García Paul S, Ciavatta Vincent T, Fidler Jonathan A, Woodbury Anna, Levy Jerrold H, Tyor William R
Research Division, Atlanta VA Medical Center, Decatur, GA, 30033, USA.
Neuroanesthesia Laboratory, Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, 30332, USA.
Neurochem Res. 2015 Nov;40(11):2220-9. doi: 10.1007/s11064-015-1711-1. Epub 2015 Sep 5.
Thrombin's role in the nervous system is not well understood. Under conditions of blood-brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. We investigated the effects of physiologically relevant concentrations of thrombin on cortical neurons using two culture-based assays. We examined thrombin's effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin's effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor-1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin's receptor in the setting of direct thrombin inhibitors could be a potential neuroprotective strategy.
凝血酶在神经系统中的作用尚未得到充分理解。在血脑屏障受损的情况下（如神经外科手术或中风），凝血酶可导致神经细胞凋亡并形成胶质瘢痕。尽管如此，已发现在脑缺血和脑出血模型中，用凝血酶进行预处理具有神经保护作用。我们使用两种基于细胞培养的检测方法，研究了生理相关浓度的凝血酶对皮质神经元的影响。我们通过对荧光标记神经元的定量分析，研究了凝血酶对神经突的影响。为了表征凝血酶对神经元存活的影响，我们用分光光度法测量了酶活性的变化。我们分别使用受体激动剂和凝血酶抑制剂，研究了凝血酶及其受体在神经保护中的作用。我们发现，低浓度的凝血酶（1 nM）可促进神经突生长和分支、提高神经元活力，并防止兴奋性毒性损伤。相比之下，较高浓度的凝血酶（100 nM）对神经元健康可能有害，这可通过神经突生长受抑制得到证明。较低浓度的凝血酶产生的神经保护作用与抗纤维蛋白溶解剂抑肽酶和直接凝血酶抑制剂阿加曲班相当。有趣的是，外源性应用物种特异性凝血酶抑制剂抗凝血酶III对神经元健康有害；这表明在我们的培养系统中，一些内源性凝血酶对于神经元的最佳健康状态是必要的。通过微摩尔浓度的凝血酶受体激动剂肽TRAP激活凝血酶受体蛋白酶激活受体-1（PAR-1），对神经元活力没有不利影响。存在一个最佳的凝血酶浓度可促进神经元健康。凝血酶的神经毒性作用不涉及PAR受体的激活，因此在直接凝血酶抑制剂的情况下，对凝血酶受体进行单独的药物调控可能是一种潜在的神经保护策略。
