The effects of 2-chloro-3-(4-acetophenyl)-amino-1,4-naphthoquinone (NQ301), an antithrombotic agent, on aggregation, binding of fibrinogen to glycoprotein (GP)Iib/IIIa complex and intracellular signals were investigated using human platelets. NQ301 significantly inhibited the collagen-, thrombin-, arachidonic acid-, thapsigargin- and calcium ionophore A23187-induced aggregation of washed human platelets with IC₅₀ values of 13.0±0.1, 11.2±0.5, 21.0±0.9, 3.8±0.1 and 46.2±0.8 μM, respectively. NQ301 also significantly inhibited FITC-conjugated fibrinogen binding to human platelet surface GPIIb/IIIa complex, but failed to inhibit the fibrinogen binding to purified GPIIb/IIIa complex. These data demonstrate that NQ301 inhibits platelet aggregation by suppression of the intracellular pathway, rather than by direct inhibition of fibrinogen-GPIIb/IIIa complex binding. NQ301 significantly inhibited the increase of cytosolic Ca²⁺ concentration and ATP secretion, and also significantly increased platelet cAMP levels in the activated platelets. These results suggest that the antiplatelet activity of NQ301 may be mediated by inhibition of cytosolic Ca²⁺ mobilization, enhancement of cAMP production and inhibition of ATP secretion in activated platelets.