Navran S S, Romstedt K, Chang J, Miller D D, Feller D R
Thromb Res. 1984 Mar 1;33(5):499-510. doi: 10.1016/0049-3848(84)90015-x.
We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes human platelet aggregation and secretion in a concentration dependent manner. The present study was undertaken to further characterize the specificity of the effects of PLC and to better understand the mechanism of the action of this inducer. A methylene-dioxybenzazepine (MDBA) analog of trimetoquinol was synthesized and tested for antiplatelet activity. MDBA (3-30 microM) inhibited PLC-induced aggregation in a concentration dependent manner. Whereas up to 200 microM MDBA did not inhibit aggregation induced by either thrombin, arachidonic acid, or U46619. Effects of PLC (0.05 U/ml) on hydrolysis of phosphatidylinositol, production of phosphatidic acid and thromboxane B2 (TXB2) synthesis were investigated using [32P]-phosphate and [14C]-arachidonic acid labeled platelets. PLC (0.05 U/ml) caused a time dependent decrease in platelet phosphatidylinositol. Up to 50% of labeled phosphatidylinositol was lost from platelets in five minutes. MDBA (3-30 microM) inhibited PLC-induced loss of phosphatidylinositol in a concentration dependent manner. An increase in phosphatidic acid was also observed in PLC-stimulated platelets. Up to 100 microM MDBA did not inhibit production of phosphatidic acid. PLC-treated platelets did not produce any TXB2. In other experiments possible protease contamination of PLC preparations was tested by incubating PLC (0.03-0.5 U/ml) with [14C]-casein. PLC in concentrations up to ten times higher than the concentrations used in aggregation studies did not cause hydrolysis of [14C]-casein, whereas more than 30% of [14C]-casein was hydrolyzed by trypsin. PLC-induced aggregation was not inhibited by up to 300 microM adenosine or ATP. In other experiments, platelet aggregation by ADP was inhibited by adenosine and ATP in a concentration dependent manner. The addition of calcium (0.5- 2.0 mM) increased aggregation by PLC in a concentration dependent manner. These findings suggest that PLC-induced activation of platelets is: (a) dependent on phosphatidylinositol hydrolysis but not on the production of phosphatidic acid, TXB2 or secretion of ADP; (b) not caused by protease contaminants; (c) calcium dependent; and (d) MDBA inhibits PLC-induced aggregation by blocking phosphatidylinositol hydrolysis.
我们之前已经表明，产气荚膜梭菌的磷脂酶C（PLC）以浓度依赖的方式引起人血小板聚集和分泌。本研究旨在进一步表征PLC作用的特异性，并更好地理解这种诱导剂的作用机制。合成了三甲氧苄喹醇的亚甲基二氧苯并氮杂䓬（MDBA）类似物，并测试其抗血小板活性。MDBA（3 - 30 microM）以浓度依赖的方式抑制PLC诱导的聚集。而高达200 microM的MDBA并不抑制凝血酶、花生四烯酸或U46619诱导的聚集。使用[32P] - 磷酸盐和[14C] - 花生四烯酸标记的血小板研究了PLC（0.05 U/ml）对磷脂酰肌醇水解、磷脂酸生成和血栓素B2（TXB2）合成的影响。PLC（0.05 U/ml）导致血小板磷脂酰肌醇随时间减少。五分钟内高达50%的标记磷脂酰肌醇从血小板中丢失。MDBA（3 - 30 microM）以浓度依赖的方式抑制PLC诱导的磷脂酰肌醇丢失。在PLC刺激的血小板中也观察到磷脂酸增加。高达100 microM的MDBA并不抑制磷脂酸的生成。经PLC处理的血小板不产生任何TXB2。在其他实验中，通过将PLC（0.03 - 0.5 U/ml）与[14C] - 酪蛋白孵育来测试PLC制剂中可能存在的蛋白酶污染。浓度比聚集研究中使用的浓度高十倍的PLC不会引起[14C] - 酪蛋白的水解，而超过30%的[14C] - 酪蛋白被胰蛋白酶水解。高达300 microM的腺苷或ATP不抑制PLC诱导的聚集。在其他实验中，腺苷和ATP以浓度依赖的方式抑制ADP诱导的血小板聚集。添加钙（0.5 - 2.0 mM）以浓度依赖的方式增加PLC诱导的聚集。这些发现表明，PLC诱导的血小板活化：（a）依赖于磷脂酰肌醇水解，但不依赖于磷脂酸、TXB2的生成或ADP的分泌；（b）不是由蛋白酶污染物引起的；（c）依赖于钙；并且（d）MDBA通过阻断磷脂酰肌醇水解来抑制PLC诱导的聚集。
