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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

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.

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.

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.

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.

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

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

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.

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.

Background Brain thrombin activity is increased following acute ischemic stroke and may play a pathogenic role through the protease-activated receptor 1 (PAR1). In order to better assess these factors, we obtained a novel detailed temporal and spatial profile of thrombin activity in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Methods Thrombin activity was measured by fluorescence spectroscopy on coronal slices taken from the ipsilateral and contralateral hemispheres 2, 5, and 24 h following pMCAo (n = 5, 6, 5 mice, respectively). Its spatial distribution was determined by punch samples taken from the ischemic core and penumbra and further confirmed using an enzyme histochemistry technique (n = 4). Levels of PAR1 were determined using western blot. Results Two hours following pMCAo, thrombin activity in the stroke core was already significantly higher than the contralateral area (11 ± 5 vs. 2 ± 1 mU/ml). At 5 and 24 h, thrombin activity continued to rise linearly (r = 0.998, p = 0.001) and to expand in the ischemic hemisphere beyond the ischemic core reaching deleterious levels of 271 ± 117 and 123 ± 14 mU/ml (mean ± SEM) in the basal ganglia and ischemic cortex, respectively. The peak elevation of thrombin activity in the ischemic core that was confirmed by fluorescence histochemistry was in good correlation with the infarcts areas. PAR1 levels in the ischemic core decreased as stroke progressed and thrombin activity increased. Conclusion In conclusion, there is a time- and space-related increase in brain thrombin activity in acute ischemic stroke that is closely related to the progression of brain damage. These results may be useful in the development of therapeutic strategies for ischemic stroke that involve the thrombin-PAR1 pathway in order to prevent secondary thrombin related brain damage.