CHARACTERIZATION OF ECTO-ATPASE ON HUMAN BLOOD-CELLS - A PHYSIOLOGICAL-ROLE IN PLATELET-AGGREGATION

Ecto-ATPase (EC 3.6.1.15) is a plasma membrane-bound enzyme which degr ades extracellular triphosphate nucleotides. Although its physiologica l function is still unclear, the enzyme obscures the study of P-2 puri noceptors (i.e. receptors for ATP and other di- and triphosphate nucle otides), since it is capable of metabolizing the pharmacological ligan ds, such as ATP, for these receptors. We characterized the ecto-ATPase activity on human blood cells with a [gamma(32)P]ATP assay and HPLC m easurements. We also determined whether ecto-ATPase activity could aff ect the anti-aggregatory role of ATP in whole human blood. The K-m for ATP of the ecto-ATPase on human blood cells was 8.5 +/- 2.3 mu M and the maximum degradation rate, at 37 degrees, was 2.7 +/- 1.1 nmol ATP/ (min x mL whole blood). In whole blood the major part of ATP was broke n down by the blood cells, predominantly by the leukocytes. ATP and UT P were broken down equally well, mainly yielding the corresponding di- and monophosphates. In search of inhibitors for the ecto-ATPase, we s tudied several analogs of ATP. 8-Bromo-ATP as well as 2'- and 3'-deoxy -ATP were substrates for the enzyme. In contrast, modification of the phosphate side chain yielded inhibitors. Subsequently, a possible role of the ecto-ATPase in platelet aggregation was verified. To assess th e role of the plasma membrane-bound enzyme, platelet aggregation was d etermined in whole blood instead of platelet-rich plasma. In the prese nce of ATP alone, an antagonist of ADP-induced platelet aggregation, s ome aggregation was still observed. As breakdown of ATP by the ecto-AT Pase leads to gradual formation of ADP, as mentioned above, we compare d the effects of a stepwise Versus bolus addition of ADP. Subsequent d osing of ADP (1.5, 2.5, 5 and 10 mu M) resulted in platelet aggregatio n but to a much smaller extent, at most approximately 60%, compared to the amount of platelet aggregation obtained with a bolus addition of ADP (10 mu M). In conclusion, human blood cells possess a high affinit y ecto-ATPase which degrades ATP as well as ATP analogs with modified base and ribose moieties. ATP analogs with a modified phosphate chain are inhibitors of the ecto-ATPase. A direct role of the ecto-ATPase ac tivity on platelet aggregation is probably small, as degradation of AT P to ADP proceeds slowly and cumulative addition of ADP to platelets i n whole blood results in a modest amount of aggregation. In view of th e need for ecto-ATPase inhibitors to characterize and classify the P-2 purinoceptors, the combination of the [gamma(32)P] assay and the HPLC system appears to be a powerful tool for screening ligands for this i nhibitory effect. Moreover, the blood cells provide an easily obtainab le human source for ecto-ATPase.