Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1-and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.
Bibliographical noteFunding Information:
Funding: This research was funded by the National Research Foundation of Korea (NRF-2018R1A6A1A03023718) and the Ministry of Education (MOE, Korea) through the fostering project of ‘Yonsei University and Industry Cooperation Complex’ supervised by the Korea Institute for Advancement of Technology (KIAT).
This research was funded bythe National ResearchFoundation of Korea (NRF-2018R1A6A1A03023718) and the Ministry of Education (MOE, Korea) through the fostering project of ?Yonsei University and Industry Cooperation Complex? supervised by the Korea Institute for Advancement of Technology (KIAT).
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All Science Journal Classification (ASJC) codes
- Molecular Biology
- Computer Science Applications
- Physical and Theoretical Chemistry
- Organic Chemistry
- Inorganic Chemistry