Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation

Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of pla telet alpha(IIb)beta(3) binding to fibrinogen. In addition to its antiplate let activity, echistatin also exhibits activating properties by inducing a switch of alpha(IIb)beta(3) conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor aff inity modulation, but it is unable to activate integrin-dependent intracell ular signals. Since proteins may exhibit a multivalent functionality as a r esult of their absorption to a substrate, in this study we evaluated whethe r immobilised echistatin is able to stimulate platelet adhesion and signall ing. The immobilisation process led to an increase of echistatin affinity f or integrin(s) expressed on resting platelets. Unlike the soluble form, imm obilised echistatin bound at comparable extent either unstimulated or ADP-a ctivated platelets. Furthermore, echistatin presented in this manner was ef fective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyr osine phosphorylated proteins resembling that of fibrinogen-attached platel ets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72(syk) and pp125(FAK). Inhibitors of platelet signa lling, such as apyrase, prostaglandin E-1, cytochalasin D and bisindolylmal eimide, while nor affecting platelet adhesion to immobilised echistatin, ab olished pp125(FAK) phosphorylation. This suggests that signals activating p rotein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125(FAK) phosphorylation. (C) 20 00 Elsevier Science B.V. All rights reserved.