The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation.
OBJECTIVE
Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated.


METHODS
The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca(2+) mobilization respectively.


RESULTS
When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca(2+) rises. Blockade of ADP P2Y(12) receptor with 2-methylthioadenosine 5'-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5'-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation.


CONCLUSIONS
These results suggest that PAR4 acts in parallel with the P2Y(12)/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.
Wu
Chin-Chung
CC
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.
Wu
Shih-Yun
SY
Liao
Chieh-Yu
CY
Teng
Che-Ming
CM
Wu
Yang-Chang
YC
Kuo
Sheng-Chu
SC
D000730:Androstadienes / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D058539:Phosphatidylinositol 3-Kinase / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology*; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013917:Thrombin / Q000502:physiology*; D000077191:Wortmannin
C412964:1-benzyl-3-(ethoxycarbonylpheny)-indazole; D000730:Androstadienes; D007191:Indazoles; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D058921:Purinergic P2Y Receptor Antagonists; D018179:Receptors, Thrombin; D058539:Phosphatidylinositol 3-Kinase; D011493:Protein Kinase C; D013917:Thrombin; C112908:protease-activated receptor 4; D002118:Calcium; D000077191:Wortmannin