Inhibition of Thrombin-Induced Vascular Endothelial Growth Factor Production in Human Neuroblastoma (NB-1) Cells by Argatroban

Abstract

Thrombin, a serine protease that plays a pivotal role in blood coagulation, wound healing, and angiogenesis, has also been implicated in the mitogenesis of various cell types. Previously, we showed that thrombin and the thrombin receptor agonist peptide (TRAP-14; SFLLRNPNDKYEPF) for protease-activated receptor 1 (PAR1) induce vascular endothelial growth factor (VEGF) secretion in PC-12 cells. In this study, we show that thrombin and TRAP-14 also stimulate VEGF secretion in the human NB-1 neuroblastoma cells. In these cells, we further show that thrombin-induced VEGF secretion was blocked by cycloheximide and actinomycin D, indicating that de novo protein synthesis is essential for this process. Reduced thrombin-induced VEGF secretion upon treatment with LY294002, calphostin C, or BAPTA, further suggests that the process is dependent on phosphatidyl-inositol-3-kinase, protein kinase C, and calcium. However, the complete loss of thrombin-induced VEGF production upon treatment with argatroban, a derivative of arginine and a potent anticoagulant/antithrombin agent, supports the notion that argatroban serves as a useful therapeutic tool for thrombin-associated pathologic conditions. Here, it appears that argatroban may be effective in controlling disorders linked to thrombin-induced VEGF production in neuronal cells.

Targeting Angiogenesis for Controlling Neuroblastoma

“…Combinatorial treatment with vinblastine, a monoclonal antibody (DC101) targeting VEGFR-2 and rapamycin (mTOR inhibitor) in both neuroblastoma cells and orthotopic xenografted mice showed significant inhibition of tumor growth, angiogenesis, and reduction in microvessel formation suggesting that this combination may be relevant to design new curative strategies against neuroblastoma [57]. Argatroban (a derivative of arginine and a potent anti-coagulant and anti-thrombin agent) serves as a useful therapeutic tool for inhibition of thrombin-induced VEGF production in human neuroblastoma (NB-1) cells, and it may be effective in controlling disorders linked to thrombin-induced VEGF production in neuronal cells [58]. …”

Thrombin-Induced VEGF Expression in Human Retinal Pigment Epithelial Cells

“…The synergy between thrombin and TGF-␤2 is likely to result from the combination of the signaling pathways unique to each stimulus. The Smad pathway by TGF-␤ 51 and the Ca ϩ2 pathway by thrombin 60 are likely candidates for the synergistic action of the two factors. Consistent with this hypothesis is our finding that 36% of thrombin-induced and 19% of thrombinϩTGF-␤2-induced, but not TGF-␤2-induced, RPE VEGF stimulation was sensitive to the calcium chelator BAPTA.…”

Protease-Activated Receptors, Apoptosis and Tumor Growth

“…Thrombin inhibition by hirudin completely abrogates this effect [88] . Moreover, a PAR-1 agonist mimics thrombin-induced angiogenesis [89] , whereas PAR-1 antagonists or inhibitors block both tumor growth [59] and angiogenesis [90] , indicating that thrombin exerts its effect in angiogenesis through PAR-1 activation. Finally, silencing PAR-1 expression led to vessel regression in a murine model of prostate cancer [60] .…”