Platelets Regulate Thrombopoietin Production: The Missing Link

Humans produce and remove ~10¹¹ platelets daily, and the rate of production can rise sharply under conditions of platelet destruction. Platelet production must be tightly regulated to avoid spontaneous bleeding if counts are low or arterial occlusion and organ damage if counts are high. Thrombopoietin (TPO), the primary regulator of megakaryopoiesis and platelet production, was first isolated and cloned in 1994. This discovery was a major milestone in medicine and led to the availability of TPO mimetics for clinical care. Hepatocytes are the primary cells responsible for the production and secretion of circulating TPO. However, mechanisms regulating circulating TPO levels have been debated for decades. In one model, circulating TPO levels are maintained solely by its uptake and metabolism by high-affinity c-Mpl receptors on platelets and megakaryocytes. In another model, circulating platelet levels are sensed, resulting in appropriately regulated levels of TPO mRNA expression in liver and bone marrow. Despite these discussions, no physiological ligand-receptor pair capable of regulating steady state TPO production has yet been identified and the mechanisms through which platelets regulate TPO production remain poorly understood. Recent studies have highlighted the role of glycan modifications on platelet surface proteins in mediating platelet clearance. The hepatic Ashwell-Morell receptor (AMR) can bind and remove platelets with reduced α2,3-linked sialic acid (desialylated platelets), such as after cold storage, during sepsis, or in St3gal4^-/- mice lacking the ST3Gal-IV sialyltransferase. The AMR is a transmembrane heteroligomeric glycoprotein complex composed of ASGPR1 (also called CLEC4H1, HL-1) and ASGPR2 (CLEC4H2, HL-2) subunits, which are highly conserved among mammalian species. However, since its discovery four decades ago, the regulatory role of the hepatic AMR remains unclear. Injection of desialylated platelets into rabbits stimulates platelet production, suggesting a possible link between desialylated platelet consumption and liver TPO secretion. We report that sialic acid loss determines platelet circulatory lifespan by triggering platelet removal by the hepatic AMR. Binding of desialylated platelets to the AMR induces hepatic TPO gene transcription and translation, thereby regulating platelet production. This conclusion is based on the following results. First, desialylated platelet uptake by the AMR regulates hepatic TPO mRNA expression in situ, as evidenced by increased (~50%) hepatic TPO mRNA levels in St3gal4^-/- mice and reduced (~50%) hepatic TPO mRNA levels in Asgr2^-/- mice lacking the AMR ASGPR2 subunit, relative to controls. Second, injection of endogenously (isolated from St3gal4^-/- and Asgr2^-/- mice) and exogenously (sialidase-treated) desialylated mouse platelets into wild-type mice stimulates hepatic TPO mRNA expression by ~50% after 12h and subsequently increases plasma TPO levels and blood platelet counts. In contrast, desialylated platelets injected into Asgr2^-/- mice have no effect on hepatic TPO mRNA content, plasma TPO levels and blood platelet counts. Third, incubation of desialylated human platelets with the hepatoma cell line HepG2 increases HepG2 TPO mRNA content by approximately three-fold after six hours in vitro. Recognition of this previously unrecognized physiological feedback mechanism illuminates a novel platelet-dependent regulatory mechanism for hepatic TPO production.