Elevating TPO production up a Notch

In this issue of Blood, Sun et al identify hepatic Notch 1 and platelet Delta-like 4 (DLL4) as key regulators of thrombopoietin (TPO) production downstream of the Ashwell-Morell receptor (AMR) or asialoglycoprotein receptor (ASGR).¹ 

Circulating blood platelets primarily function in hemostasis, initiating thrombus formation following vascular injury. Additionally, platelets participate in antimicrobial host defense, secrete cytokines, inducing inflammation, and release growth factors, aiding tissue repair. Tight regulation of platelet production is essential to prevent spontaneous bleeding if counts are low and to avoid arterial occlusion and organ damage if counts are high. The cytokine TPO, primarily produced by hepatocytes, is the primary regulator of platelet production, supporting hematopoietic stem cells and the proliferation and differentiation of bone marrow megakaryocytes responsible for generating platelets. Although the molecular mechanisms regulating circulating TPO depletion by platelets and megakaryocytes via receptor-mediated endocytosis and lysosomal degradation are well characterized, those regulating hepatic TPO production remain debated.² 

Early studies have unequivocally demonstrated that interleukin 6 (IL-6) is a potent stimulator of hepatic TPO mRNA expression, leading to a marked increase in platelet production during reactive inflammatory thrombocytosis, epithelial ovarian cancer, or selective liver irradiation. These findings provided a regulated pathway to increase platelet production during acute inflammatory responses. However, the physiological ligand-receptor pair regulating steady-state hepatic TPO mRNA production had remained elusive until 2015 when we demonstrated that desialylated platelets regulate TPO production via JAK2-STAT3 signaling by binding to the hepatic AMR.³ 

Platelets circulating within the bloodstream undergo changes to their surface glycoproteins as they age. Specifically, glycans associated with these proteins lose sialic acid, which exposes underlying galactose residues. This triggers the binding of desialylated platelets to the AMR (see figure), resulting in phosphorylation and activation of the tyrosine kinase JAK2 and phosphorylation and translocation into the nucleus of the transcription factor STAT3.³ This mechanism bears similarities with the IL-6 signaling cascade, where binding of IL-6 to its hepatic receptor IL-6R engages the signal-transducing subunit gp130, leading to the tyrosine phosphorylation of STAT3 by gp130-associated JAK1. However, the direct association of JAK2 or STAT3 with the AMR or their engagement with other signaling components remained uncertain until Sun et al identified Notch 1 as the missing link.

The Notch signaling pathway plays a crucial role in determining cell fate decisions and functions during various developmental stages.⁴ In mammals, 4 types of Notch receptors (Notch 1-4) and 5 types of ligands grouped into DLL (1, 3, and 4) and Jagged (1 and 2) families have been identified. Sun et al reveal that Notch 1 deletion in mouse hepatocytes significantly reduces plasma TPO and hepatic TPO mRNA levels, leading to decreased circulating platelet count and impaired megakaryocyte differentiation and maturation.¹ However, the overall liver ultrastructure and the basic function of hepatocytes remain unaffected. Importantly, these effects are reversible with exogenous TPO. The findings indicate that hepatic Notch 1 primarily influences the production of TPO, rather than affecting the overall health and functionality of liver cells. Additionally, Notch 1 deficiency inhibits JAK2 and STAT3 phosphorylation, further implicating Notch 1 in hepatic TPO production regulation. The study demonstrates that HES5, a gene targeted by Notch 1 signaling, facilitates the interaction between Notch 1 and TPO production by binding to phosphorylated JAK2 and STAT3 (see figure). This is supported by decreased HES5 expression in Notch 1–deficient hepatocytes.

An exciting aspect of the article is the newly identified physical association between Notch 1 and the AMR subunit ASGR1 (see figure).¹ The AMR is a heterotrimer composed of 2 ASGR1 and 1 ASGR2 subunits.⁵ ASGR1 and ASGR2 transcripts are present in different cell types, including Kupffer cells, the tissue-resident macrophages of the liver. However, the functional AMR heterotrimer is only expressed in hepatocytes. The AMR plays a crucial role in the internalization and lysosomal degradation of exogenous asialoglycoproteins. In humans, ASGR1 haploinsufficiency also associates with reduced levels of non–high-density lipoprotein cholesterol and a reduced risk of coronary artery disease.⁶ The interaction between Notch 1 and ASGR1 is crucial for proper Notch 1 signaling. AMR blockade disrupts Notch 1 signaling, which reduces the ability of hepatocytes to produce TPO.¹ The findings highlight the specialized role of the AMR and Notch 1 in regulating hepatic TPO production to ensure a steady-state megakaryocyte and platelet formation program.

Sun et al also demonstrates that DLL4, the Notch 1 ligand on desialylated platelets, activates hepatic Notch 1 signaling to increase TPO production (see figure). Blocking DLL4 inhibits Notch 1 activation, HES5 expression, JAK2 and STAT3 phosphorylation, TPO production, and subsequent platelet count increase. The findings prompt questions about the relationship between DLL4 and the von Willebrand factor receptor subunit glycoprotein (GP)Ibα on desialylated platelets, as previous studies have shown a critical role for GPIbα in hepatic TPO production.⁷ It remains unclear whether DLL4 is expressed normally on the surface of platelets in the absence of GPIbα or whether DLL4 itself undergoes desialylation during platelet aging.

Additionally, the mechanisms by which desialylated platelets physically encounter hepatocytes in vivo remain unclear. Although we and others have observed ingested platelet markers inside hepatocytes,^8,9 how platelets cross the liver sinusoidal endothelial fenestrae is uncertain. Sun et al suggest that desialylated platelets reach hepatocytes by extending finger-like filopodia. Studies have proposed that Kupffer cells contribute to platelet clearance via the integrin αMβ2 (Mac-1, CD11b/CD18), the C-type lectin 4F, or the macrophage galactose lectin.² One elegant but unexplored hypothesis is that binding of desialylated platelets to Kupffer cells leads to platelet microvesiculation, and that platelet microvesicles, rather than platelets, encounter hepatocytes in vivo.

In summary, Sun et al reveal a crucial regulatory role of Notch 1 in the hepatic TPO production downstream of the AMR. This discovery highlights the potential of targeting Notch 1 signaling to modulate TPO levels, providing a promising therapeutic option.

Conflict-of-interest disclosure: The authors declare no competing financial interests.