Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation.
Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet alpha(IIb)beta(3) binding to fibrinogen. In addition to its antiplatelet 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 affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADP-activated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72(syk) and pp125(FAK). Inhibitors of platelet signalling, such as apyrase, prostaglandin E(1), cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125(FAK) phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125(FAK) phosphorylation.
Belisario
M A
MA
Dipartimento di Biochimica e Biotecnologie Mediche, Universitá di Napoli Federico II, Via S. Pansini, n 5, 80131, Naples, Italy. belisari@cds.unina.it
Tafuri
S
S
Di Domenico
C
C
Della Morte
R
R
Squillacioti
C
C
Lucisano
A
A
Staiano
N
N
D000244:Adenosine Diphosphate; D000527:Alprostadil / Q000494:pharmacology; D001081:Apyrase / Q000494:pharmacology; D001792:Blood Platelets / Q000737:chemistry*; D002448:Cell Adhesion; D015638:Cytochalasin D / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D016023:Integrins / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins; D008301:Maleimides / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
D004791:Enzyme Inhibitors; D007211:Indoles; D016023:Integrins; D036341:Intercellular Signaling Peptides and Proteins; D008301:Maleimides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; C057986:echistatin; D015638:Cytochalasin D; D014443:Tyrosine; D000244:Adenosine Diphosphate; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D001081:Apyrase; D000527:Alprostadil; C088060:bisindolylmaleimide