Abstract
Binding fibrinogen to activated glycoprotein (GP)IIb/IIIa is the final common pathway of platelet aggregation and has become a successful target for antiplatelet therapy. In the present study, we found that a small chemical compound, 3,4-methyl-enedioxy-β-nitrostyrene (MNS), exhibited potent and broad-spectrum inhibitory effects on human platelet aggregation caused by various stimulators. Moreover, addition of MNS to human platelets that had been aggregated by ADP caused a rapid disaggregation. We demonstrated that the antiaggregatory activity of MNS is due to inhibition of GPIIb/IIIa activation by measuring the binding amount of PAC-1 in platelets. In contrast, MNS is not a direct antagonist of GPIIb/IIIa, because MNS did not affect fibrinogen binding to fixed ADP-stimulated platelets. By investigating how MNS inhibits GPIIb/IIIa activation, we found that MNS potently inhibited the activity of tyrosine kinases (Src and Syk) and prevented protein tyrosine phosphorylation and cytoskeletal association of GPIIb/IIIa and talin, but it had no direct effects on protein kinase C, Ca2+ mobilization, Ca2+-dependent enzymes (myosin light chain kinase and calpain), and arachidonic acid metabolism, and it did not affect the cellular levels of cyclic nucleotides. Therefore, MNS represents a new class of tyrosine kinase inhibitor that potently prevents GPIIb/IIIa activation and platelet aggregation without directly affecting other signaling pathways required for platelet activation. Because MNS inhibits GPIIb/IIIa functions in a manner different from GPIIb/IIIa antagonists, this feature may provide a new strategy for treatment of platelet-dependent thrombosis.
Footnotes
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This work was supported by National Science Council of Taiwan Grant NSC 94-2320-B-037-041.
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ABBREVIATIONS: GP, glycoprotein; MNS, 3,4-methylenedioxy-β-nitrostyrene; U46619, 9,11-dideoxy-9α, 11α-methanoepoxy PGF2α; PDBu, phorbol 12,13-dibutyrate; FITC, fluorescein isothiocyanate; JAK, Janus tyrosine kinase; MARCKS, myristoylated alanine-rich C kinase substrate; DMSO, dimethyl sulfoxide; PLC, phospholipase C; TBST, Tris-buffered saline supplemented with 0.1% Tween 20; AA, arachidonic acid; A23187, calcimycin; PKC, protein kinase C; PGE, prostaglandin E1; RGDS, Arg-Gly-Asp-Ser; MLCK, myosin light chain kinase; GF109203X, 3-[1-[3-(dimethyl-aminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; ML-7, 1-(5-iodonaphthalene-1-sulfonyl)homopiperazine.
- Received March 2, 2006.
- Accepted July 11, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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