The molecular mechanisms that control thrombopoiesis

Abstract

Our understanding of thrombopoiesis--the formation of blood platelets--has improved greatly in the last decade, with the cloning and characterization of thrombopoietin, the primary regulator of this process. Thrombopoietin affects nearly all aspects of platelet production, from self-renewal and expansion of HSCs, through stimulation of the proliferation of megakaryocyte progenitor cells, to support of the maturation of these cells into platelet-producing cells. The molecular and cellular mechanisms through which thrombopoietin affects platelet production provide new insights into the interplay between intrinsic and extrinsic influences on hematopoiesis and highlight new opportunities to translate basic biology into clinical advances.

Figures

Figure 1

The regulation of thrombopoietin levels. A steady-state amount of hepatic thrombopoietin (TPO) is regulated by platelet c-Mpl receptor–mediated uptake and destruction of the hormone. Hepatic production of the hormone is depicted. Upon binding to platelet c-Mpl receptors, the hormone is removed from the circulation and destroyed, which reduces blood levels. In the presence of inflammation, IL-6 is released from macrophages and, through TNF-α stimulation, from fibroblasts and circulates to the liver to enhance thrombopoietin production. Thrombocytopenia also leads to enhanced marrow stromal cell production of thrombopoietin, although the molecular mediator(s) of this effect is not yet completely understood.

Figure 2

Hematopoietic cytokine receptor architecture and mechanism of initial signaling. A stylized hematopoietic cytokine receptor is shown, depicting the 1 or 2 cytokine receptor motifs (C, Cys; WS, Trp-Ser-Xaa-Trp-Ser), the transmembrane domain, and the box1 sequence to which JAK kinases bind. Also shown are the 3 major domains of JAK kinases, the FERM domain, which binds to box1, and the kinase JH1 and regulatory JH2 domains. Finally, upon JAK activation, the site of receptor tyrosine phosphorylation is shown, which then serves as a docking site for STATs and adapter proteins (SHC or SHP2).

Figure 3

Signaling pathways activated by thrombopoietin. A stylized drawing of c-Mpl is shown in the activated (phosphorylated) form. Once phosphorylated, Tyr112 serves as a docking site for STAT3 and STAT5, both activated by thrombopoietin in megakaryocytes, which leads to production of Bcl-xL, among other antiapoptotic and pro-proliferative signaling molecules. The same site also serves to recruit SHC, which in turn recruits Grb2 and SOS (the latter a guanine nucleotide exchange factor for Ras), exchanging GTP for GDP, and thereby activating Ras. In succession, a MAPKKK (MAPK kinase kinase, e.g., Raf), a MAPKK (MAPK kinase), and the MAPK ERK1/2 or p38 MAPK are recruited and activated. As shown, Raf activation also contributes to PI3K activation. At a site proximal to Tyr112, a complex containing the phosphatase SHP2, the adapter protein Gab1, and the regulatory subunit of PI3K (p85) forms upon phosphorylation by JAK2, which recruits the kinase subunit of PI3K (p110), leading to phosphorylation of cell membrane–bound phosphoinositol4,5 biphosphate (PIP2) and thus generating phosphoinositol3,4,5 triphosphate (PIP3). PIP3 then recruits pleckstrin homology domain–containing proteins, including the Ser/Thr protein tyrosine kinase Akt. Once activated at the cell membrane, Akt phosphorylates (and inactivates) GSK3β, which also promotes cell proliferation. Akt also phosphorylates the transcription factor FOXO3a, leading to its nuclear exit and thus precluding its induction of the cell cycle inhibitor p27. Inhibition of cell signaling is also initiated by JAK activation; shown in red is the transcriptional regulation of SOCS proteins by STATs, and their subsequent blockade of signaling by preclusion of signaling molecule docking to P-Tyr residues of the receptor or their JAK-induced phosphorylation.

Figure 4

A molecular model of JAK2 JH1 and JH2 domains. Based on the tertiary structure of the dimer receptor tyrosine kinase FGF receptor-4, the model depicts the ATP-binding site (yellow), the kinase active site (orange), the activation loop of JH1 in both inactive (purple) and active (red) conformations, and the location of JH2 residue Val617 (V617). Adapted with permission from Protein Engineering (66).

Figure 5

Genetic alterations in thrombopoietin that lead to enhanced translation efficiency. The normal thrombopoietin mRNA (light blue) is spliced from 7 exons, of which 3 are shown (A). The numbered initiation codons found in the primary thrombopoietin transcript are shown as within their corresponding ORFs (e.g., the thrombopoietin ORF is dark blue and initiates from AUG8). The sites of mutation that lead to enhanced translation of the thrombopoietin transcript do so (B) by eliminating exon 3 by altered splicing (ΔE3) to create a new thrombopoietin ORF initiated by a highly efficient initiation codon (AUG5); (C) by nonsense mutation, prematurely truncating ORF7, which embeds the normal thrombopoietin ORF; or (D) by shifting the efficiently initiated ORF7 (by a single-nucleotide insertion) to now include the thrombopoietin polypeptide. Adapted with permission from Blood (82).