Gas6 is complexed to the soluble tyrosine kinase receptor Axl in human blood

Background: The vitamin K-dependent Gas6 protein (product of growth arrest specific gene 6) binds to, and activates the TAM receptor tyrosine kinases Tyro3, Axl and Mer. It has been suggested that Gas6 and the TAM receptors are important for primary platelet functions, but Gas6 cannot be found in human platelets. However, Gas6 is present in human plasma at a concentration of around 0.2 nM, which is a thousand-fold lower than that of the homologous protein S. The Axl and Mer receptors can be cleaved close to the cell membrane, yielding soluble molecules consisting of the extracellular parts of the receptors. Objective: To investigate if soluble Axl (sAxl) is present in human serum and plasma and if Gas6 circulates in complex with sAxl. Methods: We expressed recombinant sAxl, raised antibodies and developed and validated an ELISA for Axl. Serum and plasma were analyzed using ELISAs for Gas6, Axl and sAxl-Gas6 complexes. Serum was gel filtered and fractions analyzed by the different ELISAs to determine if Gas6 in serum is free or complexed. Immunoprecipitation was used to investigate binding between Gas6 and sAxl in serum. Results: sAxl is present in serum and plasma at around 0.6 nM and all Gas6 is bound to sAxl. No complexes between Gas6 and the soluble forms of Mer and Tyro3 could be detected, indicating that sAxl is the physiological binder of Gas6 in human serum. Conclusions: Gas6 in circulation is bound to sAxl suggesting circulating Gas6 to be inhibited and incapable of stimulating the TAM receptors.