/CD18 signaling through fibrin, said method comprising:
providing a research tool composition comprising: a fibrin composition adhered to a solid surface, wherein the fibrin composition comprises an exposed, pro-inflammatory, cryptic fibrin epitope capable of binding to CD11b/CD18 integrin;
contacting one or more binding partners with the research tool composition; and
isolating the binding partners that display a significant change in functional activity of the fibrin in the research tool composition;
wherein the binding partners that display the change in functional activity of the fibrin in the research tool composition are candidate agents for modulation of a biological process involving CD11b/CD18 signaling through the interaction of CD11b/CD18 integrin with fibrin.
3. The method of claim 1 , wherein the solid surface comprises glass.
4. The method of claim 1 , wherein the solid surface comprises polyethylene, polystyrene or a cyclic olefin polymer substrate.
5. The method of claim 1 , wherein the cryptic fibrin epitope is γ 377_395 .
6. The method of claim 1 , wherein the immobilizing comprises drying the fibrin composition on the solid support.
7. The method of claim 2 , wherein the solid surface comprises glass.
8. The method of claim 2 , wherein the solid surface comprises polyethylene, polystyrene or a cyclic olefin polymer substrate.
9. The method of claim 2 , wherein the cryptic fibrin epitope is γ 377_395 .
10. The method of claim 2 , wherein the providing comprises drying the fibrin composition on the solid support.
11. The method of claim 2 , wherein the method further comprises introducing cells that express CD11b/CD18 integrin to the research tool composition.
12. The method of claim 11 , wherein the cells introduced to the research tool composition comprise macrophages.