Abstract
The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count, and the level of production can be greatly increased under conditions of platelet destruction. Thrombopoietin (TPO) is the primary regulator of platelet production, supporting the survival, proliferation and differentiation of platelet precursors, bone marrow megakaryocytes. Hepatocytes are a major source of production and secretion of circulating TPO. However, mechanisms regulating circulating TPO levels have been debated for decades. Here, we provide experimental evidence that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR or asialoglycoprotein receptor), thereby regulating platelet survival and hepatic TPO levels. These conclusions are based on the following evidence: 1) Mice lacking the AMR Asgr2 subunit had increased platelet survival, compared to wild type (WT) mice. Platelets from Asgr2-null mice showed increased loss of sialic acid, as evidenced by flow cytometry using the galactose specific lectins RCAI and ECL, showing that removal of desialylated platelets by the AMR regulates in vivo platelet survival. 2) Livers isolated from Asgr2-null mice had TPO mRNA levels decreased by 40%, compared to WT mice. In contrast, liver TPO mRNA levels were increased by 30% in St3gal4-null mice lacking the sialyltransferase ST3GalIV, where desialylated platelet clearance is increased and specifically mediated by the AMR. Both plasma TPO levels and platelet TPO contents were similarly altered in both mutant mice. Thus, desialylated platelet uptake by the AMR regulated liver TPO levels. 3) Desialylated platelets isolated from St3gal4-null or Asgr2-null mice infused into WT mice increased hepatic TPO mRNA levels as early as 12h post-infusion. Plasma TPO concentrations and bone marrow megakaryocyte numbers increased in parallel with TPO mRNA levels, peaking by day 2 post-infusion, followed by new platelet release at day 10 post-infusion. In contrast, desialylated platelets infused into Asgr2-null mice had no effect on TPO mRNA synthesis, TPO plasma levels and bone marrow megakaryocyte numbers. 4) Incubation of human hepatoma cell line, HepG2 cells, with human desialylated platelets by sialidase treatment resulted in TPO mRNA expression increase by 2.2 and 2.9-fold after 4 and 6h, respectively, followed by significant increase in TPO secretion. 5) The signaling pathways activated by uptake of desialylated platelets by the AMR to induce TPO mRNA transcription were investigated in vivo and in vitro. Major polypeptides of 60-70 and 125 kDa were highly tyrosine phosphorylated in WT liver cells, as evidenced by SDS-PAGE. Using a specific antibody directed against JAK2, we identified the 125-kDa phosphoprotein as the tyrosine kinase JAK2 in mouse liver cells and human HepG2 cells. Analysis of liver samples revealed a marked reduction in JAK2 phosphorylation in Asgr2-null mice and significant increase in St3gal4-null mice. 6) The JAK1/2 inhibitor AZD1480 significantly decreased phosphorylation of JAK2, phosphorylation and translocation to the nucleus of the acute phase response transcription factor STAT3, TPO mRNA expression and TPO secretion in HepG2 cells incubated with desialylated platelets. In vivo treatment of WT mice with AZD1480 blocked TPO mRNA increase promoted by injection of endogenously desialylated platelets. Therefore we conclude that platelets desialylate as they circulate, thereby becoming the primary AMR ligand and providing a novel physiological feedback mechanism to regulate plasma TPO levels and platelet production in vivo and in vitro.
Disclosures
No relevant conflicts of interest to declare.
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