5'-Methylthioadenosine and 2',5'-dideoxyadenosine blockade of the inhibitory effects of adenosine on ADP-induced platelet aggregation by different mechanisms.
5'-Methylthioadenosine and 2',5'-dideoxyadenosine blockade of the inhibitory effects of adenosine on ADP-induced platelet aggregation by different mechanisms.
复制标题
5-甲硫腺苷和2,5-二脱氧腺苷通过不同机制阻断腺苷对ADP诱导的血小板聚集的抑制作用。
DOI：
10.1016/0006-2952(80)90363-9
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发表时间：
1980
影响因子：
5.8
通讯作者：
ParksJr,RE
中科院分区：
文献类型：
--
作者：
Agarwal,KC;ParksJr,RE
方法采集0.1体积的新鲜人血。 3.8%柠檬酸钠。通过差速离心获得富血小板血浆（PRP）和贫血小板血浆（PPP）（13）。血小板聚集通过Born 的比浊法测量(23)。按照 Steer 和 Wood (24) 的描述进行洗涤血小板膜的制备和腺苷酸环化酶的测定，不同之处在于测定混合物中排除了咖啡因。 200微升反应混合物含有Tris-HCl缓冲液（pH 8.0），30 mM； gCl2，25 mM；磷酸肌酸，B 5 mM；肌酸磷酸激酶，8-10单位；和 [n-3* P]-ATP，200 PM（II. 5 x 10 cpn/测定）。将反应混合物在振荡水浴中于38℃平衡2-3分钟，并通过添加30μl含有5-10~9蛋白质的血小板膜制剂开始反应。在零时、5分钟和10分钟时，通过添加0.2ml含有ATP(4ml)、CAMP(1mM)和十二烷基硫酸钠(2％)的溶液来终止反应。将 50 微升含有 [3H] cAMP (8,000-10,000 cpn) 的物质添加到每个测定管中以监测（32PlcAMP 回收率），并将混合物放入沸水浴中 3 分钟。向每个管中添加 0.8 ml 水后，使用 Dowex (AG 5OW-X4) 和氧化铝柱纯化 CAMP，如 Salomon 等人 (25) 所述。最终洗脱液 (4)将来自almina colLnnns的[32Pl.ml)与闪烁%nfluid混合并通过Lowry等人的方法(26)测定[32Pl和[3H1]蛋白质浓度。
MethodsFresh human blood was collected in 0.1 vol. of 3.8% sodium citrate. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were obtained by differential centrifgation (13). Platelet aggregation was measured by the turbidometric method of Born (23). The preparation of washed platelet membranes and the assay of adenylate cyclase were performed as described by Steer and Wood (24) except that caffeine was excluded from the assay mixture. Two hundred microliters of reaction mixture contained Tris-HCl buffer (pH 8.0), 30 mM; gCl2, 25 mM; creatine phos hate, B 5 mM; creatine phosphokinase, 8-10 units; and [n-3* P]-ATP, 200 PM (ll. 5 x 10 cpn/assay). The reaction mixture was equilibrated at 38 for 2-3 min in a shaking water bath and the reaction was started by the addition of 30 ul of the platelet membrane preparation containing 5-10~ 9 of protein. At zero time, 5 and 10 min, the reactions were stopped by the addition of 0.2 ml of a solution containing ATP (4 my), CAMP (1 mM) and sodium dodecylsulfate(2%). Fifty microliters containing [3H] cAMP (8,000-10,000 cpn) was added to each assay tube to monitor (32PlcAMP recovery and the mixtures were placed in a boiling water bath for 3 min. After addition of 0.8 ml of water to each tube, CAMP was purified using Dowex (AG 5OW-X4) and alumina columns as described by Salomon et al.(25). The final eluates(4 ml) from the almina colLnnns were mixed with scintilla% nfluid and counted for [32Pl and [3H1. Protein concentrations were determined by the method of Lowry et al.(26).