Abstract
Tyrosine phosphorylation of multiple platelet proteins is regulated by the integrin alpha IIb beta 3. In order to further examine integrin-regulated tyrosine phosphorylation, we have used small Arg-Gly-Asp-containing snake venom proteins (termed disintegrins) that inhibit platelet aggregation to competitively block the agonist-induced binding of fibrinogen to alpha IIb beta 3. One structurally unique disintegrin, contortrostatin (which appears to be a disulfide-linked dimer of 13.5 kDa with two Arg-Gly-Asp sites), was found to trigger signaling events typically mediated by fibrinogen cross-linking of alpha IIb beta 3, as demonstrated by tyrosine phosphorylation of the tyrosine kinase pp72syk and a 140-kDa protein. Contortrostatin and another disintegrin, multisquamatin (a monomer of 5.7 kDa with a single Arg-Gly-Asp site), did not affect thrombin-induced platelet shape change, secretion, or integrin-independent tyrosine phosphorylation; however, they inhibited aggregation and aggregation-dependent tyrosine phosphorylation of numerous proteins, including the focal adhesion kinase pp125FAK. Our results suggest that structurally distinct disintegrins have varying effects on tyrosine phosphorylation; while monomeric multisquamatin and dimeric contortrostatin both inhibit aggregation-dependent tyrosine phosphorylation, contortrostatin also possesses a unique functional activity that allows it to activate an intracellular signaling pathway leading to tyrosine phosphorylation. This activity may be involved in the function of this snake venom protein on hemostasis.
Highlights
Disintegrins Contortrostatin and brinogen and a cell surface receptor thabtelongs to the integrin family, aI& [1].cyrrn& playsa critical role in regulating the Multisquamatin Differentially Regulate Platelet Tyrosine latter two of the threewaves of agonist-induced tyrosine phosphorylation [2,3]
Buatlso signaling events mediated receptorand cytoskeletal reartion, we have used small Arg-Gly-Asp-containing snake the tyrosine kinases responsible for the integrinvenom proteinsthat inhibitplate- dependent tyrosine phosphorylation have yet to be identified, let aggregation to competitively block the agonist-in- at least three tyrosine kinases are regulated by integrins. 1)
These results suggest a possible role for tin and another disintegrin, multisquamatin, did not one andthree, for pp72"yk in wavesone and two, and for affect thrombin-induced platelet shape change, secre- ~ ~ 1in w2 ave5 thr~ee. ~
Summary
One)tyrosinephosphorylation, while both inhibited aggrega- Tyrosine phosphorylation events that are dependent on intion and theaggregation-dependent (wavethree) tyrosine phos- tegrin engagement and subsequent platelet aggregation have phorylation of ~ ~ 1 an2d p955/97~. Hibited the wave two tyrosinephosphorylation of p140 and with one of the following: 1)7E3, a monoclonal antibody t o this p50-68, which is dependent on the clusteringof aII,P3h;owever, receptor; 2) the tetrapeptide Arg-Gly-Asp-Ser, which competicontortrostatin induced the tyrosine phosphorylation of these tively interferes with fibrinogen binding; or 3) EGTA, which wave two proteins (p140 and pp7Tyk). These results suggest chelates the calcium required for integrin binding [26, 27]. In order to further investigate the effects of platelet activation inhibitors
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