Anwer K
Department of Zoological and Biomedical Sciences, Ohio University, Athens 45701.
Thromb Res. 1988 Jan 1;49(1):5-21. doi: 10.1016/0049-3848(88)90355-6.
We have shown earlier that abnormal platelet aggregation in spontaneously hypertensive rats (SHR) is not caused by prostaglandins. In this study platelets from SHR and normotensive (Wistar Kyoto, WKY) rats were used to examine the role of phosphoinositides and phosphorylation of 47,000 and 20,000 Dalton proteins in abnormal platelet activation in hypertension. Thrombin (0.05 U/ml) induced a rapid decrease in (32P)-P04 labelled phosphatidylinositol-4, 5-bisphosphate (PIP2), phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol (PI) in washed rat platelets. However, significantly greater loss of PIP2 and PI was seen in SHR platelets than in WKY platelets. For example the level of PIP2 declined by 32% in SHR platelets and only by 13% in WKY platelets at five seconds of incubation with thrombin. The loss of PI was similar in SHR and WKY platelets for the first five seconds of incubation with thrombin. However, by 15 seconds SHR platelets showed a significantly greater loss (24%) in PI than in WKY platelets (8%). Thrombin induced a 14% and 18% decrease in PIP at three seconds in WKY and SHR platelets respectively. In SHR platelets PIP level returned to the baseline in five seconds and then rose to 20% above the baseline by 30 seconds. In contrast PIP level in WKY platelets slowly reached the basal value by 30 seconds. Thrombin also produced a two- to three-fold greater accumulation of (32P)-phosphatidic acid (PA) in SHR platelets than in WKY platelets. Thrombin (0.05 U/ml) induced rapid phosphorylation of 47,000 Dalton (P47) and 20,000 Dalton (P20) proteins in both WKY and SHR platelets. Thrombin induced a four-fold greater increase in phosphorylation of P47 in SHR platelets than in WKY platelets in the first five seconds. Thrombin produced significantly greater increase in phosphorylation of P20 in SHR platelets (34% and 41%) than in WKY platelets (18% and 28%) at 5 and 15 seconds. Phosphorylation of P20 was followed by dephosphorylation in both WKY and SHR platelets. Aspirin (500 microM) did not affect phosphorylation of either P47 or P20 in SHR or WKY platelets. In other experiments prostaglandin E1 (0.5 microM), which stimulates adenylate cyclase via a guanine nucleotide regulatory protein termed Gs, caused an eighteen-fold increase in cyclic AMP level in SHR platelets as compared to a six-fold increase in WKY platelets. These data lead us to suggest that increased turnover of phosphoinositides and increased phosphorylation of P47 and P20 are involved in abnormal platelet activation in SHR platelets.
我们之前已经表明，自发性高血压大鼠（SHR）中异常的血小板聚集并非由前列腺素引起。在本研究中，使用SHR和正常血压（Wistar Kyoto，WKY）大鼠的血小板来检测磷酸肌醇以及47,000道尔顿和20,000道尔顿蛋白质的磷酸化在高血压中异常血小板激活中的作用。凝血酶（0.05 U/ml）可使洗涤后的大鼠血小板中（32P）-磷酸标记的磷脂酰肌醇-4,5-二磷酸（PIP2）、磷脂酰肌醇-4-磷酸（PIP）和磷脂酰肌醇（PI）迅速减少。然而，与WKY血小板相比，SHR血小板中PIP2和PI的损失明显更大。例如，在与凝血酶孵育5秒时，SHR血小板中PIP2水平下降了32%，而WKY血小板中仅下降了13%。在与凝血酶孵育的前5秒，SHR和WKY血小板中PI的损失相似。然而，到15秒时，SHR血小板中PI的损失（24%）比WKY血小板（8%）明显更大。凝血酶在3秒时分别使WKY和SHR血小板中的PIP减少了14%和18%。在SHR血小板中，PIP水平在5秒时恢复到基线，然后在30秒时升至基线以上20%。相比之下，WKY血小板中的PIP水平在30秒时缓慢达到基础值。凝血酶还使SHR血小板中（32P）-磷脂酸（PA）的积累比WKY血小板多两到三倍。凝血酶（0.05 U/ml）可使WKY和SHR血小板中47,000道尔顿（P47）和20,000道尔顿（P20）蛋白质迅速磷酸化。在最初的5秒内，凝血酶使SHR血小板中P47的磷酸化增加幅度比WKY血小板大4倍。在5秒和15秒时，凝血酶使SHR血小板中P20的磷酸化增加幅度（34%和41%）比WKY血小板（18%和28%）明显更大。在WKY和SHR血小板中，P20的磷酸化之后均发生去磷酸化。阿司匹林（500 microM）不影响SHR或WKY血小板中P47或P20的磷酸化。在其他实验中，前列腺素E1（0.5 microM）通过一种称为Gs的鸟嘌呤核苷酸调节蛋白刺激腺苷酸环化酶，与WKY血小板中6倍的增加相比，使SHR血小板中环磷酸腺苷水平增加了18倍。这些数据使我们认为，磷酸肌醇周转率的增加以及P47和P20磷酸化的增加与SHR血小板中异常的血小板激活有关。
