A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF
Background: Neural inflammation is linked to coagulation. Low levels of thrombin have a neuroprotective effect, mediated by activated protein C (APC). We describe a sensitive novel method for the measurement of APC activity at the low concentrations found in neural tissue. Methods: APC activity was...
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doaj-e9b39a38d4ae43a4ad3fbfd11c1a74302020-11-25T02:58:39ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-03-01212422242210.3390/ijms21072422A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSFValery Golderman0Shany G. Gofrit1Nicola Maggio2Orna Gera3Alexandra Gerasimov4Dar Laks5Joab Chapman6Efrat Shavit-Stein7Department of Neurology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, IsraelDepartment of Neurology, The Chaim Sheba Medical Center, Ramat Gan 5266202, IsraelDepartment of Neurology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, IsraelDepartment of Neurology, The Chaim Sheba Medical Center, Ramat Gan 5266202, IsraelDepartment of Neurology, The Chaim Sheba Medical Center, Ramat Gan 5266202, IsraelDepartment of Neurology, The Chaim Sheba Medical Center, Ramat Gan 5266202, IsraelDepartment of Neurology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, IsraelDepartment of Neurology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, IsraelBackground: Neural inflammation is linked to coagulation. Low levels of thrombin have a neuroprotective effect, mediated by activated protein C (APC). We describe a sensitive novel method for the measurement of APC activity at the low concentrations found in neural tissue. Methods: APC activity was measured using a fluorogenic substrate, Pyr-Pro-Arg-AMC, cleaved preferentially by APC. Selectivity was assessed using specific inhibitors and activators. APC levels were measured in human plasma, in glia cell lines, in mice brain slices following mild traumatic brain injury (mTBI) and systemic lipopolysaccharide (LPS) injection, and in cerebrospinal fluid (CSF) taken from viral meningoencephalitis patients and controls. Results: Selectivity required apixaban and alpha-naphthylsulphonylglycyl-4-amidinophenylalanine piperidine (NAPAP). APC levels were easily measurable in plasma and were significantly increased by Protac and CaCl<sub>2</sub>. APC activity was significantly higher in the microglial compared to astrocytic cell line and specifically lowered by LPS. Brain APC levels were higher in posterior regions and increased by mTBI and LPS. Highly elevated APC activity was measured in viral meningoencephalitis patients CSF. Conclusions: This method is selective and sensitive for the measurement of APC activity that significantly changes during inflammation in cell lines, animal models and human CSF.https://www.mdpi.com/1422-0067/21/7/2422activated protein C (APC)protease-activated receptor 1 (PAR1)endothelial protein C receptor (EPCR)thrombin |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Valery Golderman Shany G. Gofrit Nicola Maggio Orna Gera Alexandra Gerasimov Dar Laks Joab Chapman Efrat Shavit-Stein |
spellingShingle |
Valery Golderman Shany G. Gofrit Nicola Maggio Orna Gera Alexandra Gerasimov Dar Laks Joab Chapman Efrat Shavit-Stein A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF International Journal of Molecular Sciences activated protein C (APC) protease-activated receptor 1 (PAR1) endothelial protein C receptor (EPCR) thrombin |
author_facet |
Valery Golderman Shany G. Gofrit Nicola Maggio Orna Gera Alexandra Gerasimov Dar Laks Joab Chapman Efrat Shavit-Stein |
author_sort |
Valery Golderman |
title |
A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF |
title_short |
A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF |
title_full |
A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF |
title_fullStr |
A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF |
title_full_unstemmed |
A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF |
title_sort |
novel highly sensitive method for measuring inflammatory neural-derived apc activity in glial cell lines, mouse brain and human csf |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1661-6596 1422-0067 |
publishDate |
2020-03-01 |
description |
Background: Neural inflammation is linked to coagulation. Low levels of thrombin have a neuroprotective effect, mediated by activated protein C (APC). We describe a sensitive novel method for the measurement of APC activity at the low concentrations found in neural tissue. Methods: APC activity was measured using a fluorogenic substrate, Pyr-Pro-Arg-AMC, cleaved preferentially by APC. Selectivity was assessed using specific inhibitors and activators. APC levels were measured in human plasma, in glia cell lines, in mice brain slices following mild traumatic brain injury (mTBI) and systemic lipopolysaccharide (LPS) injection, and in cerebrospinal fluid (CSF) taken from viral meningoencephalitis patients and controls. Results: Selectivity required apixaban and alpha-naphthylsulphonylglycyl-4-amidinophenylalanine piperidine (NAPAP). APC levels were easily measurable in plasma and were significantly increased by Protac and CaCl<sub>2</sub>. APC activity was significantly higher in the microglial compared to astrocytic cell line and specifically lowered by LPS. Brain APC levels were higher in posterior regions and increased by mTBI and LPS. Highly elevated APC activity was measured in viral meningoencephalitis patients CSF. Conclusions: This method is selective and sensitive for the measurement of APC activity that significantly changes during inflammation in cell lines, animal models and human CSF. |
topic |
activated protein C (APC) protease-activated receptor 1 (PAR1) endothelial protein C receptor (EPCR) thrombin |
url |
https://www.mdpi.com/1422-0067/21/7/2422 |
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