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.

Keywords: MEG-01; PAR1; PAR4; morphological change; platelet; thrombin.

Figure 1

Endogenous expression of PAR1 and PAR4 in MEG-01 cells. (A) RT-PCR analysis for PAR1 and PAR4 in MEG-01 cells. PCR products were detected at expected product sizes as indicated. (B) Western blot analysis for PAR1 in MEG-01 cells. A2058 and PAR1 knocked out (KO) A2058 cells were used as positive and negative controls, respectively. PAR1 was knocked out by CRISPR/Cas9 in A2058 cells. All experiments were repeated three times.

Figure 2

Intracellular calcium increase in response to the activation of PAR1 and PAR4 in MEG-01 cells. Intracellular calcium increase in MEG-01 cells induced by (A) PAR1-AP (100 μM), (B) PAR4-AP (100 μM), (C) low-dose thrombin (0.5 U/mL), and (D) high-dose thrombin (5 U/mL). PAR1 and PAR4 were completely blocked by vorapaxar and BMS-986120 (BMS), respectively. Vorapaxar and BMS were applied at 10 μM for 10 min prior to treatment of PAR1-AP, PAR4-AP and thrombin (mean ± S.D., n = 3–5).

Figure 3

Morphological changes induced by PAR1 and PAR4 activation in MEG-01 cells. (A) Representative images of F-actin-stained MEG-01 cells. MEG-01 cells were treated with PAR1-AP (100 μM), PAR4-AP (100 μM), or thrombin (0.1 U/mL) for 30 min and fixed, and then labeled with phalloidin-FITC (green). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (B) Measurement of the circularity of MEG-01 cells. The circularity was measured using Lionheart software. Each dot corresponds to individual analyzed cells. A total of 20 cells were analyzed for each group (mean ± S.D., n = 20). (C) Measurement of diameter of MEG-01 cells. The diameter was measured using Lionheart software. For each group, 20 cells were analyzed (mean ± S.D., n = 20); ** p < 0.01, *** p < 0.001.

Figure 4

PAR1- and PAR4-mediated morphological changes in living MEG-01 cells. (A) Schematic diagram of experimental setup. (B) Representative images of MEG-01 cells used for analysis of circularity measurements. Cells were stained with calcein-AM (1 μg/mL) and pretreated with 10 μM of vorapaxar and BMS-986120 (BMS), and then PAR1-AP (100 μM), PAR4-AP (100 μM), or thrombin (0.1 U/mL) were applied for 30 min. Scale bar = 10 μm. (C–E) Summary of circularity. PAR1 and PAR4 were inhibited with vorapaxar and BMS-986120 (BMS) at the indicated concentrations, respectively. Thrombin-induced activation of PAR1 and PAR4 was blocked by vorapaxar (10 μM), BMS-986120 (10 μM), or both, respectively. Images were captured automatically and circularity was measured using Lionheart software (mean ± S.D., n = 4–5). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Time-course assessment of PAR1- and PAR4-mediated morphological changes. (A) Representative time-lapse images of MEG-01 cells. Cells were stained with calcein-AM (1 μg/mL) and treated with PAR1-AP (100 μM) and PAR4-AP (100 μM). Images were automatically captured by Lionheart every 30 min. Scale bar = 10 μm. (B–E) Summary of circularity. Cells were applied with PAR1-AP, PAR4-AP, and 0.1 U/mL of thrombin, and circularity was measured in time-course using Lionheart software (mean ± S.D., n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant. Not treated (NT).

Figure 6

Effect of intracellular calcium signaling on morphological changes in MEG-01 cells. (A,B) Inhibition of PAP1-AP- and PAR4-AP-induced intracellular calcium increase by 2-APB. MEG-01 cells were pre-treated with 2-APB at the indicated concentrations prior to applying 100 μM of PAR1-AP (A) or PAR4-AP (B). (C) Measurement of ionomycin (10 μM)-induced intracellular calcium increase. (D–F) Summary of morphological changes. MEG-01 cells were pre-treated with 2-APB at the indicated concentrations for 30 min prior to applying 100 μM of PAR1-AP (D) or PAR4-AP (E), and ionomycin was applied at the indicated concentrations for 30 min (F). (G) Representative images of MEG-01 cells. Cells were pretreated with 100 μM of 2-APB for 30 min, and then PAR1-AP (100 μM) and PAR4-AP (100 μM) were applied for 30 min or treated with ionomycin (10 μM) for 30 min. Calcein-AM-stained images were captured automatically and circularity was measured using Lionheart software (mean ± S.D., n = 4). Scale bar = 10 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Identification of kinases involved in PAR1- and PAR4-mediated morphological changes in MEG-01 cells. (A,B) Dot plots of primary screening results for kinase inhibitors that inhibit PAR1- and PAR4-mediated morphological changes. Validated kinase inhibitors that exhibit >80% inhibitory effects on morphological changes are classified according to the class of kinase, and one representative compound for each class is indicated. (C) Representative images of MEG-01 cells were stained with calcein-AM (1 μg/mL). Cells were pretreated with 10 μM of indicated kinase inhibitors for 30 min before application of PAR1-AP (100 μM) or PAR4-AP (100 μM) for 30 min. Scale bar = 10 μm. (D–F) Summary of circularity. Cells were pretreated with 10 μM of indicated kinase inhibitors for 30 min and then PAR1-AP (100 μM), PAR4-AP (100 μM), or thrombin (0.1 U/mL) were applied. Images were taken by Lionheart automatically and circularity was measured using Lionheart software (mean ± S.D., n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Schematic model of the underlying mechanisms involved in thrombin-induced morphological changes via PAR1 and PAR4 in MEG-01 cells.