Coagulation factor Xa activates thrombin in ischemic neural tissue

Abstract

Thrombin is involved in mediating neuronal death in cerebral ischemia. We investigated its so far unknown mode of activation in ischemic neural tissue. We used an in vitro approach to distinguish the role of circulating coagulation factors from endogenous cerebral mechanisms. We modeled ischemic stroke by subjecting rat organotypic hippocampal slice cultures to 30‐min oxygen (5%) and glucose (1 mmol/L) deprivation (OGD). Perinuclear activated factor X (FXa) immunoreactivity was observed in CA1 neurons after OGD. Selective FXa inhibition by fondaparinux during and after OGD significantly reduced neuronal death in the CA1 after 48 h. Thrombin enzyme activity was increased in the medium 24 h after OGD and this increase was prevented by fondaparinux suggesting that FXa catalyzes the conversion of prothrombin to thrombin in neural tissue after ischemia in vitro. Treatment with SCH79797, a selective antagonist of the thrombin receptor protease‐activated receptor‐1 (PAR‐1), significantly decreased neuronal cell death indicating that thrombin signals ischemic damage via PAR‐1. The c‐Jun N‐terminal kinase (JNK) pathway plays an important role in excitotoxicity and cerebral ischemia and we observed activation of the JNK substrate, c‐Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR‐1 antagonist SCH79797, decreased the level of phospho‐c‐Jun Ser73. These results indicate that FXa activates thrombin in cerebral ischemia, which leads via PAR‐1 to the activation of the JNK pathway resulting in neuronal death.

Interference with Protease-activated Receptor 1 Alleviates Neuronal Cell Death Induced by Lipopolysaccharide-Stimulated Microglial Cells through the PI3K/Akt Pathway

“…Signaling through PAR-1 appears to mediate the process of brain damage in adult animal models of subarachnoid hemorrhage (SAH)1617. Previous rodent studies have shown that the interference with this pathway in primary astrocyte cultures1819 as well as in hippocampal slice cultures20 provided a potential target for therapeutic approach in maintaining vascular integrity following brain damage in rats2122.…”

Endogenous Protease Nexin-1 Protects against Cerebral Ischemia

“…Using a blood free in vitro approach, we previously showed that thrombin protein is detected on Western blots after ischemia modelled in vitro by OGD and that specific thrombin and Factor Xa inhibitors confer neuroprotection [17,31]. The results presented here make two further points:…”

A Linear Temporal Increase in Thrombin Activity and Loss of Its Receptor in Mouse Brain following Ischemic Stroke

“…In contrast, in brain slices that are subjected to ischemic conditions such as OGD, no circulating coagulation factors are involved and the time points examined were somewhat shorter. Thrombin that is generated in the brain cells rises (14, 15) and as a result PAR1, its own receptor, may first increase as a complementary step, but these receptors are then cleaved and internalized leading to reduced levels. It is hypothesized that inhibiting the levels of thrombin during acute stroke will normalize PAR1 levels at the 2–24 hour time frame.…”

“…Activation of this receptor by low concentrations of thrombin may have neuroprotective effects, while at higher concentrations thrombin has deleterious effects (7–11). In the setting of an acute ischemic stroke, high thrombin levels have been detected in the infarct area, possibly as a consequence of blood–brain barrier opening (12, 13) or as a result of its local synthesis in the brain (14, 15) or both. Functionally, thrombin has been shown to cause synaptic dysfunction (15–19) and later, on vascular disruption, inflammatory response and neuronal damage (20–22), through the activation of PAR1 (13, 20).…”