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.

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.

A novel endovascular device for emboli rerouting: part I: evaluation in a Swine model.

BACKGROUND AND PURPOSE: The feasibility and safety of a novel endovascularly delivered tubular mesh designed to reroute emboli away from a critical artery as a means of ischemic stroke prevention was tested in vitro and in vivo. METHODS: Emboli rerouting efficacy was assessed in vitro. Perfusion through the external femoral artery that was jailed by the device, cellular proliferation rate over the jailing mesh, and the resulting tissue coverage of the orifice were assessed in the swine iliofemoral bifurcation. Device-induced embolization was assessed in a swine kidney model. RESULTS: In vitro experiments demonstrated that particles as small as 60% of the pore dimension can be rerouted by the device, although at low efficacy, and rerouting efficacy approached 100% as the particle size approached the pore dimension. Repeat assessment of flow preimplantation and at various follow-up times by Doppler ultrasound showed no significant changes in the perfusion ratio of the jailed branch to the parent artery or the jailed branch to the naive contralateral artery either as a result of device implantation or at the follow-up times. Tissue coverage over the jailed ostium was limited to approximately 12% after stabilization. Cellular proliferation rate gradually decreased to diminishing level approximately 22 weeks postimplantation. The devices implanted across the renal arteries did not demonstrate any device-induced embolization after 1 month. CONCLUSIONS: It is proposed that this device could be used to reroute emboli away from important intracranial vessels as a means of stroke prevention.