A Linear Temporal Increase in Thrombin Activity and Loss of Its Receptor in Mouse Brain following Ischemic Stroke

Bushi, Doron; Stein, Efrat Shavit; Golderman, Valery; Feingold, Ekaterina; Gera, Orna; Chapman, Joab; Tanné, David

doi:10.3389/fneur.2017.00138

Cited by 18 publications

(15 citation statements)

References 50 publications

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“…We therefore aimed to decrease the activity of nonplasmin serine proteases with α 1 ‐antitrypsin, chymostatin, and bestatin (aminopeptidase inhibitor). Moreover, we added NAPAP to inhibit thrombin, that is significantly elevated following ischemic stroke (Bushi et al., 2017). Ultimately, after controlling the nonspecific proteases, specific plasmin activity was measured as the mean difference between activity measured in samples with and without the highly specific plasmin inhibitor α 2 ‐antiplasmin.…”

Section: Resultsmentioning

confidence: 99%

Increased brain plasmin levels following experimental ischemic stroke in male mice

Mindel

Weiss

Bushi

et al. 2020

J of Neuroscience Research

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Stoke is a severe condition with a narrow treatment window and high mortality rate (Feigin et al., 2016) caused by either blockage (ischemic) or rupture (hemorrhagic) of blood vessels. The damage is often permanent and irreversible and ischemic strokes are the most prevalent type, accounting for 82%-86% of all cases (Johnson et al., 2019). The final step in the coagulation cascade is the cleavage of fibrinogen into fibrin by thrombin (Bardehle et al., 2015). Additionally, thrombin regulates fibrinolysis and enhances the synthesis of a serine protease, tPA (Mandl-Weber et al., 1999). Once a blood clot is formed, tPA binds to fibrin and converts the zymogen plasminogen, already present in the clot, into the broad-spectrum serine protease

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Section: Resultsmentioning

confidence: 99%

Increased brain plasmin levels following experimental ischemic stroke in male mice

Mindel

Weiss

Bushi

et al. 2020

J of Neuroscience Research

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“…APC activity in human plasma after activation is relatively high, and measurable even by the current routine laboratory methods. Based on our previous experience, the coagulation proteases activity in the neural tissues and cells is much lower compared to plasma, and requires a more sensitive method for detection [20][21][22][23]. In order to validate the use of the new assay in neural tissue, it was applied to N9 microglia cell culture.…”

Section: Cell Culturesmentioning

confidence: 99%

A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF

Golderman

Gofrit

Maggio

et al. 2020

IJMS

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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 CaCl2. 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.

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“…In ischemic and hemorrhagic stroke, microglia are recruited to the site of injury where they mediate neuronal death [ 54 ] and contribute to brain recovery [ 44 ]. In these settings, the upregulated brain concentrations of thrombin have been linked to neuronal damage [ 26 , 29 , 30 , 55 , 56 ]. In Alzheimer's disease and in vascular dementia, high levels of thrombin and other coagulation factors have been detected in the brain aside with the recruitment of microglia and additional inflammatory components [ 57 – 59 ].…”

Section: Discussionmentioning

confidence: 99%

Thrombin Inhibition Reduces the Expression of Brain Inflammation Markers upon Systemic LPS Treatment

et al. 2018

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Systemic inflammation and brain pathologies are known to be linked. In the periphery, the inflammation and coagulation systems are simultaneously activated upon diseases and infections. Whether this well-established interrelation also counts for neuroinflammation and coagulation factor expression in the brain is still an open question. Our aim was to study whether the interrelationship between coagulation and inflammation factors may occur in the brain in the setting of systemic inflammation. The results indicate that systemic injections of lipopolysaccharide (LPS) upregulate the expression of both inflammatory and coagulation factors in the brain. The activity of the central coagulation factor thrombin was tested by a fluorescent method and found to be significantly elevated in the hippocampus following systemic LPS injection (0.5 ± 0.15 mU/mg versus 0.2 ± 0.03 mU/mg in the control). A panel of coagulation factors and effectors (such as thrombin, FX, PAR1, EPCR, and PC) was tested in the hippocampus, isolated microglia, and N9 microglia cell by Western blot and real-time PCR and found to be modulated by LPS. One central finding is a significant increase in FX expression level following LPS induction both in vivo in the hippocampus and in vitro in N9 microglia cell line (5.5 ± 0.6- and 2.3 ± 0.1-fold of increase, resp.). Surprisingly, inhibition of thrombin activity (by a specific inhibitor NAPAP) immediately after LPS injection results in a reduction of both the inflammatory (TNFα, CXL9, and CCL1; p < 0.006) and coagulation responses (FX and PAR1; p < 0.004) in the brain. We believe that these results may have a profound clinical impact as they might indicate that reducing coagulation activity in the setting of neurological diseases involving neuroinflammation may improve disease outcome and survival.

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A Linear Temporal Increase in Thrombin Activity and Loss of Its Receptor in Mouse Brain following Ischemic Stroke

Cited by 18 publications

References 50 publications

Increased brain plasmin levels following experimental ischemic stroke in male mice

Increased brain plasmin levels following experimental ischemic stroke in male mice

A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF

Thrombin Inhibition Reduces the Expression of Brain Inflammation Markers upon Systemic LPS Treatment

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