Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments.
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 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) 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 microM) 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 microM) 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 microg/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.
Sheu
Joen R
JR
Department of Pharmacology, Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 110, Taiwan. sheujr@tmu.edu.tw
Hsiao
George
G
Shen
Ming Y
MY
Wang
Ying
Y
Lin
Kuang H
KH
Lin
Chien H
CH
Chou
Duen S
DS
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology; D000242:Cyclic AMP / Q000097:blood; D006152:Cyclic GMP / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D008668:Metallothionein / Q000494:pharmacology*; D051379:Mice; D008954:Models, Biological; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000097:blood; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013923:Thromboembolism / Q000517:prevention & control*; D013929:Thromboxane B2 / Q000097:blood
D010716:Phosphatidylinositols; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D015240:Phorbol 12,13-Dibutyrate; D013929:Thromboxane B2; D003094:Collagen; D008668:Metallothionein; D000242:Cyclic AMP; D006152:Cyclic GMP