Beukers M W, Pirovano I M, van Weert A, Kerkhof C J, IJzerman A P, Soudijn W
Leiden Amsterdam Center for Drug Research, Division of Medicinal Chemistry, The Netherlands.
Biochem Pharmacol. 1993 Dec 3;46(11):1959-66. doi: 10.1016/0006-2952(93)90637-c.
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 P2 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 [gamma 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 Km for ATP of the ecto-ATPase on human blood cells was 8.5 +/- 2.3 microM 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 broken down by the blood cells, predominantly by the leukocytes. ATP and UTP were broken down equally well, mainly yielding 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 microM) 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 microM). 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.(ABSTRACT TRUNCATED AT 400 WORDS)
胞外ATP酶（EC 3.6.1.15）是一种结合于质膜的酶，可降解细胞外三磷酸核苷酸。尽管其生理功能仍不清楚，但该酶干扰了P2嘌呤受体（即ATP及其他二磷酸和三磷酸核苷酸的受体）的研究，因为它能够代谢这些受体的药理学配体，如ATP。我们用[γ-32P]ATP测定法和高效液相色谱测量法对人血细胞上的胞外ATP酶活性进行了表征。我们还确定了胞外ATP酶活性是否会影响ATP在全血中的抗聚集作用。人血细胞上胞外ATP酶对ATP的米氏常数为8.5±2.3微摩尔，37℃时的最大降解速率为2.7±1.1纳摩尔ATP/（分钟×毫升全血）。在全血中，大部分ATP由血细胞分解，主要是白细胞。ATP和UTP分解效果相同，主要产生相应的二磷酸和单磷酸产物。为寻找胞外ATP酶的抑制剂，我们研究了几种ATP类似物。8-溴-ATP以及2'-和3'-脱氧-ATP是该酶的底物。相反，磷酸侧链的修饰产生了抑制剂。随后，验证了胞外ATP酶在血小板聚集中可能的作用。为评估这种结合于质膜的酶的作用，在全血而非富血小板血浆中测定血小板聚集。仅在ATP（一种ADP诱导的血小板聚集拮抗剂）存在的情况下，仍观察到一些聚集。如上文所述，由于胞外ATP酶对ATP的分解导致ADP逐渐形成，我们比较了逐步添加与一次性添加ADP的效果。随后给予ADP（1.5、2.5、5和10微摩尔）会导致血小板聚集，但程度要小得多，与一次性添加ADP（10微摩尔）相比，最多约为60%。总之，人血细胞具有一种高亲和力的胞外ATP酶，它能降解ATP以及碱基和核糖部分经修饰的ATP类似物。磷酸链经修饰的ATP类似物是胞外ATP酶的抑制剂。胞外ATP酶活性对血小板聚集的直接作用可能较小，因为ATP向ADP的降解进行缓慢，且在全血中向血小板累积添加ADP会导致适度的聚集。（摘要截短至400字）
