THROMBIN RECEPTOR ACTIVATION AND INTEGRIN ENGAGEMENT STIMULATE TYROSINE PHOSPHORYLATION OF THE PROTOONCOGENE PRODUCT, P95(VAV), IN PLATELETS

The vav proto-oncogene product, p95(vav) or Vav, is primarily expresse d in hematopoietic cells and has been shown to be a substrate for tyro sine kinases. Although its function is unknown, Vav shares a region of homology with DBL, an exchange factor for the Rho family of GTP-bindi ng proteins. The presence of this domain and the observation that cell s transformed with Vav display prominent stress fibers and focal adhes ions similar to those that are observed in RhoA transformed cells sugg ests that Vav may play a role in regulating the actin cytoskeleton. We have, therefore, examined Vav phosphorylation in platelets, which und ergo dramatic cytoskeletal reorganization in response to agonists. Two potent platelet agonists, thrombin (via its G protein-coupled recepto r) and collagen (via its interaction with the alpha(2) beta(1), integr in), caused Vav to become phosphorylated on tyrosine. Weaker platelet agonists, including ADP, epinephrine and the thromboxane A(2) analog, U46619, did not. The phosphorylation of Vav in response to thrombin wa s maximal within 15 s and was unaffected by aspirin, inhibitors of agg regation, or the presence of the ADP scavenger, apyrase. Vav phosphory lation was also observed when platelets became adherent to immobilized collagen (via integrin alpha(2) beta(1)), fibronectin (via integrin a lpha(5) beta(1)), and fibrinogen (via integrin alpha(IIb)beta(3)). The se results show that Vav phosphorylation by tyrosine kinases 1) occurs during platelet activation by potent agonists, 2) also occurs when pl atelets adhere to biologically relevant matrix proteins, 3) requires n either platelet aggregation nor the release of secondary agonists such as ADP and TxA(2), and 4) can be initiated by at least some members o f two additional classes of receptors, G protein-coupled receptors and integrins, providing further evidence that both of these can couple t o tyrosine kinases.