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.
Highlights
Platelet activation is an essential event in hemostasis and thrombosis [1]
Platelet activation is crucial for hemostasis and thrombosis, and signaling within platelets begins via the activation of protease-activated receptors (PARs), P2Y12, and the thromboxane receptor by thrombin, ADP, and thromboxane A2 (TxA2), respectively [1]
Signaling events underlying the platelet activation induced by these agonists commonly include the Gq-mediated activation of phospholipase C (PLC), which triggers intracellular calcium increase and granule secretion, G12/13-mediated RhoA activation, which stimulates the reorganization of the actin cytoskeleton, resulting in the formation of filopodia and lamellipodia, and PI3K activation, which is crucial for stabilizing platelet aggregates [1,5]
Summary
Platelet activation is an essential event in hemostasis and thrombosis [1]. While platelets circulate freely under resting conditions, once vascular injury occurs, platelets are activated by locally generated agonists, including thrombin, ADP, and thromboxane A2 (TxA2) [2]. Thrombin is the most potent agonist for platelet activation [1] It stimulates platelets through a subset of G protein-coupled receptors (GPCRs) known as protease-activated receptors (PARs) [3]. Human platelets express two subtypes of PARs, PAR1 and PAR4, that act as thrombin receptors [3]. These two receptors are activated upon cleavage of their N-terminus by thrombin and initiate the multiple G protein-mediated intracellular events that are required for platelet adhesion and aggregation [4]. PAR-mediated signaling transductions mainly include Gq-mediated phospholipase C (PLC) activation that triggers intracellular calcium increase and G12/13-mediated Rho activation that triggers platelet shape change, and these two synergistically contribute to platelet granule secretion and result in platelet adhesion and aggregation [5,6]
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