Hidaka H, Asano T
J Biol Chem. 1976 Dec 10;251(23):7508-16.
Contact of rat platelets with thrombin or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in the secretion of adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterases. Significant association of calcium with platelets occurred during platelet surface contact with thrombin. Thrombin concentration to induce association of calcium virtually agreed with that to release the enzyme. The finding that A-23187 (5 to 20 muM) also provoked a rapid and marked association of extracellular calcium with platelets suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between platelet and calcium. Two different phosphodiesterases, a relatively specific cyclic AMP and a relatively specific cyclic GMP phosphodiesterase were secreted from platelets into the plasma in soluble form. The amounts of the phosphodiesterases secreted were dose- or time-dependent on thrombin (0.1 to 2 units) or A-23187 (5 to 20 muM) within 30 min. The enzyme release by thrombin was completely inhibited by heparin but the release by A-23187 was not. The two phosphodiesterases secreted seemed to correspond to the two enzymes isolated from platelet homogenates in many respects. Rat platelets contained, at least, three cyclic 3':5'-nucleotide phosphodiesterases, namely, two relatively specific cyclic AMP phoshodiesterases and a relatively specific cyclic GMP phosphodiesterase which were clearly separated from each other by Sepharose 6B or DEAE-cellulose column chromatography or sucrose gradient centrifugation. The two platelet cyclic AMP phosphodiesterase (Mr = 180,000 and 280,000) had similar apparent Km values of 0.69 and 0.75 muM with different sedimentation coefficient values of 4.9 S and 7.1 S, respectively. They did not hydrolyze cyclic GMP significantly. A cyclic GMP phosphodiesterase (Mr - 260,000) exhibited abnormal kinetics for cyclic GMP with an apparent Km value of 1.5 muM and normal kinetics for cyclic AMP with a Km of 300 muM. The properties of a platelet cyclic AMP phosphodiesterase (Mr = 180,000) and a platelet cyclic GMP phosphodiesterase were found to agree with those of the two phosphodiesterases released from platelets by thrombin or A-23187. Depletion of extracellular calcium by an addition of citrate, EDTA, or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) to the blood or platelet suspension resulted in a loss of the activity of the smaller form of platelet cyclic AMP phosphodiesterase (Mr = 180,000) and addition of calcium restored the activity of this cyclic AMP phosphodiesterase. Thus, calcium seemed to be involved in the mechanism of an occurrence of this smaller form of cyclic AMP phosphodiesterase as well as the secretion of this enzyme. Contact of human platelets with thrombin also resulted in the secretion of cyclic nucleotide phosphodiesterase which was dependent on the concentration of calcium. No species difference was observed in this respect.
在细胞外钙存在的情况下，大鼠血小板与凝血酶或二价阳离子载体A - 23187接触，会导致腺苷3':5'-单磷酸（环磷酸腺苷）和鸟苷3':5'-单磷酸（环磷酸鸟苷）磷酸二酯酶的分泌。在血小板与凝血酶表面接触期间，钙与血小板发生了显著结合。诱导钙结合的凝血酶浓度实际上与释放该酶的浓度一致。A - 23187（5至20 μM）也引发细胞外钙与血小板迅速且显著的结合，这一发现表明钙向细胞内环境的动员可能至少部分解释了血小板与钙之间的这种结合。两种不同的磷酸二酯酶，一种相对特异性的环磷酸腺苷磷酸二酯酶和一种相对特异性的环磷酸鸟苷磷酸二酯酶，以可溶形式从血小板分泌到血浆中。在30分钟内，分泌的磷酸二酯酶量在凝血酶（0.1至2单位）或A - 23187（5至20 μM）作用下呈剂量或时间依赖性。凝血酶诱导的酶释放被肝素完全抑制，但A - 23187诱导的释放未被抑制。分泌的两种磷酸二酯酶在许多方面似乎与从血小板匀浆中分离出的两种酶相对应。大鼠血小板至少含有三种环3':5'-核苷酸磷酸二酯酶，即两种相对特异性的环磷酸腺苷磷酸二酯酶和一种相对特异性的环磷酸鸟苷磷酸二酯酶，它们通过琼脂糖6B或二乙氨基乙基纤维素柱色谱法或蔗糖梯度离心法彼此清晰分离。两种血小板环磷酸腺苷磷酸二酯酶（分子量分别为180,000和280,000）具有相似的表观米氏常数，分别为0.69和0.75 μM，沉降系数值不同，分别为4.9 S和7.1 S。它们对环磷酸鸟苷的水解作用不显著。一种环磷酸鸟苷磷酸二酯酶（分子量 = 260,000）对环磷酸鸟苷表现出异常动力学，表观米氏常数为1.5 μM，对环磷酸腺苷表现出正常动力学，米氏常数为300 μM。发现一种血小板环磷酸腺苷磷酸二酯酶（分子量 = 180,000）和一种血小板环磷酸鸟苷磷酸二酯酶的特性与凝血酶或A - 23187从血小板释放的两种磷酸二酯酶的特性一致。向血液或血小板悬液中添加柠檬酸盐、乙二胺四乙酸（EDTA）或乙二醇双（β - 氨基乙醚）- N,N'-四乙酸（EGTA）使细胞外钙耗尽，导致较小形式的血小板环磷酸腺苷磷酸二酯酶（分子量 = 180,000）活性丧失，添加钙可恢复该环磷酸腺苷磷酸二酯酶的活性。因此，钙似乎参与了这种较小形式的环磷酸腺苷磷酸二酯酶的产生机制以及该酶的分泌过程。人血小板与凝血酶接触也会导致环核苷酸磷酸二酯酶的分泌，且该分泌依赖于钙的浓度。在这方面未观察到物种差异。
