Thrombopoietin has an essential role in megakaryopoiesis and thrombopoiesis. To investigate the signaling processes induced by thrombopoietin, we have employed human platelets and recently demonstrated that thrombopoietin induces rapid tyrosine phosphorylation of Jak-2, Tyk2, Shc, Stat3, Stat5, p120(c-cbl) and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine phosphorylated protein in platelets stimulated by thrombopoietin is approximately 85 to 95 kD, we examined the possibility that this could be Vav, a 95-kD proto-oncogene product. Specific antisera against Vav recognized the same 95 kD protein in lysates of Jurkat cells, which are known to express Vav, and platelets, indicating that platelets have Vav. Thrombopoietin induced rapid tyrosine phosphorylation of Vav in platelets without an elevation in cytosolic free calcium concentration or activation of protein kinase C. Vav was also tyrosine phosphorylated upon treatment of platelets with thrombin, collagen, or U46619, which activate phospholipase C, leading to an increased ionized calcium concentration and activation of protein kinase C. Ionomycin or phorbol 12-myristate 13-acetate (PMA) also induces tyrosine phosphorylation of Vav, suggesting that an increase in ionized calcium concentration or activation of protein kinase C may lead to phosphorylation of Vav. Thrombopoietin also induced tyrosine phosphorylation of Vav in FDCP-2 cells, genetically engineered to express human c-Mpl (FDCP-hMpl5). However, neither ionomycin nor PMA induced an increase in tyrosine phosphorylation of Vav in FDCP-hMpl5 cells, suggesting that the calcium and protein kinase C pathways of Vav phosphorylation may be unique to platelets. Further, Vav became incorporated into the Triton X-100 insoluble 10,000 g sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. Vav was constitutively associated with a 28-kD adapter protein, Grb2, which is also incorporated into the cytoskeleton in an aggregation-dependent fashion. Lastly, we found that Vav is cleaved when there is activation of calpain, a protease that may have a role in postaggregation signaling processes. Our data suggest that thrombopoietin and other agonists may induce tyrosine phosphorylation of Vav by different mechanisms and Vav may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.