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Piperlongumine, a constituent of Piper longum L., inhibits rabbit platelet aggregation as a thromboxane A2 receptor antagonist

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Abstract

Piper longum L. has been used as a crude drug for the treatment of the disorder of peripherally poor blood circulation in Asia. In the present study, we examined the effect of piperlongumine, a constituent of P. longum L., on rabbit platelet aggregation. Piperlongumine concentration-dependently inhibited platelet aggregation induced by thromboxane A2 receptor agonist U46619, but it only slightly inhibited thrombin-induced one. Piperlongumine also inhibited U46619-induced phosphatidylinositol hydrolysis and the binding of [3H]SQ29548 to thromboxane A2 receptor with a similar concentration-dependency to the aggregation. It is assumed that piperlongumine inhibits platelet aggregation as a thromboxane A2 receptor antagonist.

Introduction

Blood vessel wall injury triggers sudden platelet activation and platelet plug formation, followed by the occurrence of blood coagulation and the formation of fibrin-containing thrombi that occludes the site of injury. These events limit vital blood loss at a site of injured tissue. However, platelet plug and thrombi may often block narrow and diseased vessels, leading to ischemia and/or destruction in vital organs (Aronow, 2004, McNicol and Israels, 2003). In addition, platelet aggregation and subsequent thrombus formation occur in coronary and cerebral arteries, causing myocardial infarction and stroke, respectively (Stoyioglou and Jaff, 2004). The platelet aggregation is regulated by a lot of physiological agonists, such as collagen, platelet-activating factor, adenosine 5′-diphosphate (ADP) and thromboxane A2 (TXA2) (McNicol and Israels, 2003).

When TXA2, a metabolite of arachidonic acid, is released from activated platelets, it binds TXA2 receptors (prostanoid TP receptor) and causes platelet shape change and aggregation (Ohkubo et al., 1996), as a positive feedback mediator. Prostanoid TP receptor interacts with heterotrimeric G proteins, Gq/11, Gi/o and G12/13 (Djellas et al., 1999, Offermanns et al., 1994, Shenker et al., 1991, Ushikubi et al., 1994). It is known that the stimulation of prostanoid TP receptor results in platelet shape change mainly through G12/13 pathway (Klages et al., 1999), and subsequent aggregation through Gq/11 pathway (Offermanns et al., 1997).

TXA2-induced platelet aggregation is important for thrombus formation in vascular beds. At present, non-steroidal anti-inflammatory drugs (NSAIDs) and a TXA2 synthase inhibitor are clinically used for reducing TXA2-mediated platelet aggregation by decreasing TXA2 synthesis in platelets, such as aspirin, indomethacin and ozagrel (Catella-Lawson et al., 2001, Kawano et al., 2001). Therefore, it is thought that the regulation of prostanoid TP receptor-mediated platelet aggregation is clinically important for thrombosis formation.

Piper longum L. has been used as a crude drug to improve intestinal disorder, asthma and peripherally poor circulation in Asia. In particular, the improving activity of the peripherally poor blood circulation is well known. On the other hand, it has been shown that platelet function affects the peripherally poor blood circulation through the platelet aggregation and thrombosis formation (Nagatome et al., 2005). P. longum L. contains various constituents, such as piperine, piperidine, pipernonaline and piperlongumine (Yang et al., 2002). Although piperine is a constituent of both P. longum L. and Piper nigrum L., piperlongumine is that of P. longum L., but not P. nigrum L.. In addition, the pharmacological effect of piperlongumine on platelet function remains to be solved. In the present study, we examined the effect of piperlongumine on rabbit platelet aggregation, and show that piperlongumine inhibits platelet aggregation as a prostanoid TP receptor antagonist.

Section snippets

Materials

Piperlongumine (Fig. 1A) was purchased from Sigma-Aldrich (St Louis, MO, U.S.A). 9,11-Dideoxy-9α,11α-epoxymethano-prostaglandin F2α (U46619) and [1S-[1α,2α (Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazine]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29548) were obtained from Cayman Chemical Company (Ann Arbor, MI, U.S.A). Thrombin was from Wako Pure Chemicals (Osaka, Japan). [3H]Inositol and [3H]SQ29548 were from Perkin Elmer Life Science (Boston, MA, U.S.A). All other

Effect of piperlongumine on U46619-induced platelet aggregation

Chemical structure of piperlongumine, a constituent of P. longum L., is shown in Fig. 1A. U46619 (3 μM), a prostanoid TP receptor agonist, caused platelet aggregation, which was completely inhibited by piperlongumine (100 μM) or SQ29548 (3 μM), a prostanoid TP receptor antagonist. However, piperlongumine only slightly inhibit thrombin (0.05 U/ml)-induced platelet aggregation (Fig. 1B). U46619 caused platelet aggregation in a concentration-dependent manner with an EC50 value of 0.47 μM (data not

Discussion

Piperlongumine is a constituent of P. longum L. which has been used as a crude drug for the treatment of the disorder of peripherally poor circulation. Because the disorder of peripherally poor circulation is occurred by a reduction of blood flow in peripheral artery, an inhibition of platelet aggregation is assumed to be one of the ways to improve the peripherally poor circulation through an inhibition of thrombus formation. TXA2 is associated with various diseases, such as thrombogenesis,

Acknowledgments

This work was supported in part by Grant-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology (No. 18058002 to N.N.) and from Japan Society for Promotion of Science (No. 18890015 to M.S.).

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