The thrombin receptor (PAR1) is associated with microtubules, mitosis and process formation in glioma cells.

The cell surface protease-activated receptor 1 (PAR1) is overexpressed in glioblastoma multiforme (GBM). We studied the function and structure of intracellular microtubule (MT) and PAR1 in a tubulin-mediated process. We found that exposure to thrombin increased the percentage of proliferative, S, and M phases cells, affected morphology, and increased process elongation. PAR1 antagonist inversely affects these measures, increases tubulin end-binding protein 3 (EB3) mRNA expression in C6 cells, and reduces EB3 comet length, track length, and duration in neuroblastoma cells. In addition, immunofluorescence staining suggests that PAR1 is in close association with the MT α-tubulin and with coagulation cascade proteins during cell division stages. Our findings support PAR1 involvement in MT dynamics.

Amino acid sequence homology of monoclonal serum free light chain dimers and tissue deposited light chains in AL amyloidosis: a pilot study.

OBJECTIVES: Diagnosis of light chain amyloidosis (AL) requires demonstration of amyloid deposits in a tissue biopsy followed by appropriate typing. Previous studies demonstrated increased dimerization of monoclonal serum free light chains (FLCs) as a pathological feature of AL. To further examine the pathogenicity of FLC, we aimed at testing amino acid sequence homology between circulating and deposited light chains (LCs). METHODS: Matched tissue biopsy and serum of 10 AL patients were subjected to tissue proteomic amyloid typing and nephelometric FLC assay, respectively. Serum FLC monomers (M) and dimers (D) were analyzed by Western blotting (WB) and mass spectrometry (MS). RESULTS: WB of serum FLCs showed predominance of either κ or λ type, in agreement with the nephelometric assay data. Abnormal FLC M-D patterns typical of AL amyloidosis were demonstrated in 8 AL-λ patients and in one of two AL-κ patients: increased levels of monoclonal FLC dimers, high D/M ratio values of involved FLCs, and high ratios of involved to uninvolved dimeric FLCs. MS of serum FLC dimers showed predominant constant domain sequences, in concordance with the tissue proteomic amyloid typing. Most importantly, variable domain sequence homology between circulating and deposited LC species was demonstrated, mainly in AL-λ cases. CONCLUSIONS: This is the first study to demonstrate homology between circulating FLCs and tissue-deposited LCs in AL-λ amyloidosis. The applied methodology can facilitate studying the pathogenicity of circulating FLC dimers in AL amyloidosis. The study also highlights the potential of FLC monomer and dimer analysis as a non-invasive screening tool for this disease.

Diagnostic Challenges and Solutions in Systemic Amyloidosis.

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.

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.

Ser77Tyr transthyretin amyloidosis in Israel: Initial manifestations and diagnostic features.

OBJECTIVE: Amyloidosis due to the transthyretin Ser77Tyr mutation (ATTRS77Y) is a rare autosomal-dominant disorder, characterized by carpal-tunnel syndrome, poly- and autonomic-neuropathy, and cardiomyopathy. However, related symptoms and signs are often nonspecific and confirmatory tests are required. We describe the age and frequency of early symptoms and diagnostic features among individuals of Jewish Yemenite descent in Israel. METHODS: Records of mutation carriers were retrospectively reviewed. ATTRS77Y diagnosis was defined by the presence of amyloid in tissue and/or amyloid-related cardiomyopathy. RESULTS: We identified the Ser77Tyr mutation at the heterozygous state in 19 amyloidosis patients (mean age at diagnosis: 62 ± 5.7 years, range 49-70) and 30 amyloid-negative carriers. The probability for disease diagnosis increased from 4.4% at age 49 to 100% at 70 and occurred earlier in males. Initial symptoms preceded diagnosis by 5 ± 3.8 years (range 0-12) and were commonly sensory changes in the extremities. Erectile dysfunction predated these in 8/13 (62%) males. In two patients cardiac preceded neurological symptoms. Two patients declined symptoms. Electrophysiological studies near the time of diagnosis indicated a median neuropathy at the wrist in 18/19 (95%) and polyneuropathy in 13/19 (68%). Skin biopsy revealed epidermal denervation in 15/16 (94%) patients. Cardiomyopathy was identified in 16/19 (84%). Sensory complaints or epidermal denervations were present in 17/30 (57%) of amyloid-negative carriers and co-occurred in 10/30 (33%). INTERPRETATION: ATTRS77Y symptoms commonly occur after age 50, but may begin earlier. Median neuropathy, skin denervation and cardiomyopathy are frequently identified. Symptoms may be absent in patients and common in amyloid-negative carriers.

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.

Modulation of the Thrombin Pathway Restores LTP in a Pilocarpine Mice Model of Status Epilepticus.

Background: Status epilepticus (SE) leads to memory impairment following a seizure, attributed to long-term potentiation (LTP) reduction. Thrombin, a coagulation factor that activates protease-activated receptor 1 (PAR1) is involved in cognitive impairment following traumatic brain injury by reducing hippocampal LTP and in seizures as seen in a SE pilocarpine-induced mice model. Thrombin pathway inhibition prevents this cognitive impairment. We evaluated the effect of thrombin pathway inhibition in the pilocarpine-induced SE mice model, on LTP, hippocampal, and serum markers for inflammation, the PAR1 pathway, and neuronal cell damage. Methods: SE was induced by injecting C57BL/6J mice with pilocarpine. Before pilocarpine injection, mice were injected with either the specific thrombin inhibitor α-NAPAP [Nα-(2-naphthalene-sulfonylglycyl)-4-amidino-DL-phenylalaninepiperidide], the PAR1 antagonist SCH79797, or vehicle-only solution. Recordings of excitatory postsynaptic potentials (EPSP) were conducted from hippocampal slices 24 h following pilocarpine injection. Hippocampal real-time PCR for the quantification of the PAR1, prothrombin, and tumor necrosis factor α (TNF-α) mRNA expression levels was conducted. Serum levels of neurofilament light chain (NfL) and TNF-α were measured by a single molecule array assay. Results: The EPSP was reduced in the pilocarpine-induced SE mice (p < 0.001). This reduction was prevented by both NAPAP and SCH79797 treatments (p < 0.001 for both treatments). Hippocampal expression of TNF-α was elevated in the pilocarpine-induced SE group compared to the control (p < 0.01), however, serum levels of TNF-α were not changed. NfL levels were elevated in the pilocarpine-induced SE group (p = 0.04) but not in the treated groups. Conclusions: Pilocarpine-induced SE reduces LTP, in a thrombin PAR1-related mechanism. Elevation of serum NfL supports neuronal damage accompanying this functional abnormality which may be prevented by PAR1 pathway modulation.

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.

Neuroprotective Effect of Glatiramer Acetate on Neurofilament Light Chain Leakage and Glutamate Excess in an Animal Model of Multiple Sclerosis.

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.

In-thrombus thrombin secretion: a new diagnostic marker of atrial fibrillation in cryptogenic stroke.

BACKGROUND: Endovascularly 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. METHODS: Clots 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. RESULTS: Thrombin activity eluted from clots of AF origin decreased significantly with time in contrast to steady levels eluted from AS origin thrombi (P<0.0001 by repeated measures ANOVA). The Cr stroke group was indistinguishable from the AF group and differed statistically from the AS group (P=0.017 by repeated measures ANOVA). In artificial clots we found a biphasic activity pattern, with initially decreasing levels of eluted thrombin (AF pattern) and then, with continuing washes, steady eluted thrombin levels (AS pattern). CONCLUSIONS: An 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.

Central and Peripheral Mechanisms in ApoE4-Driven Diabetic Pathology.

Apolipoprotein E (APOE) ε4 gene allele and type 2 diabetes mellitus (T2DM) are prime risk factors for Alzheimer's disease (AD). Despite evidence linking T2DM and apoE4, the mechanism underlying their interaction is yet to be determined. In the present study, we employed a model of APOE-targeted replacement mice and high-fat diet (HFD)-induced insulin resistance to investigate diabetic mechanisms associated with apoE4 pathology and the extent to which they are driven by peripheral and central processes. Results obtained revealed an intriguing pattern, in which under basal conditions, apoE4 mice display impaired glucose and insulin tolerance and decreased insulin secretion, as well as cognitive and sensorimotor characteristics relative to apoE3 mice, while the HFD impairs apoE3 mice without significantly affecting apoE4 mice. Measurements of weight and fasting blood glucose levels increased in a time-dependent manner following the HFD, though no effect of genotype was observed. Interestingly, sciatic electrophysiological and skin intra-epidermal nerve fiber density (IENFD) peripheral measurements were not affected by the APOE genotype or HFD, suggesting that the observed sensorimotor and cognitive phenotypes are related to central nervous system processes. Indeed, measurements of hippocampal insulin receptor and glycogen synthase kinase-3ß (GSK-3ß) activation revealed a pattern similar to that obtained in the behavioral measurements while Akt activation presented a dominant effect of diet. HFD manipulation induced genotype-independent hyperlipidation of apoE, and reduced levels of brain apoE in apoE3 mice, rendering them similar to apoE4 mice, whose brain apoE levels were not affected by the diet. No such effect was observed in the peripheral plasma levels of apoE, suggesting that the pathological effects of apoE4 under the control diet and apoE3 under HFD conditions are related to the decreased levels of brain apoE. Taken together, our data suggests that diabetic mechanisms play an important role in mediating the pathological effects of apoE4 and that consequently, diabetic-related therapy may be useful in treating apoE4 pathology in AD.

The neuro-glial coagulonome: the thrombin receptor and coagulation pathways as major players in neurological diseases.

The neuro-glial interface extends far beyond mechanical support alone and includes interactions throu-gh coagulation cascade proteins. Here, we systematically review the evidence indicating that synaptic and node of Ranvier glia cell components modulate synaptic transmission and axonal conduction by a coagulation cascade protein system, leading us to propose the concept of the neuro-glial coagulonome. In the peripheral nervous system, the main thrombin receptor protease activated receptor 1 (PAR1) is located on the Schwann microvilli at the node of Ranvier and at the neuromuscular junction. PAR1 activation effects can be both neuroprotective or harmful, depending on thrombin activity levels. Low physiological levels of thrombin induce neuroprotective effects in the Schwann cells which are mediated by the endothelial protein C receptor. High levels of thrombin induce conduction deficits, as found in experimental autoimmune neuritis, the animal model for Guillaine-Barre syndrome. In the central nervous system, PAR1 is located on the peri-synaptic astrocyte end-feet. Its activation by high thrombin levels is involved in the pathology of primary inflammatory brain diseases such as multiple sclerosis, as well as in other central nervous system insults, including trauma, neoplasms, epilepsy and vascular injury. Following activation of PAR1 by high thrombin levels the seizure threshold is lowered. On the other hand, PAR1 activation by lower levels of thrombin in the central nervous system protects against a future ischemic insult. This review presents the known structure and function of the neuro-glial coagulonome, focusing on coagulation, thrombin and PAR1 in a pathway which may be either physiological (neuroprotective) or detrimental in peripheral nervous system and central nervous system diseases. Understanding the neuro-glial coagulonome may open opportunities for novel pharmacological interventions in neurological diseases.

The Effect of Neuronal Activity on Glial Thrombin Generation.

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 < 0.0001, by ANOVA) compared to normal KCl levels. Since the sciatic nerve includes components of axons and Schwann cell myelin sheath, we continued to investigate the effect of high KCl on a Schwannoma cell line as a model for nodal Schwann cell microvilli. We demonstrated a transient decrease in thrombin activity in response to high extracellular KCl (F(1, 18) = 9.56, p = 0.0063). The major neuronal inhibitor of thrombin is PN-1, and we therefore measured the effect of high KCL on PN-1 immunofluorescence intensity. We found significantly higher PN-1 staining intensity 3 min after the application of high KCL in comparison to cells exposed to high KCL for 7 min and to cells in regular KCL (F(2, 102) = 8.4737, p < 0.0004), and this effect may explain the changes in thrombin activity. The present results support an interaction between neuronal activity and the coagulation pathway as a novel mechanism for neuron-glia crosstalk at the node of Ranvier.

Local Regulation of Thrombin Activity by Factor Xa in Peripheral Nerve Schwann Cells.

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 ±â€¯1.6 mU/ml compared to 35.2 ±â€¯6 mU/ml p = 0.02) and in Schwannoma cell line (4.5 ±â€¯0.2 mU/ml compared to 18.1 ±â€¯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 ±â€¯38 versus 1 ±â€¯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.

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.

Thrombin and protein C pathway in peripheral nerve Schwann cells.

Thrombin and activated protein C (aPC) bound to the endothelial protein C receptor (EPCR) both activate protease-activated receptor 1 (PAR1) generating either harmful or protective signaling respectively. In the present study we examined the localization of PAR-1 and EPCR and thrombin activity in Schwann glial cells of normal and crushed peripheral nerve and in Schwannoma cell lines. In the sciatic crush model nerves were excised 1h, 1, 4, and 7days after the injury. Schwannoma cell lines produced high levels of prothrombin which is converted to active thrombin and expressed both EPCR and PAR-1 which co-localized. In the injured sciatic nerve thrombin levels were elevated as early as 1h after injury, reached their peak 1day after injury which was significantly higher (24.4±4.1mU/ml) compared to contralateral uninjured nerves (2.6±7mU/ml, t-test p<0.001) and declined linearly reaching baseline levels by day 7. EPCR was found to be located at the microvilli of Schwann cells at the node of Ranvier and in cytoplasm surrounding the nucleus. Four days after sciatic injury, EPCR levels increased significantly (57,785±16602AU versus 4790±1294AU in the contralateral uninjured nerves, p<0.001 by t-test) mainly distal to the site of injury, where axon degeneration is followed by proliferation of Schwann cells which are diffusely stained for EPCR. EPCR seems to be located to cytoplasmic component of Schwann cells and not to compact myelin component, and is highly increased following injury.

Thrombin Activity and Thrombin Receptor in Rat Glioblastoma Model: Possible Markers and Targets for Intervention?

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.