In addition, they showed that heparin stabilized both glycosylated and non glycosylated VEGF A165 against chaotropic or thermal denaturation without inducing any conformational buy inhibitor change. This implies that the competition of ATP and heparin for binding to the mitogen is of biological rele vance. Further studies have to be undertaken in order to define exactly where ATP Inhibitors,Modulators,Libraries is bound providing a basis for elucidating putative complex receptor interactions. We found no evidence that ATP binding protects VEGF A165 against plasmin cleavage, as pre viously suggested for FGF 2. We therefore propose that the biological activity of the VEGF A165 ATP com plex is due to improved receptor binding, and not due to increased stability of the growth factor.
Supporting this theory, VEGF Inhibitors,Modulators,Libraries A165 failed to induce HUVEC prolif eration when the ATP concentration in the cell culture media was too low. This result corresponded to the minimal concentration of eATP required for the neuroprotective activity of Inhibitors,Modulators,Libraries NGF and FGF 2, which was determined to be approximately 1 nM. In our cell culture experiments, alkaline phosphatase was required to lower the concentration of ATP to levels that affected cell proliferation. It is feasible that high con centrations of alkaline phosphatase had independent effects, and we cannot discount the possibility of minor contamination with proteases. Alkaline phosphatase added without exogeneous VEGF A165 did not influence HUVEC viability. Therefore, we believe side effects of alka line phosphates at these concentrations are unlikely.
This is in line with investigations with other growth factors like FGF2 and NGF that demonstrated Inhibitors,Modulators,Libraries similar results when using alkaline phosphatase to lower eATP concentrations. A completely different observation was made when using another Inhibitors,Modulators,Libraries growth factor, granulocyte colony stimulat ing factor. This factor does not bind ATP and the neuroprotective activity of GCSF is not influenced by degradation of extracellular ATP by alkaline phosphatase. This is in contrast to the situation with the ATP binding growth factors FGF2 and NGF, where an extracellular ATP concentration above about 1 nM is essential for the neuroprotective activity of these growth factors. However, this is plausible in the context of our hypothesis that ATP growth factor interaction is essential for the activity of ATP binding growth factors but not important for non ATP binding growth factors like GCSF.
It is therefore reasonable to assume that the observed inhibition of HUVEC proliferation by VEGF A165 was due to the low ATP levels caused by alkaline selleck products phosphatase, which prevented the formation of the pre sumed biologically active VEGF A165 ATP complex. It is known that eATP can act synergistically with angiogenic growth factors including VEGF A165 via P2Y receptor signaling.