The role of PAR4 in thrombin-induced thromboxane production in human platelets

Chin-Chung Wu; Tsong-Long Hwang; Chang-Hui Liao; Sheng-Chu Kuo; Fang-Yu Lee; Che-Ming Teng

doi:10.1160/TH03-02-0103

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2003; 90(02): 299-316
DOI: 10.1160/TH03-02-0103

Platelets and Blood Cells

Schattauer GmbH

The role of PAR4 in thrombin-induced thromboxane production in human platelets

Authors

Chin-Chung Wu

¹Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung,
Tsong-Long Hwang

²Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan
Chang-Hui Liao

²Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan
Sheng-Chu Kuo

³Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung,
Fang-Yu Lee

⁴Yung-Shin Pharmaceutical Industry Co. Ltd, Taichung
Che-Ming Teng

⁵Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan

Abstract

Summary

There are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [³H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca²⁺]_i and activation of phospholipase A₂. These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.

Keywords

Protease-activated receptor - thrombin - thromboxane - platelets - YD-3

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Referenzen

References
1 Hemker HC. Thrombin generation, an essential step in haemostasis and thrombosis. In: Bloom A.L., Forbes, C.D., Thomas, D.P., Tuddenham, E.G.D., eds. Haemostasis and Thrombosis. Edinburgh, U.K., Churchill Livingstone. 1994; 477-90.
2 Vu TKH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional throm-bin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-68.
3 Vu TKH, Wheaton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 674-7.
4 Chen J, Ishii M, Wang L, Ishii K, Coughlin SR. Thrombin receptor activation: confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intramolecular liganding mode. J Biol Chem 1994; 269: 16041-5.
5 Dery O, Corvera CU, Steinhoff M, Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol 1999; 274: C1429-52.
6 Coughlin SR. How the protease thrombin talks to cells?. Proc Natl Acad Sci USA 1999; 96: 11023-7.
7 Lau LF, Pumiglia K, Cote YP, Feinstein MB. Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators. Biochem J 1994; 303: 391-400.
8 Brass LF, Vassallo RR, Belmonte E, Ahuja M, Cichowski K, Hoxie JA. Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. J Biol Chem 1992; 267: 13795-8.
9 Huang DT, Vu TK, Wheaton VI, Ishii K, Coughlin SR. Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation. J Clin Invest 1992; 89: 1350-3.
10 Kahn ML, Zheng YW, Huang W. et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-4.
11 Xu WF, Andersen H, Whitmore TE. et al. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 1998; 95: 6642-6.
12 Kahn ML, Nakanishi-Mastsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptor 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999; 103: 879-87.
13 FitzGerald GA. Mechanisms of platelet activation: thromboxane A₂ as an amplifying signal for other agonists. Am J Cardiol 1991; 68: 11B-15B.
14 Siess W, Grunberg B, Luber K, Vauti F, Wil-helm B. Platelet signal transduction mechanisms induced by eicosanoids. Eicosanoids 1992; 5: S13-S15.
15 Kramer RM, Roberts EF, Hyslop PA, Utter-back BG, Hui KY, Jakubowski JA. Differential activation of cytosolic phospholipase A2 (cPLA₂) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA₂ independent of the mitogen-activated protein kinases ERK1/2. J Biol Chem 1995; 270: 14816-23.
16 Henriksen RA, Samokhin GP, Tracy PB. Thrombin-induced thromboxane synthesis by human platelets. Properties of an anion binding exosite I-independent receptor. Arterioscler Thromb Vasc Biol 1997; 17: 3519-26.
17 Henriksen RA, Hanks V K. PAR4 agonist peptide AYPGKF stimulates thromboxane production by human platelets. Arterioscler Thromb Vasc Biol 2002; 22: 861-6.
18 Wu CC, Hwang TL, Liao CH. et al. Selective inhibition of protease-activated receptor 4-dependent platelet activation by YD-3. Thromb Haemost 2002; 87: 1026-33.
19 Lee FY, Lien JC, Huang LJ. et al. Synthesis of 1-benzyl-3-(5’-hydroxymethyl- 2’-furyl) inda-zole analogues as novel antiplatelet agents. J Med Chem 2001; 44: 3746-9.
20 Wu CC, Huang SW, Hwang TL, Kuo SC, Lee FY, Teng CM. YD-3, a novel inhibitor of protease-induced platelet activation. Br J Pharma-col 2000; 130: 1289-96.
21 Born GVR. Quantitative investigations into the aggregation of blood platelets. J Physiol 1962; 162: 67-72.
22 Shapiro MJ, Weiss EJ, Faruqi TR, Coughlin SR. Protease-activated receptor 1 and 4 are shutoff with distinct kinetics after activation by thrombin. J Biol Chem 2000; 275: 25216-21.
23 Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD. Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest 1997; 100: 350-6.
24 Merritt JE, McCarthy SA, Davies MPA, Moores KE. Use of fluo-3 to measure cytosolic Ca²⁺ in platelets and neutrophils. Biochem J 1990; 269: 513-9.
25 Zhang HC, Derian CK, Andrade-Gordon P. et al. Discovery and optimization of a novel series of thrombin receptor (PAR-1) antagonists: potent, selective peptide mimetics based on indole and indazole templates. J Med Chem 2001; 44: 1021-4.
26 Andrade-Gordon P, Maryanoff BE, Derian CK. et al. Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor. Proc Natl Acad Sci USA 1999; 96: 12257-62.
27 Channon JY, Leslie CC. A calcium-dependent mechanism for associating a soluble arachidonoyl- hydrolyzing phospholipase A₂ with membrane in the macrophage cell line RAW 264.7. J Biol Chem 1990; 265: 5409-13.
28 Clark JD, Lin LL, Kriz RW. et al. A novel arachidonic acid-selective cytosolic PLA₂ contains a Ca²⁺-dependent translocation domain with homology to PKC and GAP. Cell 1991; 65: 1043-51.
29 Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev 2001; 53: 245-82.
30 Covic L, Gresser AL, Kuliopulos A. Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets. Biochemistry 2000; 39: 5458-67.
31 Pollock WK, Rink TJ. Thrombin and ionomycin can raise platelet cytosolic Ca²⁺ to micro-molar levels by discharge of internal Ca²⁺stores: studies using fura-2. Biochem Biophys Res Commun 1986; 139: 308-14.
32 Rittenhouse-Simmons S, Russell FA, Deyki D. Mobilization of arachidonic acid in human platelets. Kinetics and Ca²⁺ dependency. Bio-chim Biophys Acta 1977; 488: 370-80.
33 Kramer RM, Roberts EF, Manetta JV, Hyslop PA, Jakubowski JA. Thrombin-induced phosphorylation and activation of Ca²⁺-sensitive cytosolic phospholipase A₂ in human platelets. J Biol Chem 1993; 268: 26796-804.
34 Börsch-Haubold AG, Kramer RM, Watson SP. Cytosolic phospholipase A₂ is phosphorylated in collagen- and thrombin-stimulated human platelets independent of protein kinase C and mitogen-activated protein kinase. J Biol Chem 1995; 270: 25885-92.
35 Kramer RM, Roberts EF, Um SL. et al. p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A₂ (cPLA₂) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA₂ . J Biol Chem 1996; 271: 27723-9.
36 Börsch-Haubold AG, Ghomashchi F, Pasquet S. et al. Phosphorylation of cytosolic phospholipase A₂ in platelets is mediated by multiple stress-activated protein kinase pathways. Eur J Biochem 1999; 265: 195-203.
37 Sabri A, Guo J, Elouardighi H, Darrow AL, Andrade-Gordon P, Steinberg SF. Mechanisms of protease-activated receptor-4 actions in cardiomyocytes. Role of Src tyrosine kinase. J Biol Chem 2003; 278: 11714-20.
38 Covic L, Singh C, Smith H, Kuliopulos A. Role of the PAR4 thrombin receptor in stabilizing platelet-platelet aggregates as revealed by a patient with Hermansky-Pudlak syndrome. Thromb Haemost 2002; 87: 722-7.
39 Magocsi M, Sarkadi B, Kovacs T, Gardos G. Thrombin-induced activation of calcium transport pathways and their role in platelet functions. Biochim Biophys Acta 1989; 984: 88-96.
40 Massini P, Luscher EF. On the significance of the influx of calcium ions into stimulated human blood platelets. Biochim Biophys Acta 1976; 436: 652-63.