Heparan sulfate proteoglycan is essential to thrombin-induced calcium transients and nitric oxide production in aortic endothelial cells

Thrombin induces Ca²⁺ transients and subsequent nitric oxide (NO) production in vascular endothelial cells.Thrombin cleaves protease-activated receptors, resulting in activation of intracellular signals, but it is not clarified how the extracellular thrombin stays around the cells to exert its enzyme activities. This study aimed to investigate the possible involvement of heparin sulfate proteoglycan (HSPG) in the effects of thrombin on vascular endothelium. Heparinase III completely removed the polysaccharide chain of HSPG in bovine aortic endothelial cells (BAECs).Thrombin induced Ca²⁺ transients in control BAECs, but not in heparinase III-treated BAECs. In contrast, ATP induced Ca²⁺ transients both in control and heparinase III-treated BAECs. Thrombin that was pre-incubated with heparin also failed to induced Ca²⁺ transients in BAECs. Furthermore, thrombin-induced NO production, as assessed with DAF-2 fluorescence, was suppressed in heparinase III-treated BAECs and by the pre-incubation of thrombin with heparin. ATP-induced NO production was, however, not affected in heparinase III-treated BAECs. These results indicate that it is essential for thrombin to bind to the polysaccharide chain of HSPG for inducing Ca ²⁺ transients and NO production in BAECs.