The effects of thrombin, a blood coagulation serine protease, were studied in rat hippocampal slices, in an attempt to comprehend its devastating effects when released into the brain after stroke and head trauma. Thrombin acting through its receptor, protease-activated receptor 1 (PAR1), produced a long-lasting enhancement of the reactivity of CA1 neurons to afferent stimulation, an effect that saturated the ability of the tissue to undergo tetanus-induced long-term potentiation. This effect was mediated by activation of a PAR1 receptor, because it was shared by a PAR1 agonist, and was blocked by its selective antagonist. An independent effect of thrombin involved the lowering of the threshold for generating epileptic seizures in CA3 region of the hippocampus. Thus, the experiments in a slice mimicked epileptic and cognitive dysfunction induced by thrombin in the brain, and suggest that these effects are mediated by activation of the PAR1 receptor.

Minimal traumatic brain injury (mTBI) is associated with the existence of retrograde amnesia and microscopic bleeds containing activated coagulation factors. In an mTBI model, we report that thrombin induces amnesia through its receptor protease-activated receptor 1 (PAR-1). Thrombin activity was significantly elevated (32 %, p < 0.05) 5 min following mTBI compared to controls. Amnesia was assessed by the novel object recognition test in mTBI animals and in animals injected intracerebroventricularly (ICV) with either thrombin or a PAR-1 agonist 1 h after the acquisition phase. Saline-injected controls had a preference index of over 0.3 while mTBI animals and those injected with thrombin or the PAR-1 agonist spent equal time with both objects indicating no recall of the object presented to them 24 h previously (p < 0.05). Co-injecting a PAR-1 antagonist (SCH79797) completely blocked the amnestic effects of mTBI, thrombin, and the PAR-1 agonist. Long-term potentiation, measured in hippocampal slices 24 h after mTBI, ICV thrombin or the PAR-1 agonist, was significantly impaired and this effect was completely reversed by the PAR-1 antagonist. The results support a crucial role for PAR-1 in the generation of amnesia following mTBI, revealing a novel therapeutic target for the cognitive effects of brain trauma.

Inflammatory demyelinating diseases of peripheral nerves are associated with altered nerve conduction and with activation of the coagulation pathway. Thrombin mediates many of its effects through protease-activated receptor 1 (PAR-1). We examined the possibility that thrombin may mediate conduction abnormalities through PAR-1 on rat sciatic nerve. PAR-1 was found to be present by both RT-PCR and Western blot analysis of the sciatic nerve. Activation of PAR-1 by a specific peptide agonist caused a 3-fold increase in phosphorylated extracellular signal-regulated kinase (ERK) in the sciatic nerve indicating the existence of functional receptors in the nerve. By confocal immunofluoresence microscopy of the sciatic nerve using anti-PAR-1 antibody and double staining for the paranodal marker contactin-associated protein 1 (Caspr1) or the nodal markers gliomedin and ezrin, the receptor was localized predominantly to myelin microvilli at the node of Ranvier. Thrombin and the PAR-1-specific agonist were applied to exposed rat sciatic nerve and their effects on nerve conduction were measured. Thrombin at concentrations of 100 and 200 U/ml and PAR-1 agonists 150 and 300 muM produced a conduction block within 30 min of application. This effect was maintained for at least 1 h and was reversible by washing. The function of the nodal non-compacted myelin is not well known. The current results implicate this structure and PAR-1 activation in the pathogenesis of conduction block in inflammatory and thrombotic nerve diseases.

J. Neurochem. (2011) 119, 460–473. Abstract We studied the localization, activation and function of protease‐activated receptor 1 (PAR‐1) at the CNS synapse utilizing rat brain synaptosomes and slices. Confocal immunofluoresence and transmission electron microscopy in brain slices with pre‐embedding diaminobenzidine (DAB) immunostaining found PAR‐1 predominantly localized to the peri‐synaptic astrocytic endfeet. Structural confocal immunofluorescence microscopy studies of isolated synaptosomes revealed spherical structures stained with anti‐PAR‐1 antibody which co‐stained mainly for glial‐filament acidic protein compared with the neuronal markers synaptophysin and PSD‐95. Immunoblot studies of synaptosomes demonstrated an appropriate major band corresponding to PAR‐1 and activation of the receptor by a specific agonist peptide (SFLLRN) significantly modulated phosphorylated extracellular signal‐regulated kinase. A significant membrane potential depolarization was produced by thrombin (1 U/mL) and the PAR‐1 agonist (100 μM) and depolarization by high K+ elevated extracellular thrombin‐like activity in the synaptosomes preparation. The results indicate PAR‐1 localized to the peri‐synaptic astrocytic endfeet is most likely activated by synaptic proteases and induces cellular signaling and modulation of synaptic electrophysiology. A protease mediated neuron‐glia pathway may be important in both physiological and pathological regulation of the synapse.