Morii N, Teru-uchi T, Tominaga T, Kumagai N, Kozaki S, Ushikubi F, Narumiya S
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
J Biol Chem. 1992 Oct 15;267(29):20921-6.
In the accompanying paper (Nemoto, Y., Namba, T., Teru-uchi, T., Ushikubi, F., Morii, N., and Narumiya, S. (1992) J. Biol. Chem. 267, 20916-20920), we have identified rhoA protein as the sole substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human blood platelets. Here we examined the role of rhoA protein in platelet functions. C3 exoenzyme added to washed platelets dose- and time-dependently ADP-ribosylated rhoA protein in situ in the cells. Concomitant with this modification, inhibition of thrombin-induced platelet aggregation was observed. This inhibition was not reversed by washing the treated platelets, but was not found when C3 exoenzyme was pretreated with mouse monoclonal anti-C3 exoenzyme antibody. C3 exoenzyme treatment did not affect thrombin-induced inositol 1,4,5-trisphosphate production. Secretion of preloaded [14C]serotonin was delayed by the enzyme treatment, but the extent of the secretion was not influenced. In addition, the enzyme treatment did not change the expression of the glycoprotein IIb-IIIa complex on the platelet surface. The enzyme treatment also suppressed platelet aggregation induced by phorbol myristate acetate. These results suggest that rhoA protein plays a role mainly in the aggregation process downstream from receptor-phospholipase C coupling. This, together with the previous finding that rhoA protein modulates stress fiber formation in cultured fibroblasts (Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell Biol. 111, 1001-1007), suggests that rhoA protein regulates the assembly of actin filaments and the avidity of the platelet integrin (glycoprotein IIb-IIIa) in the aggregation process.
在随附论文（根本洋、难波彻、照内彻、串久史、守井直、成宫修司，《生物化学杂志》，第267卷，第20916 - 20920页，1992年）中，我们已确定rhoA蛋白是肉毒杆菌C3 ADP - 核糖基转移酶（C3外切酶）在人血小板中的唯一底物蛋白。在此，我们研究了rhoA蛋白在血小板功能中的作用。添加到洗涤过的血小板中的C3外切酶在细胞内对rhoA蛋白进行剂量和时间依赖性的ADP - 核糖基化。伴随这种修饰，观察到凝血酶诱导的血小板聚集受到抑制。这种抑制作用在洗涤处理过的血小板后不会逆转，但在用小鼠单克隆抗C3外切酶抗体预处理C3外切酶时未观察到。C3外切酶处理不影响凝血酶诱导的肌醇1,4,5 - 三磷酸的产生。酶处理使预加载的[14C]血清素的分泌延迟，但分泌程度不受影响。此外，酶处理不改变血小板表面糖蛋白IIb - IIIa复合物的表达。酶处理还抑制了佛波酯肉豆蔻酸酯诱导的血小板聚集。这些结果表明，rhoA蛋白主要在受体 - 磷脂酶C偶联下游的聚集过程中起作用。这与之前发现rhoA蛋白调节培养成纤维细胞中应力纤维形成的结果（帕特森、塞尔夫、加勒特、贾斯特、阿克托里斯、霍尔，《细胞生物学杂志》，第111卷，第1001 - 1007页，1990年）一起表明，rhoA蛋白在聚集过程中调节肌动蛋白丝的组装和血小板整合素（糖蛋白IIb - IIIa）的亲和力。
