Thrombin-Induced Release Of Adenine Nucleotides From Cultured Human Endothelial Cells

The effect of thrombin on the release of adenine nucleotides from cultured human umbilical vein endothelium was studied. When endothelial cell monolayers were incubated with ³H-adenosine for two hours at 37°C, fifty percent of the radioactivity was incorporated into the cell. Thin-layer radiochromatography revealed that greater than 90% of the tritium was associated with ADP and ATP.

Addition of thrombin to the monolayers resulted in the release of tritium into the medium. This effect was dose-dependent and saturable, with a maximal response seen at 1 × 10^-8 M thrombin. Diisopropylphosphoryl thrombin (DIP-thrombin) , which is enzymatically inactive, did not cause a release of tritium. Blocking the high-affinity thrombin binding sites which are present on the endothelial cell surface was done with DIP-thrombin. Saturating these binding sites by greater than 95% with a fifty-fold excess of DIP-thrombin had no effect on thrombin-induced release, indicating that the high-affinity binding sites are not involved in the release of adenine nucleotides by thrombin.