Ware J A, Saitoh M, Smith M, Johnson P C, Salzman E W
Department of Medicine, Harvard-Thorndike Laboratory, Boston, Massachusetts.
Am J Physiol. 1989 Jan;256(1 Pt 1):C35-43. doi: 10.1152/ajpcell.1989.256.1.C35.
The protein kinase C activators phorbol ester 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (DAG) cause platelet aggregation, secretion, and a rise in aequorin-indicated cytoplasmic Ca2+ ([Ca2+]i), but the importance of this action to platelet activation by these agonists has not been established. We found that the previous addition of PMA or DAG either enhanced or inhibited the platelet response if thrombin was subsequently added, depending on the latter's concentration. The effects of PMA or DAG on the response to thrombin were obtained only if the agonists were added in concentrations sufficient to elevate [Ca2+]i themselves. A [Ca2+]i rise also occurred after the second agonist (thrombin), but its magnitude did not necessarily correlate with subsequent aggregation, secretion, or the activation of protein kinase C as reported by the phosphorylation of a 47-kDa protein (p47). The protein kinase C inhibitor sphingosine inhibited aggregation and p47 phosphorylation caused by PMA or DAG alone or with thrombin, but the [Ca2+]i rise in response to the first agonist was not affected. PMA-induced aggregation and p47 phosphorylation were inhibited by quin2, which also inhibited protein kinase C activity in a cell-free system. We conclude that a rise in aequorin-indicated [Ca2+]i is necessary for PMA or DAG to activate platelets or to alter the subsequent platelet response to thrombin; this [Ca2+]i rise may be a prerequisite for activation of protein kinase C.
蛋白激酶C激活剂佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）和1 -油酰基- 2 -乙酰甘油（DAG）可引起血小板聚集、分泌以及水母发光蛋白指示的细胞质Ca2+（[Ca2+]i）升高，但这种作用对这些激动剂激活血小板的重要性尚未确定。我们发现，如果随后添加凝血酶，根据其浓度，预先添加PMA或DAG可增强或抑制血小板反应。只有当激动剂以足以自身升高[Ca2+]i的浓度添加时，PMA或DAG才会对凝血酶反应产生影响。在添加第二种激动剂（凝血酶）后也会出现[Ca2+]i升高，但其幅度不一定与随后的聚集、分泌或47 kDa蛋白（p47）磷酸化所报告的蛋白激酶C激活相关。蛋白激酶C抑制剂鞘氨醇可抑制由PMA或DAG单独或与凝血酶共同引起的聚集和p47磷酸化，但对第一种激动剂引起的[Ca2+]i升高没有影响。PMA诱导的聚集和p47磷酸化被喹2抑制，喹2在无细胞系统中也抑制蛋白激酶C活性。我们得出结论，水母发光蛋白指示的[Ca2+]i升高是PMA或DAG激活血小板或改变随后血小板对凝血酶反应所必需的；这种[Ca2+]i升高可能是蛋白激酶C激活的先决条件。
