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Antiphospholipid syndrome (APS) affects the brain by both hypercoagulation and immunological mechanisms. APS is characterized by several autoantibodies binding to a thrombolytic complex including beta-2-glycoprotein I (β2-GPI) and annexin A2 (ANXA2). Teriflunomide, an oral drug for the treatment of multiple sclerosis (MS), has a cytostatic effect on B cells and is therefore a potential antibody-targeting treatment for APS. In this study, we assessed the effect of teriflunomide in two APS mouse models by inducing autoantibody formation against β2-GPI and ANXA2 in female BALB/c mice. The ANXA2 model displayed a behavioral change suggesting an anti-anxiety effect in open field and forced swim tests, early in the course of the disease. This effect was normalized following teriflunomide treatment. Conversely, behavioral tests done later during the study demonstrated depression-like behavior in the ANXA2 model. No behavioral changes were seen in the β2-GPI model. Total brain IgG levels were significantly elevated in the ANXA2 model but not in the teriflunomide treated group. No such change was noted in the brains of the β2-GPI model. High levels of serum autoantibodies were induced in both models, and their levels were not lowered by teriflunomide treatment. Teriflunomide ameliorated behavioral changes in mice immunized with ANXA2 without a concomitant change in serum antibody levels. These findings are compatible with the effect of teriflunomide on neuroinflammation. Teriflunomide ameliorated behavioral and brain IgG levels in mice immunized with ANXA2 without a concomitant change in serum antibody levels. These findings are compatible with an effect of teriflunomide on the IgG permeability to the brain and neuroinflammation.

Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research.

Background. Due to the interactions between neuroinflammation and coagulation, the neural effects of lipopolysaccharide (LPS)-induced inflammation (1 mg/kg, intraperitoneal (IP), n = 20) and treatment with the anti-thrombotic enoxaparin (1 mg/kg, IP, 15 min, and 12 h following LPS, n = 20) were studied in C57BL/6J mice. Methods. One week after LPS injection, sensory, motor, and cognitive functions were assessed by a hot plate, rotarod, open field test (OFT), and Y-maze. Thrombin activity was measured with a fluorometric assay; hippocampal mRNA expression of coagulation and inflammation factors were measured by real-time-PCR; and serum neurofilament-light-chain (NfL), and tumor necrosis factor-α (TNF-α) were measured by a single-molecule array (Simoa) assay. Results. Reduced crossing center frequency was observed in both LPS groups in the OFT (p = 0.02), along with a minor motor deficit between controls and LPS indicated by the rotarod (p = 0.057). Increased hippocampal thrombin activity (p = 0.038) and protease-activated receptor 1 (PAR1) mRNA (p = 0.01) were measured in LPS compared to controls, but not in enoxaparin LPS-treated mice (p = 0.4, p = 0.9, respectively). Serum NfL and TNF-α levels were elevated in LPS mice (p < 0.05) and normalized by enoxaparin treatment. Conclusions. These results indicate that inflammation, coagulation, neuronal damage, and behavior are linked and may regulate each other, suggesting another pharmacological mechanism for intervention in neuroinflammation.

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.

Many coagulation factor proteases are increased in the brain during ischemic stroke. One of these proteases is plasmin. In this study we established a novel method for direct quantitative measurement of plasmin activity in male mouse brain slices using a sensitive fluorescent substrate in the presence of specific protease inhibitors. In both the ischemic and contralateral hemispheres, plasmin activity increased 3, 6, and 24 hr following stroke in comparison to healthy mice (F(3, 72) = 39.5, p

Coagulation mechanisms are critical for maintaining homeostasis in the central nervous system (CNS). Thrombin, an important player of the coagulation cascade, activates protease activator receptors (PARs), members of the G-protein coupled receptor family. PAR1 is located on neurons and glia. Following thrombin activation, PAR1 signals through the extracellular signal-regulated kinase pathway, causing alterations in neuronal glutamate release and astrocytic morphological changes. Similarly, the anticoagulation factor activated protein C (aPC) can cleave PAR1, following interaction with the endothelial protein C receptor. Both thrombin and aPC are expressed on endothelial cells and pericytes in the blood-brain barrier (BBB). Thrombin-induced PAR1 activation increases cytosolic Ca2+ concentration in brain vessels, resulting in nitric oxide release and increasing F-actin stress fibers, damaging BBB integrity. aPC also induces PAR1 activation and preserves BBB vascular integrity via coupling to sphingosine 1 phosphate receptors. Thrombin-induced PAR1 overactivation and BBB disruption are evident in CNS pathologies. During epileptic seizures, BBB disruption promotes thrombin penetration. Thrombin induces PAR1 activation and potentiates N-methyl-D-aspartate receptors, inducing glutamate-mediated hyperexcitability. Specific PAR1 inhibition decreases status epilepticus severity in vivo. In stroke, the elevation of brain thrombin levels further compromises BBB integrity, with direct parenchymal damage, while systemic factor Xa inhibition improves neurological outcomes. In multiple sclerosis (MS), brain thrombin inhibitory capacity correlates with clinical presentation. Both thrombin inhibition by hirudin and the use of recombinant aPC improve disease severity in an MS animal model. This review presents the mechanisms underlying the effects of coagulation on the physiology and pathophysiology of the CNS.

Amyloidosis refers to a clinically heterogeneous group of disorders characterized by the extracellular deposition of amyloid proteins in various tissues of the body. To date, 42 different amyloid proteins that originate from normal precursor proteins and are associated with distinct clinical forms of amyloidosis have been described. Identification of the amyloid type is essential in clinical practice, since prognosis and treatment regimens both vary according to the particular amyloid disease. However, typing of amyloid protein is often challenging, especially in the two most common forms of amyloidosis, i.e., the immunoglobulin light chain amyloidosis and transthyretin amyloidosis. Diagnostic methodology is based on tissue examinations as well as on noninvasive techniques including serological and imaging studies. Tissue examinations vary depending on the tissue preparation mode, i.e., whether it is fresh-frozen or fixed, and they can be carried out by ample methodologies including immunohistochemistry, immunofluorescence, immunoelectron microscopy, Western blotting, and proteomic analysis. In this review, we summarize current methodological approaches used for the diagnosis of amyloidosis and discusses their utility, advantages, and limitations. Special attention is paid to the simplicity of the procedures and their availability in clinical diagnostic laboratories. Finally, we describe new methods recently developed by our team to overcome limitations existing in the standard assays used in common practice.

Background Inflammation and coagulation are linked and pathogenic in neuroinflammatory diseases. Protease-activated receptor 1 (PAR1) can be activated both by thrombin, inducing increased inflammation, and activated protein C (aPC), inducing decreased inflammation. Modulation of the aPC-PAR1 pathway may prevent the neuroinflammation associated with PAR1 over-activation. Methods We synthesized a group of novel molecules based on the binding site of FVII/aPC to the endothelial protein C receptor (EPCR). These molecules modulate the FVII/aPC-EPCR pathway and are therefore named FEAMs—Factor VII, EPCR, aPC Modulators. We studied the molecular and behavioral effects of a selected FEAM in neuroinflammation models in-vitro and in-vivo. Results In a lipopolysaccharide (LPS) induced in-vitro model, neuroinflammation leads to increased thrombin activity compared to control (2.7 ± 0.11 and 2.23 ± 0.13 mU/ml, respectively, p = 0.01) and decreased aPC activity (0.57 ± 0.01 and 1.00 ± 0.02, respectively, p p p p = 0.02). In a minimal traumatic brain injury (mTBI) induced neuroinflammation in-vivo model in mice, increased thrombin activity, PAR1 activation, and TNF-α levels were measured. Additionally, significant memory impairment, as indicated by a lower recognition index in the Novel Object Recognition (NOR) test and Y-maze test (NOR: 0.19 ± 0.06, -0.07 ± 0.09, p = 0.03. Y-Maze: 0.50 ± 0.03, 0.23 ± 0.09, p = 0.02 control and mTBI, respectively), as well as hypersensitivity by hot-plate latency (16.6 ± 0.89, 12.8 ± 0.56 s, control and mTBI, p = 0.01), were seen. FEAM prevented most of the molecular and behavioral negative effects of neuroinflammation in-vitro and in-vivo, most likely through EPCR-PAR1 interactions. Conclusion FEAM is a promising tool to study neuroinflammation and a potential treatment for a variety of neuroinflammatory diseases.

Status epilepticus (SE) is a life-threatening condition characterized by ongoing seizure activity which can lead to severe brain damage and death if not treated properly. Recent work suggests that alterations in blood-brain barrier (BBB) function and subsequent cortical exposure to coagulation factors may initiate, promote, and/or sustain SE. This suggestion is based on the observation that the serine protease thrombin, which plays a fundamental role in the blood coagulation cascade, increases neural excitability through the activation of protease-activated receptor 1 (PAR1). However, it remains unclear whether systemic inhibition of thrombin asserts "anti-epileptic" effects in vivo. We here used the pilocarpine model of SE in adult 3-month-old male mice to address the question whether intraperitoneal injection of the thrombin inhibitor α-NAPAP (0.75 mg/kg) counters SE. Indeed, pharmacological inhibition of thrombin ameliorates the behavioral outcome of pilocarpine-induced SE. Similar results are obtained when the thrombin receptor PAR1 is pharmacologically blocked using intraperitoneal injection of SCH79797 (25 μg/kg) prior to SE induction. Consistent with these results, an increase in thrombin immunofluorescence is detected in the hippocampus of pilocarpine-treated animals. Moreover, increased hippocampal serine protease activity is detected 90 min after SE induction, which is not observed in animals treated with α-NAPAP prior to SE induction. Together, these results corroborate and extend recent studies suggesting that novel oral anticoagulants which target thrombin (and PAR1) may assert anti-epileptic effects in vivo. KEY MESSAGES: Systemic thrombin/PAR1-inhibition ameliorates anticoagulants behavioral seizures. Status epilepticus increases thrombin levels in the hippocampus. Increased serine protease activity in the hippocampus after status epileptic. Anti-epileptic potential of clinically used anticoagulants must be evaluated.

Introduction Antiphospholipid syndrome (APS) is an autoimmune disorder manifested by thromboembolic events, recurrent spontaneous abortions and elevated titers of circulating antiphospholipid antibodies. In addition, the presence of antiphospholipid antibodies seems to confer a fivefold higher risk for stroke or transient ischemic attack. Although the major antigen of APS is β2 glycoprotein I, it is now well established that antiphospholipid antibodies are heterogeneous and bind to various targets. Recently, antibodies to Annexin A2 (ANXA2) have been reported in APS. This is of special interest since data indicated ANXA2 as a key player in fibrinolysis. Therefore, in the present study we assessed whether anti-ANXA2 antibodies play a pathological role in thrombosis associated disease. Materials and Methods Mice were induced to produce anti-ANXA2 antibodies by immunization with ANXA2 (iANXA2) and control mice were immunized with adjuvant only. A middle cerebral artery occlusion stroke model was applied to the mice. The outcome of stroke severity was assessed and compared between the two groups. Results Our results indicate that antibodies to ANXA2 lead to a more severe stroke as demonstrated by a significant larger stroke infarct volume (iANXA2 133.9 ± 3.3 mm3 and control 113.7 ± 7.4 mm3; p = 0.017) and a more severe neurological outcome (iANXA2 2.2 ± 0.2, and control 1.5 ± 0.18; p = 0.03). Conclusions This study supports the hypothesis that auto-antibodies to ANXA2 are an independent risk factor for cerebral thrombosis. Consequently, we propose screening for anti-ANXA2 antibodies should be more widely used and patients that exhibit the manifestations of APS should be closely monitored by physicians.

Background Ischemic stroke is a common and debilitating disease with limited treatment options. Protease activated receptor 1 (PAR1) is a fundamental cell signaling mediator in the central nervous system (CNS). It can be activated by many proteases including thrombin and plasmin, with various down-stream effects, following brain ischemia. Methods A permanent middle cerebral artery occlusion (PMCAo) model was used in PAR1 KO and WT C57BL/6J male mice. Mice were evaluated for neurological deficits (neurological severity score, NSS), infarct volume (Tetrazolium Chloride, TTC), and for plasmin and thrombin activity in brain slices. Results Significantly low levels of plasmin and thrombin activities were found in PAR1 KO compared to WT (1.6±0.4 vs. 3.2±0.6 ng/μl, p3, p Conclusions PAR1 KO mice have smaller infarcts, with lower thrombin and plasmin activity levels. These findings may suggest that modulation of PAR1 is a potential target for future pharmacological treatment of ischemic stroke.

Diabetic peripheral neuropathy (DPN) is a disabling common complication of diabetes mellitus (DM). Thrombin, a coagulation factor, is increased in DM and affects nerve function via its G-protein coupled protease activated receptor 1 (PAR1). Methods: A novel PAR1 modulator (PARIN5) was designed based on the thrombin PAR1 recognition site. Coagulation, motor and sensory function and small fiber loss were evaluated by employing the murine streptozotocin diabetes model. Results: PARIN5 showed a safe coagulation profile and showed no significant effect on weight or glucose levels. Diabetic mice spent shorter time on the rotarod (p &lt, 0.001), and had hypoalgesia (p &lt, 0.05), slow conduction velocity (p &lt, 0.0001) and reduced skin innervation (p &lt, 0.0001). Treatment with PARIN5 significantly improved rotarod performance (p &lt, 0.05), normalized hypoalgesia (p &lt, 0.05), attenuated slowing of nerve conduction velocity (p &lt, 0.05) and improved skin innervation (p &lt, 0.0001). Conclusion: PARIN5 is a novel pharmacological approach for prevention of DPN development, via PAR1 pathway modulation.

BackgroundEndovascularly retrieved clots are a potential resource for diagnosing stroke etiology, which may influence secondary prevention treatment. In this study we measured thrombin activity eluted by serially washing clots.MethodsClots were retrieved from 68 patients with acute ischemic stroke, freshly frozen and classified by standard criteria into proven atrial fibrillation (AF, 18 patients), atherosclerotic origin (AS, 15 patients), cryptogenic stroke (Cr, 17 patients) and other known causes (18 patients). Thawed clot samples were washed by transferring them into 1 mL buffer in seven hourly cycles and a fluorescent substrate assay was used to measure secreted thrombin activity. The clots were also examined histologically. Artificial fibrin and red blood cell-rich clots were similarly assayed for wash-eluted thrombin activity as an external control.ResultsThrombin activity eluted from clots of AF origin decreased significantly with time in contrast to steady levels eluted from AS origin thrombi (PConclusionsAn assay measuring the change in thrombin in clots retrieved during acute stroke endovascular thrombectomy procedures may serve as a diagnostic marker of the origin of the clot. The suggested mechanism for these differences may be the clot location before its retrieval, with high blood flow causing thrombin washout in atherosclerotic clots, in contrast to atrium appendage low blood flow retaining high thrombin levels.

Mild traumatic brain injury (mTBI) is common and associated with cognitive impairment. Stress and mTBI are known to modulate the neural function. The present study aims at exploring the effect of prior stress exposure on cognitive function following mTBI.Eight weeks old male ICR mice were subjected to either stress induced by forced swimming stress alone, stress followed by an immediate mTBI, or stress followed by 30 min break and then mTBI. We had two control groups: SHAM group - a control group which was not exposed to stress nor to mTBI and control mTBI group - a control group which was exposed only to TBI with no stress. Mice were weighed prior and at 12, 24 h and 1 week following interventions. Motor evaluation was conducted by rotarod. Behavioral changes were evaluated using open field, Y maze, elevated plus maze and staircase tests, at 12 h and 1 week following interventions. Brain levels of NMDAR subunits (R1, R2A, R2B), GABAStress alone, mTBI alone, and stress followed by immediate mTBI resulted in a significant weight loss compared to control (p 0.05). Stress 30 min prior to mTBI had a protective effect on weight (p = 0.14 compared to control). The stress and mTBI alone groups showed reduced time at the center of the open field arena 1 week after intervention (p 0.05 for both). Time in the novel arm of the Y maze was significantly shorter in the mTBI and stress followed by delayed mTBI (p = 0.02). Immediate stress prior to mTBI had normalized times in the novel arm (p = 0.95 compared to control). Combination of stress and mTBI significantly modified NMDAR subunits levels (increased NMDAR1, p 0.008, decreased NMDAR2A p = 0.02) as well as increased MR levels (p = 0.04).Exposure to stress prior to mTBI may improve the cognitive consequences of mTBI. These data may point towards a novel, unexpected role of stress as a possible resilience mechanism in the setting of mTBI.

Thrombin through its receptor PAR-1 plays an important role in the peripheral nervous system. PAR-1 is located at the microvilli of Schwann cells at the node of Ranvier, and thrombin is generated by the coagulation system on these glial structures. In the present study, we examined the link between neuronal activity and modulation of thrombin generation by glial Schwann cells. Thrombin activity was assessed in sciatic nerves in reaction to high KCl as a model of neuronal activity. We demonstrated a significant transient effect of high KCL on thrombin activity (F(5, 20) = 42.65, p

Glia cells are involved in upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Protease activated receptor 1 (PAR1) pathway is related to brain pathologies. Brain PAR1 is located on peri-synaptic astrocytes, adjacent to pyramidal motor neurons, suggesting possible involvement in ALS. Brain thrombin activity in superoxide dismutase 1 (SOD1) mice was measured using a fluorometric assay, and PAR1 levels by western blot. PAR1 was localized using immunohistochemistry staining. Treatment targeted PAR1 pathway on three levels, thrombin inhibitor TLCK (N-Tosyl-Lys-chloromethylketone), PAR1 antagonist SCH-79797 and the Ras intracellular inhibitor FTS (S-trans-trans-farnesylthiosalicylic acid). Mice were weighed and assessed for motor function and survival. SOD1 brain thrombin activity was increased (p &lt, 0.001) particularly in the posterior frontal lobe (p = 0.027) and hindbrain (p &lt, 0.01). PAR1 levels were decreased (p &lt, 0.001, brain, spinal cord, p &lt, 0.05). PAR1 and glial fibrillary acidic protein (GFAP) staining decreased in the cerebellum and cortex. SOD1 mice lost weight (&ge, 17 weeks, p = 0.047), and showed shorter rotarod time (&ge, 14 weeks, p &lt, 0.01). FTS 40mg/kg significantly improved rotarod scores (p &lt, 0.001). Survival improved with all treatments (p &lt, 0.01 for all treatments). PAR1 antagonism was the most efficient, with a median survival improvement of 10 days (p &lt, 0.0001). Our results support PAR1 pathway involvement in ALS.

Axonal and neuronal pathologies are a central constituent of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), induced by the myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide. In this study, we investigated neurodegenerative manifestations in chronic MOG 35–55 induced EAE and the effect of glatiramer acetate (GA) treatment on these manifestations. We report that the neuronal loss seen in this model is not attributed to apoptotic neuronal cell death. In EAE-affected mice, axonal damage prevails from the early disease phase, as revealed by analysis of neurofilament light (NFL) leakage into the sera along the disease duration, as well as by immunohistological examination. Elevation of interstitial glutamate concentrations measured in the cerebrospinal fluid (CSF) implies that glutamate excess plays a role in the damage processes inflicted by this disease. GA applied as a therapeutic regimen to mice with apparent clinical symptoms significantly reduces the pathological manifestations, namely apoptotic cell death, NFL leakage, histological tissue damage, and glutamate excess, thus corroborating the neuroprotective consequences of this treatment.

Thrombin is a coagulation factor implicated in various pathological and physiological processes in the brain, exerting beneficial and deleterious effects in a concentration-dependent manner. Measurement of thrombin activity levels in pathological animal models is needed and in some cases, because of technical considerations, only frozen samples are available. In the current study, we used a quantitative method to evaluate thrombin activity in fresh and frozen brain sections of 43 male and female adult healthy mice. We stratified data per brain section, brain hemisphere, and mouse sex. We found lower thrombin activity in frozen sections compared with fresh sections, falling within levels considered central nervous system protective in previous studies. The results suggest that fresh section thrombin activity levels in healthy mice can be extrapolated from frozen brain sections. In addition, we found varying thrombin activity across the brain sections, with maximal activity in the olfactory system and hippocampus-containing sections. Thrombin activity did not vary between males and females, or between the right and the left hemispheres, in a statistically significantly manner.

Stress has a profound effect on ability to express neuronal plasticity, learning, and memory. Likewise, epileptic seizures lead to massive changes in brain connectivity, and in ability to undergo long term changes in reactivity to afferent stimulation. In this study, we analyzed possible long lasting interactions between a stressful experience and reactivity to pilocarpine, on the ability to produce long term potentiation (LTP) in a mouse hippocampus. Pilocarpine lowers paired pulse potentiation as well as LTP in CA1 region of the mouse hippocampal slice. When stress experience precedes exposure to pilocarpine, it protects the brain from the lasting effect of pilocarpine. When stress follows pilocarpine, it exacerbates the effect of the drug, to produce a long lasting reduction in LTP. These changes are accompanied by a parallel change in blood corticosterone level. A single exposure to selective mineralo- or gluco-corticosterone (MR and GR, respectively) agonists and antagonists can mimic the stress effects, indicating that GR's underlie the lasting detrimental effects of stress whereas MRs are instrumental in counteracting the effects of stress. These studies open a new avenue of understanding of the interactive effects of stress and epileptic seizures on brain plasticity.

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.

Minimal traumatic brain injury (mTBI) is partially defined by the existence of retrograde amnesia and is associated with microscopic bleeds containing activated coagulation factors. In a previous study, we have found that mTBI immediately releases thrombin-like activity in the brain, which induces amnesia by activating protease-activated receptor 1 (PAR-1) and blocking long-term potentiation (LTP). In the present study, we assessed the effects of mTBI on thrombin and PAR-1 levels in the brain using the same model. After the immediate elevation, thrombin activity returned to baseline 1 h post-trauma and increased again 72 h later (42% relative to control; p 0.005). These changes were associated with a significant increase in PAR-1 levels 24 (17%; p 0.05) and 72 h (20%; p 0.05) post-trauma. Interestingly, the late elevation in thrombin-like activity was also associated with elevation of the major central nervous system thrombin inhibitor, protease nexin-1, 72 h post-mTBI (10%; p 0.005). When thrombin was injected into brain ventricles, an increased sensitivity to seizure-like activity was detected at 72 h post-mTBI. The results are compatible with astrocyte activation post-mTBI resulting in increased thrombin secretion, PAR-1 expression, and seizure sensitivity.

Although thrombin has an important role in both central and peripheral nerve diseases, characterization of the anatomical distribution of its proteolytic activity has been limited by available methods. This study presents the development, challenges, validation and implementation of a novel histochemical method for visualization of thrombin activity in the nervous system. The method is based on the cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MβNA by thrombin to liberate free 4-methoxy-2-naphthylamine (4MβNA). In the presence of 5-nitrosalicylaldehyde, free 4MβNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of thrombin activity. The sensitivity of the method was determined in vitro using known concentrations of thrombin while the specificity was verified using a highly specific thrombin inhibitor. Using this method we determined the spatial distribution of thrombin activity in mouse brain following transient middle cerebral artery occlusion (tMCAo) and in mouse sciatic nerve following crush injury. Fluorescence microscopy revealed well-defined thrombin activity localized to the right ischemic hemisphere in cortical areas and in the striatum compared to negligible thrombin activity contralaterally. The histochemical localization of thrombin activity following tMCAo was in good correlation with the infarct areas per triphenyltetrazolium chloride staining and to thrombin activity measured biochemically in tissue punches (85 ± 35 and 20 ± 3 mU/ml, in the cortical and striatum areas respectively, compared to 7 ± 2 and 13 ± 2 mU/ml, in the corresponding contralateral areas; mean ± SEM; p

Interest in cannabis and its related cannabinoids THC and CBD for use as anti-convulsant therapy has been progressively increasing. While the destigmatization of cannabis and cannabis related research have progressed in the last few decades, there are still many questions that remain unanswered. This review seeks to summarize the progress made in cannabis research in the past four decades and to identify possible directions for future research that are critical for the development of cannabinoid-based therapy in epilepsy.

Organophosphates (OP) are a major threat to the health of soldiers and civilians due to their use as chemical weapons in war and in terror attacks. Among the acute manifestations of OP poisoning, status epilepticus (SE) is bearing the highest potential for long-term damages. Current therapies do not prevent brain damage and seizure-related brain injuries in OP-exposed humans. Thrombin is a serine protease known to have a fundamental function in the clotting cascade. It is highly expressed in the brain where we have previously found that it regulates synaptic transmission and plasticity. In addition, we have found that an excess of thrombin in the brain leads to hyperexcitability and therefore seizures through a glutamate-dependent mechanism. In the current study, we carried out in vitro, ex vivo, and in vivo experiments in order to determine the role of thrombin and its receptor PAR-1 in paraoxon-induced SE. Elevated thrombin activity was found in the brain slices from mice that were treated (in vitro and in vivo) with paraoxon. Increased levels of PAR-1 and pERK proteins and decreased prothrombin mRNA were found in the brains of paraoxon-treated mice. Furthermore, ex vivo and in vivo electrophysiological experiments showed that exposure to paraoxon causes elevated electrical activity in CA1 and CA3 regions of the hippocampus. Moreover, a specific PAR-1 antagonist (SCH79797) reduced this activity. Altogether, these results reveal the importance of thrombin and PAR-1 in paraoxon poisoning. In addition, the results indicate that thrombin and PAR-1 may be a possible target for the treatment of paraoxon-induced status epilepticus.

Thrombin and its protease-activated receptor 1 (PAR1) are potentially important in peripheral nerve inflammatory diseases. We studied the role of thrombin and PAR1 in rat experimental autoimmune neuritis (EAN), a model of the human Guillain-Barré syndrome (GBS). EAN was induced by bovine peripheral myelin with complete Freund's adjuvant (CFA). Thrombin activity in the sciatic nerves, clinical scores and rotarod performance were measured. Thrombin activity in the sciatic nerve was elevated in EAN compared to CFA control rats (sham rats) (p ≤ 0.004). The effect of blocking the thrombin-PAR1 pathway was studied using the non-selective thrombin inhibitor N-Tosyl-Lys-chloromethylketone (TLCK), and the highly specific thrombin inhibitor N-alpha 2 naphtalenesulfonylglycyl 4 amidino-phenylalaninepiperidide (NAPAP). In-vitro TLCK and NAPAP significantly inhibited specific thrombin activity in EAN rats sciatics (p

A transient ischemic episode causes a reduction in evoked EPSPs in hippocampal slices, followed by an NMDA dependent LTP. We explored the relations between ischemic LTP (iLTP) and the more conventional tetanic LTP (tLTP) in CA1 region of slices along the dorsal/ventral axis of the hippocampus. Dorsal hippocampal (DH) slices produced a much larger iLTP than their ventral hippocampal (VH) counterparts. In both regions, iLTP and tLTP shared the same NMDA mediated potentiation, such that one LTP saturated the ability of the other treatment to generate LTP. The smaller LTP in VH was correlated with a lower NMDA-mediated EPSP, and a parallel lower density of NMDA receptors. Calcium permeable AMPA receptors did not contribute to the DH/VH disparity. We conclude that a differential distribution of NMDA receptor subunits along the septotemporal axis of the hippocampus controls the diverse ability to evoke iLTP and tLTP in the two regions and may underlie their characteristic behavioral outputs as well as their differential sensitivity to ischemia.

Epilepsy is a devastating disease, with cognitive and emotional consequences that are not curable. In recent years, it became apparent that cannabinoids help patients to cope with epilepsy. We have studied the effects of cannabidiol (CBD) on the ability to produce long term potentiation (LTP) in stratum radiatum of CA1 region of the mouse hippocampus. Exposure to seizure-producing pilocarpine reduced the ability to generate LTP in the slice. Pre-exposure to CBD prevented this effect of pilocarpine. Furthermore, CBD caused a marked increase in ability to generate LTP, an effect that was blocked by calcium store antagonists as well as by a reduction in serotonin tone. Serotonin, possibly acting at a 5HT1A receptor, or fenfluramine (FFA), which causes release of serotonin from its native terminals, mimicked the effect of CBD. It is proposed that CBD enhances non-NMDA LTP in the slice by facilitating release of serotonin from terminals, consequently ameliorating the detrimental effects of pilocarpine.

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.

High-grade gliomas constitute a group of aggressive CNS cancers that have high morbidity and mortality rates. Despite extensive research, current therapeutic approaches enable survival beyond 2 years in rare cases only. Thrombin and its main CNS target, protease-activated receptor-1, have been implicated in tumor progression and brain edema. Our aim was to study protease-activated receptor-1 (PAR-1) protein expression and thrombin-like activity levels in both in vitro and in vivo models of glioblastoma and correlate them with the volume of the surrounding edema. We measured the presence of PAR-1 protein using fluorescence immunohistochemistry and assessed thrombin activity in various glial and non-glial cell lines and in a CNS-1 glioma rat model using a thrombin-specific fluorescent assay. Thrombin activity was found to be highly elevated in various high-grade glioma cell lines as well as in non-glial malignant cell lines. In the CNS-1 glioma model, the level of PAR-1 fluorescence in the tumor was significantly elevated compared to adjacent regions of reactive gliosis or distant brain areas. The elevated level of thrombin activity observed in the high-grade glioma positively correlated with tumor-induced brain edema. In conclusion, thrombin is secreted from glioma cells and PAR-1 may be a new biological marker for high-grade gliomas.

Factor Xa (FXa) plays a critical role in the coagulation cascade by generation of thrombin. During focal ischemia thrombin levels increase in the brain tissue and cause neural damage. This study examined the hypothesis that administration of the FXa inhibitor, apixaban, following focal ischemic stroke may have therapeutic potential by decreasing brain thrombin activity and infarct volume. Male mice were divided into a treated groups that received different doses of apixaban (2, 20, 100 mg/kg administered I.P.) or saline (controls) immediately after blocking the middle cerebral artery (MCA). Thrombin activity was measured by a fluorescence assay on fresh coronal slices taken from the mice brains 24 hr following the MCA occlusion. Infarct volume was assessed using triphenyltetrazolium chloride staining. A high dose of apixaban (100 mg/kg) significantly decreased thrombin activity levels in the ipsilateral hemisphere compared to the control group (Slice#5, p = .016; Slice#6, p = .016; Slice#7, p = .016; Slice#8, p = .036; by the nonparametric Mann-Whitney test). In addition, treatment with apixaban doses of both 100 mg/kg (32 ± 8% vs. 76 ± 7% in the treatment vs. control groups respectively; p = .005 by the nonparametric Mann-Whitney test) and 20 mg/kg (43 ± 7% vs. 76 ± 7% in the treatment vs. control groups respectively; p = .019 by the nonparametric Mann-Whitney test) decreased infarct volumes in areas surrounding the ischemic core (Slices #3 and #8). No brain hemorrhages were observed either in the treated or control groups. In summary, I.P. administration of high dose of apixaban immediately after MCA occlusion decreases brain thrombin activity and reduces infarct size.

Thrombin through its receptor plays an important role in the peripheral nervous system (PNS) but the pathways leading to its generation there are not known. In the blood, activated factor X (FXa) which is formed from factor X (FX) by tissue factor (TF) and factor VII (FVII), cleaves prothrombin into thrombin. We here studied these factors in vivo in mouse sciatic nerve and in vitro in a Schwannoma cell line and provide mRNA, immunoblot and immunohistochemistry evidence that FX and FXa are expressed in the normal and injured peripheral nerve and in Schwannoma cells. Furthermore, TF and FVII were localized histologically to the node of Ranvier in the sciatic nerve. Adding exogenous FXa increased the thrombin levels in sciatic nerve (11.6 ± 1.6 mU/ml compared to 35.2 ± 6 mU/ml p = 0.02) and in Schwannoma cell line (4.5 ± 0.2 mU/ml compared to 18.1 ± 0.5 mU/ml p 0.001), indicating a large reserve of prothrombin. In the injured nerve, FX mRNA was upregulated 1 day after injury compared to normal nerve (103 ± 38 versus 1 ± 0.3 FOI p 0.001). FXa protein levels increased 1 h after the injury and then decreased significantly at 1 and 2 days following injury despite an increase in its precursor, FX. Injecting the selective FXa inhibitor apixaban immediately upon injury decreased thrombin activation and improved motor function after nerve injury. The results localize the extrinsic coagulation pathway and FXa to the PNS, suggesting a critical role for FXa in PNS thrombin formation and the possible therapeutic use of selective FXa inhibitors in nerve injuries.