Lysyl-phenylalanine

The role of protease-activated receptor (PAR)4 in thrombin-induced platelet aggregation has been studied, and PAR4 blockade is thought to be useful as a new and promising approach in antiplatelet therapy in humans. In recent years, studies have been conducted to clarify the role of PAR4 in the host defense against invading microorganisms and pathogen-induced inflammation; however, to date, the role of PAR4 in mediating the LPS-induced inflammatory repertoire in macrophages remains to be elucidated. Here, we investigated the effects of the synthetic PAR4 agonist peptide (PAR4-AP) AYPGKF-NH2 on the phagocytosis of zymosan-FITC particles; NO, ROS, and iNOS expression; and cytokine production in C57/BL6 macrophages cocultured with PAR4-AP/LPS. The PAR4-AP impaired LPS-induced and basal phagocytosis, which was restored by pharmacological PAR4 blockade. Coincubation with the PAR4-AP/LPS enhanced NO and ROS production and iNOS expression; decreased IL-10, but not TNF-α, in the culture supernatant; and increased translocation of the p65 subunit of the proinflammatory gene transcription factor NF-κ-B. Our results provide evidence for a complex mechanism and new approach by which PAR4 mediates the macrophage response triggered by LPS through counter-regulating the phagocytic activity of macrophages and innate response mechanisms implicated in the killing of invading pathogens.

Objectives: The inhibitory effects of P2Y12 receptor antagonist on PAR1- and PAR4-activating peptide (AP)-induced platelet aggregation have not been fully elucidated. The present study aimed to investigate the inhibitory effects of P2Y12 receptor antagonist on PAR1- and PAR4-AP-induced platelet aggregation using platelet-rich plasma (PRP) from individuals including patients with stroke or transient ischemic attack (TIA). Materials and methods: PRP was given to 10 healthy individuals pretreated in vitro with cangrelor, then stimulated with adenosine diphosphate (ADP), PAR4-AP, or PAR1-AP. Moreover, 20 patients were enrolled from 148 consecutive patients with acute ischemic stroke or TIA admitted to our institute between December 2017 and April 2019. PRP obtained from each patient before and >7 days after initiation of clopidogrel was similarly stimulated with these agonists. Platelet aggregation was measured using an automatic coagulation analyzer in all participants. Results: In healthy individuals, ADP- and PAR4-AP-induced platelet aggregations were significantly inhibited depending on the cangrelor concentration in vitro, while PAR1-AP-induced platelet aggregation was slightly inhibited. In patients with stroke or TIA, clopidogrel inhibited ADP-induced platelet aggregation at all concentrations, and significantly inhibited PAR4-AP-induced platelet aggregation at 50 µmol/L of PAR4-AP (p<0.05), especially in 5 patients who showed high reactivity to PAR4-AP. PAR1-AP-induced platelet aggregation was also slightly inhibited. Conclusions: We showed significant inhibitory effects on PAR4-AP-induced platelet aggregation by clopidogrel in patients with stroke or TIA who had high reactivity to PAR4-AP.

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.