Received for publication August 8, 2006.
Revised December 6, 2006.
Accepted for publication December 6, 2006.

Protease activated receptors differentially regulate human platelet activation through a phosphatidic acid-dependent pathway

Abstract

Pathological conditions such as coronary artery disease are clinically controlled via therapeutic regulation of platelet activity. Thrombin, through PAR1 and PAR4, plays a central role in regulation of human platelet function as it is known to be the most potent activator of human platelets. Currently, direct thrombin inhibitors used to block platelet activation result in unwanted side effects of excessive bleeding. An alternative therapeutic strategy would be to inhibit PAR-mediated intra-cellular platelet signaling pathways. To elucidate the best target, we are studying differences between the two platelet thrombin receptors, PAR1 and PAR4, in mediating thrombin's action. In this study we show that platelet activation by PAR1-activating peptide (PAR1-AP) requires a phospholipase D (PLD)-mediated phosphatidic acid (PA) signaling pathway. We show that this PAR1-specific PA-mediated effect is not regulated through differential granule secretion following PAR-induced platelet activation. Perturbation of this signaling pathway via inhibition of lipid phosphate phosphatase-1 (LPP-1) by propranolol or inhibition of the phosphatidylcholine (PC)-derived phosphatidic acid (PA) formation by PLD with a primary alcohol significantly attenuated platelet activation by PAR1-AP. Platelet activation by thrombin or PAR4-AP was insensitive to these inhibitors. Further, activation of Rap1 was significantly attenuated by these inhibitors following stimulation by PAR1-AP, but not thrombin or PAR4-AP. Since PA metabolites such as diacylglycerol play an important role in intracellular signaling, identifying crucial differences in PA-regulation of PAR-induced platelet activation may lead to a greater understanding of the role of PAR1 versus PAR4 in progression of thrombosis.

Key words: Thrombin/PAR, Phospholipase C's, Phospholipase D's, IP3/DAG, Protein Kinase C, G protein regulation, Ras, Cdc42, rho, rac, other small G proteins, Cholesterol metabolism/lipoproteins, Platelets

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