ETHANOL‐INSOLUBLE ADENINE NUCLEOTIDES IN PLATELETS AND THEIR POSSIBLE ROLE IN PLATELET FUNCTION
ETHANOL‐INSOLUBLE ADENINE NUCLEOTIDES IN PLATELETS AND THEIR POSSIBLE ROLE IN PLATELET FUNCTION
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血小板中的乙醇不溶性腺嘌呤核苷酸及其在血小板功能中的可能作用
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发表时间：
1972
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通讯作者：
H. Holmsen
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作者：
H. Holmsen
腺嘌呤核苷酸在细胞内和细胞外的血小板功能中起重要作用（最近的综述见参考文献1）。关于血小板释放反应的研究，血小板释放反应是指储存在血小板颗粒中的物质迅速排出到细胞外膜的一种血小板功能。已经揭示了血小板腺嘌呤核苷酸的两个代谢不同的池。因此，在人血小板中，几乎60%的血小板腺嘌呤核苷酸储存在亚细胞颗粒中，并在释放反应期间被挤出。这些核苷酸构成代谢无活性或储存池，因为它们不能在体外被放射性核苷酸前体（32 P-正磷酸盐、3 H或1%-腺苷和腺嘌呤）标记。在释放过程中由血小板保留的核苷酸可以在体外被前体标记，并构成代谢池，其存在于血小板细胞质、线粒体和膜中。在释放反应过程中，代谢ATP通过IMP不可逆地分解为次黄嘌呤，这是一个被认为能使物质从颗粒中挤出的过程，这就是为什么消耗的ATP量被称为“释放能量ATP”。在ATP-次黄嘌呤转化中负责第一次攻击ATP的酶尚不清楚，尽管可收缩的、ATP酶展示蛋白血栓素蛋白已经被发现。的确，格雷特在寻找一种可解释血小板释放反应的爆炸性的收缩蛋白时发现了血栓素蛋白。在我们对血小板腺嘌呤代谢的大多数研究中，6*8-10的核苷酸已经用含有5 mM EDTA的43%乙醇提取。在爱尔兰的实验中，完全支持我们的两池区室化的概念，高氯酸被用于核苷酸提取。我们和爱尔兰的结果之间存在一个显著的差异：乙醇提取的代谢（放射性）ADP比高氯酸少（25-50%），与3 - 4（高氯酸）相比，在代谢池中的ATP/ADP比约为7-10（乙醇）的提取物。米尔斯和米纳斯用两种方法测定了几个物种血小板中的ATP和ADP水平：1）乙醇提取（与我们的方法相似但不相同），然后用荧光素酶系统测定核苷酸，2）高氯酸提取和偶联酶系统测定核苷酸。他们用第一种方法获得的ATP和ADP值显著低于第二种方法，但ATP/ADP比值差异不大。这些发现表明，43%乙醇不能从血小板中完全提取腺嘌呤核苷酸，也许乙醇提取后，一些代谢核苷酸仍与细胞的不溶性部分结合，高氯酸可以将其释放出来。本文证明了血小板乙醇不溶物的HC 104提取物中存在代谢性ATP和ADP，以及这些核苷酸在胶原和凝血酶诱导的释放反应中的行为。
Adenine nucleotides play an important role in platelet function, both intracellularly and extracellularly (for a recent review, see Ref. 1 ) . Studies on the platelet release reaction, a platelet function whereby substances stored in the platelets’ granules are rapidly extruded to the cell’s env i r~nmen t ,~ .~ have revealed two metabolically different pools of platelet adenine n u ~ l e o t i d e s . ~ ~ Thus, in human platelets almost 60% of the total platelet adenine nucleotides is stored in subcellular granules and is extruded during the release reaction. These nucleotides constitute the metabolically inactive or storage pool, since they cannot be labeled by radioactive nucleotide precursors ( 32P-orthophosphate, 3Hor ‘%-adenosine and adenine) in vitro. The nucleotides retained by the platelets during release can be labeled by the precursors in vitro and constitute the metabolic pool, which is present in platelet cytoplasm, mitochondria, and membranes. During the release reaction, metabolic ATP is broken down irreversibly via IMP to hypoxanthine, a process thought to energize the extrusion of materials from the granules, which is why the amounts of ATP consumed have been referred to as “release energy ATP”. The enzyme responsible for the first attack on ATP in the ATP-hypoxanthine conversion is not known, although the contractible, ATPase-displaying protein thrombosthenin has been Indeed, Grette’ discovered thrombosthenin in search of a contractile protein that could explain the explosive nature of the platelet release reaction. In most of our studies on platelet adenine metabolism,6*8-10 the nucleotides have been extracted by 43% ethanol containing 5 mM EDTA. In Ireland’s s t ~ d i e s , ~ which fully support our concept of two-pool compartmentalization, perchloric acid was used for nucleotide extraction. One striking difference exists between our and Ireland’s results: Ethanol extracts less (25-50% ) metabolic (radioactive) ADP than perchloric acid, giving extracts with ATP/ADP ratios in the metabolic pool of about 7-10 (ethanol) as compared with 3 4 (perchloric acid). Mills and Thomas’’ have assayed ATP and ADP levels in platelets from several species with two methods: 1) ethanol extraction (similar to but not identical with our method), followed by nucleotide determination with the luciferinluciferase system, and 2) perchloric acid extraction and nucleotide determination by a coupled enzyme system. They obtained significantly lower values with the first method than with the second for both ATP and ADP, but the ATP/ADP ratios were not much different. These findings indicate that 43% ethanol does not give complete extraction of adenine nucleotides from platelets, and perhaps some metabolic nucleotides remain bound to the insoluble part of the cells after ethanol extraction from which it can be set free by perchloric acid. This paper demonstrates the presence of metabolic ATP and ADP in HC104-extracts of ethanol insoluble matter in platelets, and the behavior of such nucleotides during the release reaction induced by collagen and thrombin.