Characterization of ECTO-ATP ase on human blood cells : A physiological role in platelet aggregation?

Abstract

Ecto-ATPase (EC 3.6.1.15) is a plasma membrane-bound enzyme which degrades extracellular triphosphate nucleotides. Although its physiological function is still unclear, the enzyme obscures the study of P 2 purinoceptors (i.e. receptors for ATP and other di- and triphosphate nucleotides), since it is capable of metabolizing the pharmacological ligands, such as ATP, for these receptors. We characterized the ecto-ATPase activity on human blood cells with a [ γ 32P]ATP assay and HPLC measurements. We also determined whether ecto-ATPase activity could affect 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 μM and the maximum degradation rate, at 37°, was 2.7 ± 1.1 nmol ATP/(min × mL whole blood). In whole blood the major part of ATP was broken down by the blood cells, predominantly by the leukocytes. ATP and UTP were broken down equally well, mainly yeilding the corresponding di- and monophosphates. In search of inhibitors for the ecto-ATPase, we studied 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 the role of the plasma membrane-bound enzyme, platelet aggregation was determined in whole blood instead of platelet-rich plasma. In the presence of ATP alone, an antagonist of ADP-induced platelet aggregation, some aggregation was still observed. As breakdown of ATP by the ecto-ATPase leads to gradual formation of ADP, as mentioned above, we compared the effects of a stepwise versus bolus addition of ADP. Subsequent dosing of ADP (1.5, 2.5, 5 and 10 μM) resulted in platelet aggregation 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 μM). In conclusion, human blood cells possess a high affinity 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 activity on platelet aggregation is probably small, as degradation of ATP to ADP proceeds slowly and cumulative addition of ADP to platelets in whole blood results in a modest amount of aggregation. In view of the need for ecto-ATPase inhibitors to characterize and classify the P 2 purinoceptors, the combination of the [ γ 32P]ATP assay and the HPLC system appears to be a powerful tool for screening ligands for this inhibitory effect. Moreover, the blood cells provide an easily obtainable human source for ecto-ATPase.