gms | German Medical Science

Objective: Ca²⁺ is known as cofactor of cell death after intracerebral hemorrhage (ICH). Thrombin has dose depending neuroprotective or neurotoxic effects. The role of ATP in cell death and cell protection remains controversial. The objective of this study was to determine the role of thrombin and ATP in cell death after the incubation of human astrocytes with different blood components in vitro.

Method: Human astrocytes were incubated with human arterial blood, human erythrocyte lysate, human blood plasma, ATP, and thrombin. Native CSF was used as control. Cytosolic Ca²⁺ concentrations were measured by fura-2 fluorescence. Protease-activated receptor (PAR) 1 and PAR-4 were blocked with FR 171113 and tcY-NH2. The PAR-1 agonist TRAP-6 was used for pretreatment of astrocytes. ATP sensitive P2 receptors were blocked with suramin (SUR). Apoptosis and necrosis were quantified by staining the cultured astrocytes with Hoechst33342 and propidium iodide.

Results: Human plasma and thrombin caused a single broad Ca²⁺ spike. An elevated cell death rate was observed 48 hours after incubation (33% vs. 6%; P ≤ 0.001). High dosage of thrombin (> 500 nM) induced early cell death after 5h. Erythrocyte lysate and ATP caused a single small Ca²⁺ spike. ATP induced cell death in human astrocytes in concentrations ≥ 1 mM. Erythrocyte lysate and lower ATP concentrations did not trigger cell death. Human arterial blood induced a double peaked Ca²⁺ spike, and similar cell death rates compared to human plasma were observed. After pretreatment with TRAP-6 lower cell death rates were found (26% vs. 33%; P ≤ 0.001). After blocking of PAR-1 no significant differences in cell death rates were observed. Blockage of PAR-4 induced significant lower cell death rates (24% vs. 33%; P ≤ 0.001). Blockade of ATP sensitive P2 receptors with SUR inhibited the initial calcium peak after incubation with erythrocyte lysate, ATP, or arterial blood, but no effect on cell death rates were observed.

Conclusions: Extracellular ATP concentrations, after incubation with blood components in vitro, were not high enough to induce early cell death. Thrombin and human plasma induced delayed cell death after experimental ICH. PAR-4 antagonists could be a target for the prevention of secondary cell death after spontaneous ICH.