On the mechanism of the spermine-exerted inhibition of a-thrombin-induced platelet activation

Previous reports have shown that various amines inhibited platelet activation, but no definitive conclusions on their action mechanism were drawn. We have further investigated the action of spermine on platelet responses evoked by α-thrombin and other agonists. Spermine inhibited in a concentration-dependent manner (1–10 mM), and more efficiently than spermidine and putrescine, the α-thrombin-induced (1.5 nM) platelet activation. Spermine added at a concentration that inhibited completely aggregation only partially affected the thrombin-induced increase in cytosolic Ca2+ concentration, protein phosphorylation, and ATP secretion. The polyamine had little effect on the morphology of resting platelets, as measured by electron microscopy, thrombin hydrolytic activity, and fibrinogen clotting capacity but decreased the thrombin binding to platelets and isolated glycocalicin. Spermine partially inhibited the aggregation elicited by ADP, vasopressin, platelet-activating factor, thrombin receptor-activating peptide, fluoroaluminate, ionomycin, and dioctanoylglycerol but did not affect the cytosolic Ca2+ increase induced by these agonists. The polyamine bound to both glycocalicin and platelets, and it inhibited the fibrinogen binding to stimulated platelets. The amount of 14C-spermine bound to resting cells decreased in the presence of the glycoprotein GPIb-antibody LJIB1, whereas the polyamine bound to activated platelets, which was higher than that tied to resting cells, was markedly reduced by LJCP8 or decorsin, a GPIIb/IIIa antibody and antagonist-peptide, respectively. These results indicate that spermine specifically inhibits the thrombin binding to GPIb of resting platelets and the fibrinogen binding to GPIIb/IIIa (integrin αIIbβ3) of activated platelets.