SYNTHESIS OF PROSTACYCLIN FROM PLATELET-DERIVED ENDOPEROXIDES BY CULTURED HUMAN-ENDOTHELIAL CELLS
SYNTHESIS OF PROSTACYCLIN FROM PLATELET-DERIVED ENDOPEROXIDES BY CULTURED HUMAN-ENDOTHELIAL CELLS
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DOI：
10.1172/jci109967
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发表时间：
1980-01-01
影响因子：
15.9
通讯作者：
BROEKMAN, MJ
中科院分区：
文献类型：
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作者：
MARCUS, AJ;WEKSLER, BB;BROEKMAN, MJ
阿司匹林处理的内皮细胞从纯化的前列腺素[PG]内过氧化物PGH 2合成前列环素（PGI 2）。为了确定阿司匹林处理的内皮细胞是否产生PGI 2从内过氧化物释放的刺激血小板，[3 H]花生四烯酸预标记的血小板与钙离子载体A 23187 [calcimycin]，凝血酶，或胶原蛋白在阿司匹林处理的内皮细胞悬浮液的存在下，在aggregometer比色皿反应。该方法允许以[3 H]6-酮-PGF 1 α的形式对[3 H] PGI 2进行薄层放射色谱定量。和[3 H]血栓烷[TX] A2作为[3 H] TXB 2，以及分析同一样品中的血小板聚集反应。在阿司匹林处理的内皮细胞的存在下，对所有3种药物的血小板聚集反应被抑制。[3 H]6-酮-PGF 1 α在用所有3种试剂刺激后，从合并的细胞悬浮液的上清液中回收。PGI_2的产生顺序为离子载体>凝血酶>胶原。血小板[~ 3 H] TXB_2的回收量明显减少阿司匹林处理的内皮细胞。在单独的实验中，6-酮-PGF 1 α.用放射免疫法测定TXB_2，结果与放射标记法一致。6-酮-PGF 1 α的量通过放射免疫测定法测量的PGI 2的量足以抑制血小板聚集。当200，000个血小板/μ l与3000-6000个阿司匹林处理的内皮细胞/μ l组合时获得这些结果。在更高的血小板水平下，6-酮-PGF 1 α的比例。TXB 2降低，血小板聚集。对照研究表明，阿司匹林处理的内皮细胞不能合成PGI 2从外源性放射性或内源性花生四烯酸时，凝血酶刺激。内皮细胞悬液只能使用来自刺激血小板的内过氧化物。因此，在实验条件下，可以通过2种独立的方法证明内皮细胞从活化血小板衍生的内过氧化物产生PGI 2。这些实验条件包括：增强血小板-内皮细胞的接近性，如在搅拌的细胞悬浮液中可获得的;使用增加的内皮细胞/血小板比率;以及在放射性标记实验中利用高比活度的花生四烯酸。当用凝血酶刺激未用阿司匹林处理的血小板和内皮细胞的混合物时，超过2倍的6-酮-PGF 1 α。比单独刺激内皮细胞时形成。显然，内皮细胞可以在很大程度上利用血小板内过氧化物形成PGI 2。
Aspirin-treated endothelial cells synthesize prostacyclin (PGI2) from the purified prostaglandin [PG] endoperoxide PGH2. To ascertain whether aspirin-treated endothelial cells produce PGI2 from endoperoxides released by stimulated platelets, [3H]arachidonic acid-prelabeled platelets were reacted in aggregometer cuvettes with the Ca ionophore A 23187 [calcimycin], thrombin, or collagen in the presence of aspirin-treated endothelial cell suspensions. This procedure permitted thin-layer radiochromatographic quantitation of [3H]PGI2 as [3H]6-keto-PGF1.alpha. and [3H]thromboxane [TX] A2 as [3H]TXB2, as well as analysis of platelet aggregation responses in the same sample. In the presence of aspirin-treated endothelial cells, platelet aggregation in response to all 3 agents was inhibited. [3H]6-keto-PGF1.alpha. was recovered from the supernates of the combined cell suspensions after stimulation by all 3 agents. The order of PGI2 production initiated by the stimuli was ionophore > thrombin > collagen. The amounts of platelet [3H]TXB2 recovered were markedly reduced by the addition of aspirin-treated endothelial cells. In separate experiments, 6-keto-PGF1.alpha. and TXB2 were quantitated by radioimmunoassay; the results paralleled those obtained with the use of radiolabeling. The quantity of 6-keto-PGF1.alpha. measured by radioimmunoassay represented amounts of PGI2 sufficient to inhibit platelet aggregation. These results were obtained when 200,000 platelets/.mu.l were combined with 3000-6000 aspirin-treated endothelial cells/.mu.l. At higher platelet levels the proportion of 6-keto-PGF1.alpha. to TXB2 decreased and platelet aggregation occurred. Control studies indicated that aspirin-treated endothelial cells could not synthesize PGI2 from exogenous radioactive or endogenous arachidonate when stimulated with thrombin. The endothelial cell suspensions could only have used endoperoxides from stimulated platelets. Thus, under the experimental conditions, production by endothelial cells of PGI2 from endoperoxides derived from activated platelets could be demonstrated by 2 independent methods. These experimental conditions included: enhanced platelet-endothelial cell proximity, as attainable in stirred cell suspensions; use of increased endothelial cell/platelet ratios; and utilization of arachidonate of high specific activity in radiolabeling experiments. When a mixture of platelets and endothelial cells that were not treated with aspirin was stimulated with thrombin, more than twice as much 6-keto-PGF1.alpha. was formed than when endothelial cells were stimulated alone. Apparently endothelial cells can utilize platelet endoperoxides for PGI2 formation to a significant extent.