Shukla S D, Franklin C C, Carter M G
Biochim Biophys Acta. 1987 Jul 6;929(2):134-41. doi: 10.1016/0167-4889(87)90168-6.
Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.
尽管血小板激活因子（或乙酰甘油醚磷酸胆碱；AGEPC）和凝血酶在物理化学性质和作用机制上存在差异，但二者作为血小板刺激剂，均可诱导血小板中的磷酸肌醇转换。因此，我们研究了这些试剂对磷酸肌醇磷酸二酯酶的刺激作用，并探讨它们是否引起相同或不同磷酸肌醇池的水解。在多种实验方案下，用凝血酶和/或AGEPC刺激[3H]肌醇标记的兔血小板，并以磷脂酶C介导产生的放射性肌醇单磷酸（IP）、肌醇二磷酸（IP2）和肌醇三磷酸（IP3）作为参数。AGEPC（1×10⁻⁹ M）在5秒时使[3H]IP3瞬时达到最大值（增加5至6倍），随后下降。凝血酶（2 U/ml）引起[3H]IP3增加的速率比AGEPC慢得多；5秒时增加2倍，30秒时增加5倍，2至5分钟时最大增加6至8倍。与AGEPC相比，凝血酶刺激产生的[3H]IP2和[3H]IP高出数倍。当将凝血酶和AGEPC一起添加到血小板中时，除了在早期时间点增加未达到最大值外，没有证据表明肌醇多磷酸水平有累加性增加。当在凝血酶预处理后的不同时间间隔添加AGEPC时，未观察到[3H]IP3比单独使用凝血酶或AGEPC时的最大值有额外增加。在另一组实验中，通过使用选定浓度的凝血酶（分别为0.1 U和0.3 U）使[3H]IP3出现亚最大增加（约为最大值的1/4和1/2），然后添加AGEPC（1×10⁻⁹ M）5秒。[3H]IP3的增加再次接近单独使用凝血酶或AGEPC时所见的最大水平。得出的结论是，凝血酶和AGEPC在兔血小板中对磷酸肌醇磷酸二酯酶（磷脂酶C）的激活方式不同，并且这两种刺激对磷脂酶C的刺激通过水解共同的磷脂酰肌醇4,5-二磷酸池导致IP3的产生。
