Tyro3, Axl, and Mertk receptors differentially participate in platelet activation and thrombus formation

Junsong Zhou,Qingxian Lu,Yukio Ozaki,Aizhen Yang,Raymond B Birge,Katsue Suzuki-Inoue,Yi Wu,Yucan Wang,Fengwu Chen,Zhenzhen Zhao,Viralkumar Davra

doi:10.1186/s12964-018-0308-0

Abstract

BackgroundPreviously, several studies have shown that Tyro3, Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis. However, the role of individual receptors is not fully understood.MethodsUsing single receptor-deficient platelets from TAM knockout mice in the C57BL/6 J strain, we performed a knockout study using single TAM-deficient mice. We treated platelets isolated from TAM knockout mice with the Glycoprotein VI (GPVI) agonists convulxin, poly(PHG), and collagen-related triple-helical peptide (CRP), as well as thrombin for in-vitro experiments. We used a laser-induced cremaster arterial injury model for thrombosis experiments in vivo.ResultsDeficiency of the tyrosine kinase receptors, Axl or Tyro3, but not Mertk, inhibited aggregation, spreading, JON/A binding, and P-selectin expression of platelets in vitro. In vivo, platelet thrombus formation was significantly decreased in Axl−/− and Tyro3−/− mice, but not in Mertk−/− mice. Upon stimulation with glycoprotein VI (GPVI) agonists, tyrosine phosphorylation of signaling molecules, including spleen tyrosine kinase (Syk) and phospholipase C-γ2 (PLCγ2), was decreased in Axl−/− and Tyro3−/− platelets, but not in Mertk−/− platelets. While platelet aggregation induced by agonists did not differ in the presence or absence of the Gas6 neutralizing antibody, the platelet aggregation was inhibited by anti-Axl or anti-Tyro3 neutralizing antibodies antibody, but not the anti-Mertk antibody. Additionally, the recombinant extracellular domain of Axl or Tyro3, but not that of Mertk, also inhibited platelet aggregation.ConclusionsThese data suggest that Axl and Tyro3, but not Mertk, have an important role in platelet activation and thrombus formation, and mechanistically may do so by a pathway that regulates inside to outside signaling and heterotypic interactions via the extracellular domains of TAMs.

Highlights

Several studies have shown that Axl and Mertk (Tyro3), Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis
Backgroud Tyro3, Axl, and Mertk comprise the members of the TAM family of receptor tyrosine kinases that participate in a number of important physiological functions that include the clearance of apoptotic cells, resolution of inflammation, as well as platelet aggregation and clot formation
Angelillo-Scherrer et al have shown that deficiency of Growth arrest-specific gene 6 (Gas6) and TAM receptors causes platelet dysfunction and protects mice against thrombosis, and that TAM receptors are important in platelet activation [23, 24]

Summary

Methods

Using single receptor-deficient platelets from TAM knockout mice in the C57BL/6 J strain, we performed a knockout study using single TAM-deficient mice. Preparation of washed platelets All studies on human platelets were performed after approval by the Institution Review Board. Platelets were prepared as described previously [30, 31]. After centrifugation at 250 x g for 20 min, platelet-rich plasma (PRP) was collected and gel-filtered on a Sepharose 2B column equilibrated in a Tyrode-albumin solution. Blood was collected into ACD and was diluted (1:3) with modified Tyrode’s buffer (137 mM NaCl, 20 mM HEPES, 5.6 mM glucose, 1 g L− 1 BSA, 1 mM MgCl2, 2.7 mM KCl, and 3.3 mM NaH2PO4, pH 7.4). Blood was centrifuged at 230 g for 10 min, to obtain platelet-rich plasma (PRP). Washed platelets were prepared by centrifuging PRP at 980 g for 15 min and platelet pellets were resuspended in modified Tyrode’s buffer

Results

Discussion

Conclusion