PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells

Abstract

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.

MicroRNA‐26a alleviates tubulointerstitial fibrosis in diabetic kidney disease by targeting PAR4

“…PAR4 is expressed in several types of cells and is an important mediator that facilitates direct communication between proteases and intracellular signalling mechanisms 33,34 . Previous studies have demonstrated that PAR4 can activate a variety of pro‐fibrotic signalling pathways, including MAPK, PIK3‐Akt, STAT and NF‐κB signalling pathways 35,36 . PAR4 is also involved in the pathophysiology of organ fibrosis, though few studies regarding this role of PAR4 have been reported.…”

“… 33 , 34 Previous studies have demonstrated that PAR4 can activate a variety of pro‐fibrotic signalling pathways, including MAPK, PIK3‐Akt, STAT and NF‐κB signalling pathways. 35 , 36 PAR4 is also involved in the pathophysiology of organ fibrosis, though few studies regarding this role of PAR4 have been reported. Joshi et al.…”

Thrombopoietin-independent generation of platelet-like particles from megakaryoblastic cells

“…These stimuli are phorbol diesters 22 , plant extracts with tumor-promoting activity 23 , valproic acid 13 , 15 and TPO 24 , 25 . Moreover, the MEG-01 cell line has been used as an in vitro model for elucidating the mechanism underlying dengue virus-mediated thrombocytopenia 16 and thrombin-activated platelets 26 . Despite its utility, the cell morphology and cell cycle during the differentiation of MEG-01 cells remain uncharacterized owing to the heterogeneity of the cell population.…”