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We observed that human rhabdomyosarcoma (RMS) cells highly express a tissue factor that promotes thrombin formation, which indirectly and directly affects RMS progression. First, we found that thrombin activates platelets to generate microvesicles (PMV), which transfer to RMS cells' alpha2beta3 integrin and increase their adhesiveness to endothelial cells. Accordingly, RMS cells covered with PMVs showed higher metastatic potential after i.v. injection into immunodeficient mice. Furthermore, PMVs activate mitogen-activated protein kinase (MAPK)p42/44 and AKT to chemoattract RMS cells. We also found that RMS cells express functional protease-activated receptor-1 (PAR1) and PAR3 and respond to thrombin stimulation by MAPKp42/44 and MAPKp38 phosphorylation. To our surprise, thrombin did not affect RMS proliferation or survival; it inhibited the chemotactic and adhesive properties of RMS cells. However, when PAR1-specific agonist thrombin receptor-activating peptide 6 was used, which does not activate PAR3, selective PAR1 stimulation enhanced RMS proliferation. To learn more on the role of PAR1 and PAR3 antagonism in RMS proliferation and metastasis, we knocked down both receptors by using a short hairpin RNA strategy. We found that although thrombin does not affect growth of PAR1(-/-) cells, it stimulated the proliferation of PAR3(-/-) cells. More importantly, PAR3(-/-) cells, in contrast to PAR1(-/-) ones, formed larger tumors in immunodeficient mice. We conclude that thrombin is a novel underappreciated modulator of RMS metastasis and that we have identified a novel role for PAR3 in thrombin signaling.
rhabdomyosarcomas; thrombin; microvesicles; metastases; chemotaxis
...AGG ACT CGC AAG TGC GG-3 (for PAR3), and inserting the double-strand oligonucleotide in the peptide RNA-U6.1/Neo plasmid (GenScript Co). RMS cells were transfected using Lipofectamine 2000 (Invitrogen). Isolation of PMVs Human peripheral blood platelets...
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