中国神经再生研究(英文版) ›› 2021, Vol. 16 ›› Issue (10): 2086-2092.doi: 10.4103/1673-5374.308098

• 观点:退行性病与再生 • 上一篇    下一篇

神经变性和再生标志物:检测人脑脊髓液中凝血酶和活化蛋白C的新型高灵敏度方法

  

  • 出版日期:2021-10-15 发布日期:2021-03-19

Markers for neural degeneration and regeneration: novel highly sensitive methods for the measurement of thrombin and activated protein C in human cerebrospinal fluid

Alexandra Gerasimov1, Valery Golderman2, Shany Guly Gofrit1, Shay Anat Aharoni1, Daniela Noa Zohar1, Ze’ev Itsekson-Hayosh1, Tsviya Fay-Karmon1, 3, Sharon Hassin-Baer1, 2, 3, 4, Joab Chapman1, 2, 4, 5, 6, Nicola Maggio1, 2, 4, 7, Efrat Shavit-Stein1, 2  *#br#   

  1. 1Department of Neurology, The Chaim Sheba Medical Center, Ramat Gan, Israel; 2Department of Neurology and Neurosurgery, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3The Movement Disorders Institute, Chaim Sheba Medical Center, Ramat Gan, Israel; 4Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel; 5Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 6Robert and Martha Harden Chair in Mental and Neurological Diseases, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 7Talpiot Medical Leadership Program, The Chaim Sheba Medical Center, Ramat Gan, Israel
  • Online:2021-10-15 Published:2021-03-19
  • Contact: Efrat Shavit-Stein, PhD, efrat.shavit.stein@gmail.com.

摘要:

凝血酶是一种促凝丝氨酸蛋白酶,与神经退行性疾病有关,而其间接抑制剂活化蛋白CaPC)被认为具有神经保护作用。虽然在血液中可检测到凝血酶和aPC活性的水平,但该指标在脑脊髓液(CSF)中的测定尚需探讨。为建立一种特异性和灵敏的酶促法测定CSF中的凝血酶和aPC活性,(1)以10例疑似常压性脑积水(NPH)患者CSF作为对照组,而7例中枢神经系统(CNS)感染的患者作为急性神经炎研究组和1例创伤性腰椎穿刺后的CSF样本( LP)作为阳性对照组。在存在内肽酶和氨基肽酶抑制剂的情况下,通过特异性蛋白水解切割释放的荧光来检测凝血酶和aPC活性,以确保其特异性。凝血酶和丝氨酸蛋白酶抑制剂NAPAPPMSF验证了该方法的特异性;(2)评估了CSF对凝血酶活性的抑制作用和特定凝血酶抑制剂的水平。与NPH对照相比,CNS感染患者的CSF中的凝血酶和aPC活性明显更高,表明这些因素参与了神经炎症。在存在已知凝血酶浓度的情况下,CNS感染患者的CSF凝血酶活性水平较高,而NPH患者则较低;(3Western印迹检测CSF中内源性凝血酶抑制剂蛋白酶nexin 1PN1)、淀粉样前体蛋白(APP)和抗凝血酶IIIATIII)水平显示,APP在感染性CSF中显着升高;(4)该研究找到了一种新颖且灵敏的方法用于检测脑脊液中凝血酶和aPC的活性,有助于反映凝血与神经系统神经变性与再生的关系。

https://orcid.org/0000-0003-3373-8397 (Efrat Shavit-Stein)

Abstract: Inflammation and coagulation are tightly interconnected in the pathophysiology of neuronal diseases. Thrombin, a pro-coagulant serine protease is associated with neurodegeneration and its indirect inhibitor, activated protein C (aPC), is considered neuroprotective. While levels of thrombin and aPC activity are readily measured in the blood, similar assays in the cerebrospinal fluid (CSF) have not been described. The aim of this study was to establish a specific and sensitive enzymatic assay to measure both thrombin and aPC activity in the CSF. CSF was collected from 14 patients with suspected normal pressure hydrocephalus served as a control group, while seven patients with central nervous system infections served as an acute neuro-inflammatory study group and one sample of CSF following traumatic lumbar puncture served as a positive control. Thrombin and aPC activities were measured by fluorescence released by specific proteolytic cleavage in the presence of endopeptidase and amino-peptidase inhibitors to ensure specificity. Specificity of the method was verified by thrombin and serine-protease inhibitors N-alpha-((2-naphthylsulfinyl)glycyl)-DL-p-amidinophenylalanylpiperidine and phenylmethanesulfonyl fluoride. Inhibition of thrombin activity by CSF samples and levels of specific thrombin inhibitors were also assessed. Thrombin and aPC activities were reliably measured and were significantly higher in the CSF of patients with central nervous system infections compared to normal pressure hydrocephalus controls, suggesting the involvement of these factors in neuro-inflammation. CSF thrombin activity levels in the presence of known thrombin concentration were high in patients with central nervous system infections, and low in normal pressure hydrocephalus patients. Quantification of endogenous thrombin inhibitors protease nexin 1, amyloid precursor protein and anti-thrombin III in CSF by western blot indicated a significant elevation of amyloid precursor protein in infectious CSF. In conclusion, this study describes a novel and sensitive assay aimed at the detection of thrombin and aPC activity in CSF. This method may be useful for measuring these factors that reflect degenerative and protective influences of coagulation on neurological disorders. The study procedure was approved by the Ethics Committee of the Chaim Sheba Medical Center (approval No. 4245-17-SMC) on October 18, 2018.

Key words: activated protein C, cerebrospinal fluid, infection, inflammation, normal pressure hydrocephalus, thrombin

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