Nakamura K, Kambayashi J, Suga K, Hakata H, Mori T
Thromb Res. 1985 Jun 1;38(5):513-25. doi: 10.1016/0049-3848(85)90184-7.
Phospholipase C was purified 110 fold from human platelets. The activity of the enzyme was totally dependent upon Ca2+. The activity of the enzyme was markedly enhanced in the presence of arachidonic acid and was strongly inhibited by aminoglycoside antibiotics. The enzyme hydrolyzed endogenous polyphosphoinositides in addition to PI in Ca2+ dependent manner, suggesting the involvement of this enzyme in stimulus-linked rapid hydrolysis of polyphosphoinositides in platelets. The stimulation by thrombin of 32P-labelled human platelets induced about 30% decrease in 32P-TPI and about 220% increase in 32P-PA at the first 10 sec. The degree of hydrolysis of TPI was dependent upon the amount of agonist and it was not affected by the extracellular concentration of Ca2+. The changes in 32P-phospholipids in thrombin-stimulated platelets in the absence of Ca2+ were inhibited in a dose dependent manner by preincubation with relatively higher amount of quin 2 AM. The inhibition was completely overcome by an addition of CaCl2 to the suspending buffer. By such treatment in the absence of extracellular Ca2+, the intracellular Ca2+ concentration was significantly lowered below the basal level (less than 100 nM). Those observations suggest that TPI breakdown in thrombin-stimulated platelets is primary mediated by the agonist receptor coupling and requires at least the basal level of intracellular Ca2+.
磷脂酶C从人血小板中纯化了110倍。该酶的活性完全依赖于Ca2+。在花生四烯酸存在下，该酶的活性显著增强，并且受到氨基糖苷类抗生素的强烈抑制。该酶以Ca2+依赖的方式除了水解磷脂酰肌醇（PI）外，还水解内源性多磷酸肌醇，这表明该酶参与血小板中刺激相关的多磷酸肌醇快速水解。凝血酶刺激32P标记的人血小板在最初10秒内导致32P-TPI降低约30%，32P-PA增加约220%。TPI的水解程度取决于激动剂的量，并且不受细胞外Ca2+浓度的影响。在无Ca2+的情况下，凝血酶刺激的血小板中32P-磷脂的变化在与相对大量的喹吖因-AM预孵育时以剂量依赖的方式受到抑制。通过向悬浮缓冲液中添加CaCl2，这种抑制被完全克服。通过在无细胞外Ca2+的情况下进行这种处理，细胞内Ca2+浓度显著降低至基础水平以下（低于100 nM）。这些观察结果表明凝血酶刺激的血小板中TPI的分解主要由激动剂受体偶联介导，并且至少需要细胞内Ca2+的基础水平。
