PARIN5, a Novel Thrombin Receptor Antagonist Modulates a Streptozotocin Mice Model for Diabetic Encephalopathy.

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.

Compliance compromises an interventional study on iron supplementation in female combatants.

INTRODUCTION: Low iron levels are related to overuse injuries, poor physical performance and cognitive impairments in female recruits. The aim of this study was to evaluate iron supplement compliance in female combatants during basic training, and its effect on haemoglobin (Hgb), ferritin and injuries. METHODS: 329 female recruits to light infantry units filled induction questionnaires regarding smoking status, previous overuse injuries and iron deficiency. Blood was drawn for Hgb and ferritin. Subjects with ferritin levels below 20 ng/mL were considered iron depleted and were prescribed a ferrous fumarate supplement. After 4 months of basic training, the subjects completed a follow-up questionnaire regarding overuse injuries, reasons for failure to complete basic training and compliance with iron supplementation. Blood tests were repeated. RESULTS: Mean ferritin levels declined during training (from 18.1±18.2 to 15.3±9.6, p=0.01). Compliance with iron supplementation was observed in 26 (26.3%) of the subjects. In compliant subjects, Hgb levels remained constant and ferritin levels increased by 2.9±5.4 (p=0.07). The main reasons for reported non-compliance were forgetfulness, 26 (35.6%), and gastrointestinal side effects, 17 (23.3%). Injuries during training were not found to be associated with iron status. Smokers had a significantly higher rate of reported injuries prior to training (p<0.01). CONCLUSIONS: Ferritin levels decline during training. Compliance with iron supplementation is low. Iron supplementation has a significant effect on ferritin levels, even in the non-compliance group. Injuries were not related to iron status in this group. Further research is needed in order to clarify the most appropriate iron supplementation method.

LPS-Induced Coagulation and Neuronal Damage in a Mice Model Is Attenuated by Enoxaparin.

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.

Characterization of Meningitis and Meningoencephalitis in the Israeli Defense Forces From 2004 to 2015: A Population-Based Study.

Background: Meningitis and meningoencephalitis (MME) are potential medical emergencies. Mandatory reporting of all MME cases in the Israel Defense Force (IDF) allows accurate characterization of MME incidence and course. In the present study, we described the epidemiology of MME in soldiers. Methods: Medical charts of 860,000 combat and non-combat soldiers serving during the years 2004-2015, accounting for 2,256,060 patient years, were retrospectively evaluated. The diagnosis of MME was based on signs of meningeal irritation and a count of > 5 white blood cells (WBC) in the cerebrospinal fluid (CSF). Data on the diagnosis of bacterial or aseptic MME, significant sequelae, and associated mortality were collected. Results: Approximately 273 cases of MME were diagnosed. Overall, MME incidence was 12.1/100,000 patient-years. Bacterial and viral pathogens were identified in 31/273 (11.4%) and 52/273 (19%) cases, respectively. Combat soldiers had higher incidence of bacterial meningitis [14/40 (35%) vs. 31/212 (14.6%); p = 0.002] and meningoencephalitis [13/40 (32.5%) vs. 33/212 (15.6%); p = 0.023] compared to non-combat soldiers. Their clinical presentation was more severe, including confusion [10/40 (25%) vs. 22/212 (10.4%); p = 0.018], focal neurological deficits [12/40 (30%) vs. 11/212 (5.2%); p < 0.0001], and status epilepticus [3/40 (7.5%) vs. 0/212 (0.0%); p < 0.01]. Mortality among combat soldiers was higher [5/40 (15%) vs. 1/212 (0.5%); p < 0.001]. N. meningitidis was the most frequently isolated bacteria, despite universal preventative vaccination. Conclusion: The incidence of bacterial MME in the IDF is higher than in the civilian population. Combat soldiers present with higher incidence of meningoencephalitis and bacterial meningitis.

Factor VII, EPCR, aPC Modulators: novel treatment for neuroinflammation.

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 < 0.0001). In addition, increased phosphorylated extracellular regulated kinase (pERK) (0.99 ± 0.13, 1.39 ± 0.14, control and LPS, p < 0.04) and protein kinase B (pAKT) (1.00 ± 0.09, 2.83 ± 0.81, control and LPS, p < 0.0002) levels indicate PAR1 overactivation, which leads to increased tumor necrosis factor-alpha (TNF-α) level (1.00 ± 0.04, 1.35 ± 0.12, control and LPS, 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.

Teriflunomide normalizes anti-anxiety effect in anti-ANXA2 APS mice model teriflunomide in anti-ANXA2 mice model.

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 (ß2-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 ß2-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 ß2-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 ß2-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.

Neurocoagulation from a Mechanistic Point of View in the Central Nervous System.

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.

Complement and Coagulation System Crosstalk in Synaptic and Neural Conduction in the Central and Peripheral Nervous Systems.

Complement and coagulation are both key systems that defend the body from harm. They share multiple features and are similarly activated. They each play individual roles in the systemic circulation in physiology and pathophysiology, with significant crosstalk between them. Components from both systems are mapped to important structures in the central nervous system (CNS) and peripheral nervous system (PNS). Complement and coagulation participate in critical functions in neuronal development and synaptic plasticity. During pathophysiological states, complement and coagulation factors are upregulated and can modulate synaptic transmission and neuronal conduction. This review summarizes the current evidence regarding the roles of the complement system and the coagulation cascade in the CNS and PNS. Possible crosstalk between the two systems regarding neuroinflammatory-related effects on synaptic transmission and neuronal conduction is explored. Novel treatment based on the modulation of crosstalk between complement and coagulation may perhaps help to alleviate neuroinflammatory effects in diseased states of the CNS and PNS.

Ischemic stroke in PAR1 KO mice: Decreased brain plasmin and thrombin activity along with decreased infarct volume.

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, p<0.05 and 17.2±1.0 vs. 21.2±1.0 mu/ml, p<0.01, respectively) along with a decreased infarct volume (178.9±14.3, 134.4±13.3 mm3, p<0.05). 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.

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

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.

Prolonged Systemic Inflammation Alters Muscarinic Long-Term Potentiation (mLTP) in the Hippocampus.

The cholinergic system plays a fundamental role in learning and memory. Pharmacological activation of the muscarinic receptor M1R potentiates NMDA receptor activity and induces short-term potentiation at the synapses called muscarinic LTP, mLTP. Dysfunction of cholinergic transmission has been detected in the settings of cognitive impairment and dementia. Systemic inflammation as well as neuroinflammation has been shown to profoundly alter synaptic transmission and LTP. Indeed, intervention which is aimed at reducing neuroinflammatory changes in the brain has been associated with an improvement in cognitive functions. While cognitive impairment caused either by cholinergic dysfunction and/or by systemic inflammation suggests a possible connection between the two, so far whether systemic inflammation affects mLTP has not been extensively studied. In the present work, we explored whether an acute versus persistent systemic inflammation induced by LPS injections would differently affect the ability of hippocampal synapses to undergo mLTP. Interestingly, while a short exposure to LPS resulted in a transient deficit in mLTP expression, a longer exposure persistently impaired mLTP. We believe that these findings may be involved in cognitive dysfunctions following sepsis and possibly neuroinflammatory processes.

Treatment of Diabetic Neuropathy with A Novel PAR1-Targeting Molecule.

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 <0.001), and had hypoalgesia (p <0.05), slow conduction velocity (p <0.0001) and reduced skin innervation (p <0.0001). Treatment with PARIN5 significantly improved rotarod performance (p <0.05), normalized hypoalgesia (p <0.05), attenuated slowing of nerve conduction velocity (p <0.05) and improved skin innervation (p <0.0001). CONCLUSION: PARIN5 is a novel pharmacological approach for prevention of DPN development, via PAR1 pathway modulation.

The role of thrombin in the pathogenesis of diabetic neuropathy.

Diabetic neuropathy is common and disabling despite glycemic control. Novel neuroprotective approaches are needed. Thrombin and hypercoagulability are associated with diabetes and nerve conduction dysfunction. Our aim was to study the role of thrombin in diabetic neuropathy. We measured thrombin activity by a biochemical assay in streptozotocin (STZ)-induced diabetic neuropathy in male Sprague-Dawley rats. Neuropathy severity was assessed by thermal latency and nerve conduction measures. Thermal latencies were longer in diabetic rats, and improved with the non-specific serine-protease inhibitor Tosyl-L-lysine-chloromethyl ketone (TLCK) treatment (p<0.01). The tail nerve of diabetic rats showed slow conduction velocity (p˂0.01), and interestingly, increased thrombin activity was noted in the sciatic nerve (p˂0.001). Sciatic nodes of Ranvier and the thrombin receptor, protease activated receptor 1 (PAR1) reactivity showed abnormal morphology in diabetic animals by immunofluorescence staining (p<0.0001). Treatment of diabetic animals with either the specific thrombin inhibitor, N-alpha 2 naphtalenesulfonylglycyl alpha-4 amidino-phenylalaninepiperidide (NAPAP) or TLCK preserved normal conduction velocity, (p˂0.01 and p = 0.01 respectively), and prevented disruption of morphology (p˂0.05 and p˂0.03). The results establish for the first time an association between diabetic neuropathy and excessive activation of the thrombin pathway. Treatment of diabetic animals with thrombin inhibitors ameliorates both biochemical, structural and electrophysiological deficits. The thrombin pathway inhibition may be a novel neuroprotective therapeutic target in the diabetic neuropathy pathology.

Association between inflammatory back pain features, acute and structural sacroiliitis on MRI, and the diagnosis of spondyloarthritis.

OBJECTIVES: To evaluate the association between inflammatory back pain (IBP) features, acute and structural MRI findings suggestive of sacroiliitis, and diagnosis of spondyloarthritis (SpA). METHODS: Data from 224 patients who underwent MRI for suspected sacroiliitis (2005-2015) was retrospectively reviewed by an expert rheumatologist for the presence of IBP features and for clinical standard of reference diagnosis. A telephone questionnaire was performed in cases of missing data. Acute and structural MRI parameters were scored by an experienced radiologist for the presence of sacroiliitis using the Assessment of Spondyloarthritis International Society (ASAS) criteria, Berlin score, and observer's global impression (GI) scores. Association between IBP features and MRI scores, and odds ratio for SpA diagnosis, were calculated. RESULTS: One hundred ninety-three subjects were included (119 F:74 M, mean age 39.7 ± 15.6, mean follow-up 49 ± 18 months). Fifty-two (26.9%) subjects were diagnosed with SpA. IBP scores were significantly higher in SpA patients (p < 0.001). IBP, ASAS, and GI MRI scores were significantly associated with the SpA diagnosis (p < 0.001 for all). The presence of night pain and morning stiffness was significantly associated with sacroiliac-joints' bone marrow edema (BME, p < 0.05). Sensitivity for diagnosis of SpA was high for IBP (96%) and low for the MRI parameters (26.9-57.4%), and specificity was low for IBP (32%) and high for the MRI parameters (88.3-94.3%). CONCLUSIONS: The presence of IBP features is highly associated with diagnosis of SpA and correlates with MRI BME, all probably reflect inflammation. The combination of IBP and MRI should be the cornerstone in the clinician's final diagnosis of SpA.

A Novel Compound Targeting Protease Receptor 1 Activators for the Treatment of Glioblastoma.

Data from human biopsies, in-vitro and in-vivo models, strongly supports the role of thrombin, and its protease-activated receptor (PAR1) in the pathology and progression of glioblastoma (GBM), a high-grade glial tumor. Activation of PAR1 by thrombin stimulates vasogenic edema, tumor adhesion and tumor growth. We here present a novel six amino acid chloromethyl-ketone compound (SIXAC) which specifically inhibits PAR1 proteolytic activation and counteracts the over-activation of PAR1 by tumor generated thrombin. SIXAC effects were demonstrated in-vitro utilizing 3 cell-lines, including the highly malignant CNS-1 cell-line which was also used as a model for GBM in-vivo. The in-vitro effects of SIXAC on proliferation rate, invasion and thrombin activity were measured by XTT, wound healing, colony formation and fluorescent assays, respectively. The effect of SIXAC on GBM in-vivo was assessed by measuring tumor and edema size as quantified by MRI imaging, by survival follow-up and brain histopathology. SIXAC was found in-vitro to inhibit thrombin-activity generated by CNS-1 cells (IC50 = 5 × 10-11M) and significantly decrease proliferation rate (p < 0.03) invasion (p = 0.02) and colony formation (p = 0.03) of these cells. In the CNS-1 GBM rat animal model SIXAC was found to reduce edema volume ratio (8.8 ± 1.9 vs. 4.9 ± 1, p < 0.04) and increase median survival (16 vs. 18.5 days, p < 0.02 by Log rank Mental-Cox test). These results strengthen the important role of thrombin/PAR1 pathway in glioblastoma progression and suggest SIXAC as a novel therapeutic tool for this fatal disease.