Thrombin, a coagulation protease, plays important roles in physiological function such as cell migration and proliferation. Protease-activated receptors 1 (PAR 1) and thrombomodulin (TM) are two different types of thrombin receptors. Comparison of thrombin-PAR1 as to their ability to induce proliferation, thrombin-TM complex seems to restrains cell proliferation. This study was designed to improve the understanding of these cross-talks between thrombin-TM and thrombin-PAR1. 7.5nM thrombin induced transient phosphorylation of ERKs in HEK293 cells with a peak at 20 min, where as overexpression of TM in HEK293 cells sustained the phosphorylation of ERKs. Increased level of ERKs phosphorylation remained on activation state at 4 hours after thrombin stimulation. We conducted a proteome analysis with cytosolic protein from PAR1-expressed human embryonic kidney 293 (HEK293) or TM- transfected HEK293 (HEK293TM) cells. We analyzed the cytosolic proteins profile of HEK293 cell through LC-MS/MS and successfully identified 558 proteins. Differentially expressed proteins in response of TM-expressed were identified by gel-based proteomics and DIGE analysis. Twenty-five protein spots associated with TM-mediated transformation were identified. These proteins were related to cell structure and motility function processing with PANTHER classification system. For thrombin-stimulated HEK293cells, proteomic analysis reproducibly revealed 51 significantly thrombin-responsive protein spots, 18 in HEK293 and 33 in HEK293TM cells. Bioinformatic annotations indicated that this set of proteins is enriched with cell structure, motility, metabolism, signal transduction, protein modification and protein post-translational processes. Protein selective identified in HEK293TM cells were analyzed by western blotting and immunohistochemistry. Actin was confirmed to be localized majored in the cell membrane of HEK293TM cells. In conclusion, we established a comparative subproteomics approach and can provide valuable information for understanding the activities of thrombin with two receptors.
