gms | German Medical Science

Patients with malignant gliomas often suffer from thrombembolic events, suggesting a causal link between these tumors and disturbed hemostasis. Thrombin is well known as a coagulation factor but can also act on various cells via protease-activated receptors (PAR). In this study, we investigated the impact of thrombin on biological aggressiveness of malignant gliomas.

To study actively transcribed PAR genes, we isolated mRNA from U373 glioblastoma cells followed by reverse transcription into cDNA and PCR with primers for all human PAR’s. GADPH, mock RNA, and water served as controls. Immunochemistry on U373 cells was performed with polyclonal PAR1 antibodies visualized by fluorescent secondary antibodies. The function of PAR1 was tested by calcium imaging in Fura-2-loaded cells at 340 nm and 380 nm, respectively, after short application of thrombin and thrombin inhibitors. To determine the influence on glioma proliferation, thrombin, thrombin inhibitors, as well as PAR-agonistic and antagonistic peptides were added to glioblastoma cells for 24 hours. The WST-1 and the 5-bromo-2’-deoxy-uridine incorporation assays were employed to measure effects on glioma metabolism and DNA synthesis.

First, U373 glioblastoma cells expressed mRNA for PAR1 but not for other PAR types. Second, immunochemistry proved a cluster-like expression of PAR1 protein on the cellular membrane. Third, stimulation of the cells with thrombin resulted in a dose-dependent increase of intracellular calcium. The thrombin inhibitors, hirudin and PPACK suppressed the rise of intracellular calcium. Fourth, addition of thrombin to glioblastoma cells for 24 hours significantly increased their metabolism by up to 80% over untreated controls, as determined by WST-1 conversion. This effect could be blocked by thrombin inhibitors. Fifth, application of thrombin also significantly increased DNA synthesis, as measured by incorporation of BrdU. This increase was similarly suppressed by thrombin inhibitors. Moreover, a stimulation of tumor cells with PAR1 agonist peptide resulted in enhanced proliferation, which could be prevented by coincubation with PAR1 antagonist peptide. In contrast, treatments with agonist peptides for other PAR’s did not influence the proliferation at all.

We learned from these experiments, that aggressive biology of human U373 glioblastoma cells is clearly influenced by thrombin via action on protease-activated receptors type 1.