Lysophosphatidic acid (LPA) is a ligand for LPA 1-3 of the endothelial differentiation gene family G-protein-coupled receptors, and LPA 4 -8 is related to the purinergic family G-protein-coupled receptor. Because the structure-activity relationship (SAR) of GPR92/LPA 5 is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed mutagenesis of LPA 5 residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA 5 were identified as follows: R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA 5 using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates Ͼ Ͼ N-arachidonoylglycine. These results confirm LPA 5 to be a bona fide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA 5 agonists. A pharmacophore model of LPA 5 binding requirements was developed for in silico screening, which identified two non-lipid LPA 5 antagonists. Because LPA 5 transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA 5 antagonists inhibit platelet activation. The present results suggest that selective inhibition of LPA 5 may provide a basis for future anti-thrombotic therapies.Lysophosphatidic acid (LPA, 2 1-radyl-2-hydroxy-sn-3-glycero phosphate) specifically interacts with several protein targets that regulate physiological and pathophysiological processes (1-3). LPA targets include specific G-protein-coupled receptors (GPCRs) that mediate a wide variety of biological effects, including cell proliferation (4), cell survival (5), cell migration (6), and platelet aggregation (7,8). GPCRs are the largest family of transmembrane receptors and represent targets of many therapeutics (9). Eight LPA-specific mammalian GPCRs, LPA 1-8 , have been identified to date (10 -12). Among the eight LPA receptors, LPA 1 , LPA 2 , and LPA 3 are members of the endothelial differentiation gene (EDG) family (13), and the transmembrane domains of human LPA 1-3 show 81% homology with each other (14). The five other members of the EDG family are specific for the related lysophospholipid sphingosine 1-phosphate (S1P). The structural foundation for LPA selectivity over S1P has been linked to a single amino acid at position 3.29, a conserved glutamine in the LPA-specific and glutamate in the S1P-specific members of the EDG family (14 -16). However, the recently identified non-EDG family LPA receptors, LPA 4 /p2y9 (13), LPA 5 /GPR92 (17, 18), LPA 6 /GPR87 (19), LPA 7 /p2y5 (12), and LPA 8 /p2y10 (10), are more closely related to the purinoreceptor gene cluster and share less than 20% amino acid...

Lysophosphatidic acid (LPA), a component of mildly-oxidized LDL and the lipid rich core of atherosclerotic plaques, elicits platelet activation. LPA is the ligand of G protein-coupled receptors (GPCR) of the EDG family (LPA 1-3 ) and the newly identified LPA 4-7 subcluster. LPA 4 , LPA 5 and LPA 7 increase cellular cAMP levels that would induce platelet inhibition rather than activation. In the present study we quantified the mRNA levels of the LPA 1-7 GPCR in human platelets and found a rank order LPA 4 =LPA 5 >LPA 7 >LPA 6 =LPA 2 >>LPA 1 >LPA 3 . We examined platelet shape change using a panel of LPA receptor subtype-selective agonists and antagonists and compared them with their pharmacological profiles obtained in heterologous LPA 1-5 receptor expression systems. Responses to different natural acyl and alkyl species of LPA, and octyl phosphatidic acid analogs, alpha-substituted phosphonate analogs, N-palmitoyl-tyrosine phosphoric acid, N-palmitoyl-serine phosphoric acid were tested. All of these compounds elicited platelet activation and also inhibited LPA-induced platelet shape change after pre-incubation, suggesting that receptor desensitization is likely responsible for the inhibition of this response. Fatty acid free albumin (10 µM) lacking platelet activity completely inhibited platelet shape change induced by LPA with an IC 50 of 1.1 µM but had no effect on the activation of LPA 1,2,3,&5 expressed in endogenously non-LPA-responsive RH7777 cells. However, albumin reduced LPA 4 activation and shifted the dose-response curve to the right. LPA 5 transiently expressed in RH7777 cells showed preference to alkyl-LPA over acyl-LPA that is similar to that in platelets. LPA did not increase cAMP levels in platelets. In conclusion, our results with the pharmacological compounds and albumin demonstrate that LPA does not induce platelet shape change simply through activation of LPA 1-5, and the receptor(s) mediating LPA-induced platelet activation remains elusive.

Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and 2-arachidonoylglycerol (2-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, 2-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). 2-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of 2-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of 2-AG (20 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by 2-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by 2-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A2 (TXA2) antagonist daltroban. In addition, platelet activation induced by 2-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to 2-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and 2 (CB2) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that 2-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA2. CB1, CB2 or non-CB1/CB2 receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that pharmacological CB1- and CB2-receptor ligands will not affect platelets and platelet-dependent progression and complications of cardiovascular diseases.

BackgroundPlatelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. By using the Rac1-specific inhibitor NSC23766, we have recently found that Rac1 is a central component of a signaling pathway that regulates dephosphorylation and activation of the actin-dynamising protein cofilin, dense and α-granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets.ObjectivesTo study whether NSC23766 inhibits stimulus-induced platelet secretion and aggregation in blood.MethodsHuman platelet aggregation and ATP-secretion were measured in hirudin-anticoagulated blood and platelet-rich plasma (PRP) by using multiple electrode aggregometry and the Lumi-aggregometer. Platelet P-selectin expression was quantified by flow cytometry.ResultsNSC23766 (300 μM) inhibited TRAP-, collagen-, atherosclerotic plaque-, and ADP-induced platelet aggregation in blood by 95.1%, 93.4%, 92.6%, and 70%, respectively. The IC50 values for inhibition of TRAP-, collagen-, and atherosclerotic plaque-, were 50 ± 18 μM, 64 ± 35 μM, and 50 ± 30 μM NSC23766 (mean ± SD, n = 3-7), respectively. In blood containing RGDS to block integrin αIIbβ3-mediated platelet aggregation, NSC23766 (300 μM) completely inhibited P-selectin expression and reduced ATP-secretion after TRAP and collagen stimulation by 73% and 85%, respectively. In ADP-stimulated PRP, NSC23766 almost completely inhibited P-selectin expression, in contrast to aspirin, which was ineffective. Moreover, NSC23766 (300 μM) decreased plaque-stimulated platelet adhesion/aggregate formation under arterial flow conditions (1500s-1) by 72%.ConclusionsRac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in blood stimulated by a wide array of platelet agonists including atherosclerotic plaque. By specifically inhibiting platelet secretion, the pharmacological targeting of Rac1 could be an interesting approach in the development of future antiplatelet drugs.

Atherosclerosis has an important inflammatory component. Macrophages accumulating in atherosclerotic arteries produce prostaglandin E(2) (PGE(2)), a main inflammatory mediator. Platelets express inhibitory receptors (EP(2), EP(4)) and a stimulatory receptor (EP(3)) for this prostanoid. Recently, it has been reported in ApoE(-/-) mice that PGE(2) accumulating in inflammatory atherosclerotic lesions might contribute to atherothrombosis after plaque rupture by activating platelet EP(3), and EP(3) blockade has been proposed to be a promising new approach in anti-thrombotic therapy. The aim of our investigation was to study the role of PGE(2) in human atherosclerotic plaques on human platelet function and thrombus formation. Plaque PGE(2) might either activate or inhibit platelets depending on stimulation of either EP(3) or EP(4), respectively. We found that the two EP(3)-antagonists AE5-599 (300 nM) and AE3-240 (300 nM) specifically and completely inhibited the synergistic effect of the EP(3)-agonist sulprostone on U46619-induced platelet aggregation in blood. However, these two EP(3)-antagonists neither inhibited atherosclerotic plaque-induced platelet aggregation, GPIIb/IIIa exposure, dense and alpha granule secretion in blood nor reduced plaque-induced platelet thrombus formation under arterial flow. The EP(4)-antagonist AE3-208 (1-3 μM) potentiated in combination with PGE(2) (1 μM) ADP-induced aggregation, demonstrating that PGE(2) enhances platelet aggregation when the inhibitory EP(4)-receptor is inactivated. However, plaque-induced platelet aggregation was not augmented after platelet pre-treatment with AE3-208, indicating that plaque PGE(2) does not stimulate the EP(4)-receptor. We found that PGE(2) was present in plaques only at very low levels (15 pg PGE(2)/mg plaque). We conclude that PGE(2) in human atherosclerotic lesions does not modulate (i.e. stimulate or inhibit) atherothrombosis in blood after plaque rupture.