Turnell A S, Brant D P, Brown G R, Finney M, Gallimore P H, Kirk C J, Pagliuca T R, Campbell C J, Michell R H, Grand R J
Centre for Clinical Research in Immunology and Signalling, Medical School, University of Birmingham, U.K.
Biochem J. 1995 Jun 15;308 ( Pt 3)(Pt 3):965-73. doi: 10.1042/bj3080965.
The mechanism by which thrombin and prothrombin control neurite retraction was studied in Ad12E1HER10 human neuroepithelial cells. Morphological changes in differentiated cells were apparent within minutes of the addition of very low concentrations of thrombin (3 pM). Higher concentrations (2 nM) of prothrombin were required to elicit a similar response. Doses of thrombin and prothrombin sufficient to cause neurite retraction stimulated protein tyrosine kinase activity. Protein tyrosine kinase activation also correlated positively with thrombin- and prothrombin-induced phosphoinositide 3-kinase activation and InsP6 dephosphorylation. However, thrombin-stimulated Ins(1,4,5)P3 generation and intracellular Ca2+ mobilization only occurred at concentrations in excess of those needed to induce retraction. No fluctuations in Ins(1,4,5)P3 were detected after stimulation with prothrombin, and no rapid synchronized release of Ca2+ was observed, even at very high concentrations. Prothrombin did, however, cause small oscillations in the intracellular Ca2+ concentration, similar to those produced by low concentrations of thrombin, after approximately 30 min. We conclude that prothrombin- and thrombin-induced neurite retractions are not dependent on PtdIns(4,5)P2 and Ca2+ mobilization, but are more probably mediated through an effector mechanism involving protein tyrosine kinase activation. No intracellular Ca2+ mobilization, protein tyrosine kinase activity or neurite retraction was observed after treatment of cells with proteolytically inactive mutant thrombin (S205-->A). Prothrombin-mediated intracellular Ca2+ mobilization and neurite retraction were inhibited by hirudin, which was shown to interact with thrombin but not prothrombin. It is concluded that cleavage of prothrombin to thrombin is a necessary prerequisite for biological activity on differentiated Ad12E1HER10 cells and that differences in agonist concentration are capable of coupling the thrombin receptor to different pathways within the cell.
在Ad12E1HER10人神经上皮细胞中研究了凝血酶和凝血酶原控制神经突回缩的机制。在添加极低浓度的凝血酶（3 pM）后几分钟内，分化细胞的形态变化就很明显。需要更高浓度（2 nM）的凝血酶原才能引发类似反应。足以引起神经突回缩的凝血酶和凝血酶原剂量刺激了蛋白酪氨酸激酶活性。蛋白酪氨酸激酶激活也与凝血酶和凝血酶原诱导的磷脂酰肌醇3-激酶激活以及InsP6去磷酸化呈正相关。然而，凝血酶刺激的Ins(1,4,5)P3生成和细胞内Ca2+动员仅在超过诱导回缩所需浓度时才会发生。用凝血酶原刺激后未检测到Ins(1,4,5)P3的波动，即使在非常高的浓度下也未观察到Ca2+的快速同步释放。然而，凝血酶原在大约30分钟后确实引起细胞内Ca2+浓度的小振荡，类似于低浓度凝血酶产生的振荡。我们得出结论，凝血酶原和凝血酶诱导的神经突回缩不依赖于磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P2）和Ca2+动员，而更可能是通过涉及蛋白酪氨酸激酶激活的效应机制介导的。用蛋白水解无活性的突变凝血酶（S205→A）处理细胞后，未观察到细胞内Ca2+动员、蛋白酪氨酸激酶活性或神经突回缩。水蛭素抑制了凝血酶原介导的细胞内Ca2+动员和神经突回缩，已证明水蛭素与凝血酶相互作用但不与凝血酶原相互作用。得出的结论是，凝血酶原裂解为凝血酶是对分化的Ad12E1HER10细胞具有生物活性的必要前提，并且激动剂浓度的差异能够将凝血酶受体与细胞内的不同途径偶联。
