Inhibitory Mechanisms of Metallothionein on Platelet Aggregation in In Vitro and Platelet Plug Formation in In Vivo Experiments

Abstract

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 μM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 μM) inhibited phosphoinositide breakdown in [³H]-inositol-labeled platelets, intracellular Ca⁺² mobilization in Fura-2 AM-loaded platelets, and thromboxane A₂ formation stimulated by collagen. In addition, metallothionein (4 and 8 μM) significantly Increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 μM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 μg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A₂ formation, thereby leading to inhibition of intracellular Ca⁺² mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.

Keywords

metallothionein platelet aggregation phospholipase C cyclic GMP protein kinase C

Get full access to this article

View all access options for this article.

References

Hamer

. Metallothionein. Annu Rev Biochem 55: 913–951, 1986.

Kagi

JHR

. Overview of metallothionein. Methods Enzymol 205: 613–626, 1991.

Cai

, Koropatnick

, Cherian

. Metallothioneins protects DNA from copper-induced but not iron-induced cleavage in vitro. Chem Biol Interact 96: 143–155, 1995.

Kaji

, Yamamoto

, Suzuki

, Miyajima

, Fujiwara

, Sakamoto

, Koizumi

Induction of metallothionein by thrombin in cultured vascular endothelial and smooth muscle cells. Biol Pharm Bull 18: 1272–1274, 1995.

Sugiura

, Nakamura

Metallothionein in platelets. Int Arch Allergy Immunol 103: 341–348, 1994.

Sheu

, Chao

, Yen

, Huang

. In vivo antithrombotic effect of triflavin, an Arg-Gly-Asp containing peptide on platelet plug formation in mesenteric microvessels of mice. Thromb Haemostas 72: 617–621, 1994.

Sheu

, Lee

, Lin

, Kan

, Lin

, Hung

, Lee

, Yen

. The antiplatelet activity of PMC, a potent α-tocopherol analogue, is mediated through inhibition of cyclo-oxygenase. Br J Pharmacol 127: 1206–1212, 1999.

Born

GVR

, Cross

. The aggregation of blood platelets. J Physiol 168: 178–195, 1963.

Sheu

, Teng

, Huang

. Triflavin, an RGD-containing antiplatelet peptide, binds to GP IIIa of ADP-stimulated platelets. Biochem Biophys Res Commun 189: 1236–1242, 1992.

10.

Sheu

, Lin

, Peng

, Huang

. Triflavin, an Arg-Gly-Asp-containing peptide, inhibits the adhesion of tumor cells to matrix protein via binding to multiple integrin receptors expressed on human hepatoma cells. Proc Soc Exp Biol Med 213: 71–79, 1996.

11.

Huang

, Sheu

, Teng

. Mechanism of action of a potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom. Thromb Haemostas 66: 489–493, 1991.

12.

Grynkiewicz

, Poenie

, Tsien

. A new generation of Ca⁺² indicator with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.

13.

Karniguian

, Legrand

, Caen

. Prostaglandins: specific inhibition of platelet adhesion to collagen and relationship with cyclic AMP level. Prostaglandins 23: 437–457, 1982.

14.

Grabarek

, Raychowdhury

, Ravid

, Kent

, Newman

, Ware

. Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells. J Biol Chem 267: 10011–10017, 1992.

15.

Sheu

, Hung

, Wu

, Ma

, Kan

, Lin

, Luk

, Yen

. Reduction in lipopolysaccharide-induced thrombocytopenia by triflavin in a rat model of septicemia. Circulation 99: 3056–3062, 1999.

16.

Broekman

, Ward

, Marcus

. Phospholipid metabolism in phosphatidyl inositol, phosphatic acid and lysophospholipids. J Clin Invest 66: 275–283, 1980.

17.

Siess

, Lapetina

. Platelet aggregation induced by alpha 2-adrenoceptor and protein kinase C activation. A novel synergism. Biochem J 263: 377–385, 1989.

18.

Charo

, Feinman

, Detwiler

. Inhibition of platelet secretion by an antagonist of intracellular calcium. Biochem Biophys Res Commun 72: 1462–1467, 1976.

19.

Kirk

, Creba

, Downes

, Michell

. Hormone-stimulated metabolism of inositol lipids and its relationship to hepatic receptor function. Biochem Soc Transact 9: 377–379, 1981.

20.

Berridge

. Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem J 212: 249–258, 1983.

21.

Hornby

. Evidence that prostaglandin endoperoxides can induce platelet aggregation in the absence of thromboxane A₂ production. Biochem Pharmacol 31: 1158–1160, 1982.

22.

McKean

, Smith

, Silver

. Formation of lysophosphatidylcholine in human platelets in response to thrombin. J Biol Chem 256: 1522–1524, 1981.

23.

Kraft

, Anderson

. Phorbol esters increase the amount of Ca⁺², phospholipid-dependent protein kinase associated with plasma membrane. Nature 301: 621–623, 1983.

24.

Murohara

, Parkinson

, Waldman

, Lefer

. Inhibition of nitric oxide biosynthesis promotes P-selectin expression in platelets. Role of protein kinase C. Arterioscler Thromb Vasc Biol 15: 2068–2075, 1995.

25.

McDonald

, Murad

Nitric oxide and cyclic GMP signaling. Proc Soc Exp Biol Med 211: 1–6, 1996.