Gas6 Receptors, Tyro3, Axl and Mer, Differentially Participate in Glycoprotein VI-Mediated Platelet activation

Abstract 364

Growth arrest-specific gene 6 (Gas6) has three receptors on platelets - Tyro3, Axl and Mer. It has been shown that these three receptors are equally important in platelet activation (J Clin Invest. 2005;115:237–46), and they synergizes with ADP receptor for platelet activation, but not with collagen receptor (J Thromb Haemost. 2010;8:1797–808). Considering the fact that Tyro3, Axl or Mer plays a distinct role in the function of endothelial cells and leukocytes, we hypothesized that these three receptors play specific role in platelet activation, and examined the role of each receptor in Gycoprotein VI (GPVI)-mediated platelet activation using single receptor knockout mice. In response to 100 ng/ml convulxin or 5 ng/ml poly(PHG), a GPVI-specific synthetic peptide, the aggregation of Axl^−/− or Tyro3^−/− platelets was almost completely prevented. However, the aggregation of Mer^−/− platelets was only delayed, and still could reach the maximum level. In the presence of 100 ng/ml convulxin or 5 ng/ml poly(PHG), the spreading of Axl^−/− or Tyro3^−/− platelets, but not Mer^−/− platelets, on fibrinogen-coated surfaces was markedly inhibited. Moreover, convulxin-stimulated P-selectin expression and JON/A binding were both significantly decreased in Axl^−/− and Tyro3^−/− platelets, but not in Mer^−/− platelets. Upon stimulation with 100 ng/ml convulxin, tyrosine phosphorylation of Syk, linker for activation of T cells (LAT), SLP-76(SH2 domain-containing leukocyte protein of 76) and phospholipase C (PLCγ2) was markedly reduced in the Axl^−/− or Tyro3^−/− platelets, but not in the Mer^−/− platelets. Interestingly, anti-Axl or anti-Tyro3 antibody (20 mg/ml) completely blocked convulxin-stimulated human platelet aggregation, however, the neutralizing anti-Gas6 antibody (60 mg/ml) did not have any effect, suggesting the homodimer formation of Axl or Tyro3 is involved in GPVI-mediated platelet activation. As detected by flow cytometry, there was no difference in the expression of GPVI and integrin aIIbb3 on membrane surface among Tyro-3^−/−, Axl^−/−, Mer^−/− and wild type platelets. In conclusion, Gas6 receptors are not equally important in platelet activation, Axl and Tyro3 play more critical role than Mer in GPVI-mediated platelet activation. The trans-interaction of Axl or Tyro3 mediates cell-cell contact-dependent activation, which facilitates the activation of GPVI in platelets. Thus, Axl and Tyro3 may serve as better targets than Mer for the development of new antiplatelet agents.