Suppression of ATP diphosphohydrolase/CD39 in human vascular endothelial cells

Vascular ATP diphosphohydrolase/CD39 is an endothelial cell membrane protei n with both ecto-ATPase and ecto-ADPase activities. Suppression of constitu tive CD39 expression may result in elevated concentrations of ATP and ADP a t the vascular interface that could predispose to thrombosis and inflammati on. To study the effects of suppression of CD39 synthesis, stable 25-base a ntisense chimeric oligonucleotides targeting sequences at the 5' region of CD39 were designed. Transfection of these stable oligomers into cultured hu man endothelial cells resulted in dramatic decreases in levels of CD39 mRNA transcripts. Following transfection with antisense oligonucleotides, total ADPase activity fell from 26.0 +/- 3.1 in control cultures to 9.5 +/- 3.4 nmol of P-i min(-1) (mg of protein)(-1) (p < 0.005); suppression of CD39 pr otein expression was also observed by Western blotting. Decreases in ATP di phosphohydrolase activity were associated with increases in concentrations of extracellular purine nucleotides released following stimulation of endot helial cells. Rates of initial hydrolysis of extracellular ATP released fro m purinergic agonist-stimulated endothelial cells decreased from 17.9 +/- 5 .0 to 4.8 +/- 0.5 pmol min(-1) per 10(6) cells (p < 0.005) in antisense tra nsfected cells. Therefore, CD39 regulates extracellular ATP concentrations and may be an important modulator of purinergic receptor activity in vascul ar endothelial cells.