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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Exp Hematol. 2010 Feb 24;38(5):384–391. doi: 10.1016/j.exphem.2010.02.007

F104S c-Mpl responds to a transmembrane domain-binding thrombopoietin receptor agonist: Proof of concept that selected receptor mutations in congenital amegakaryocytic thrombocytopenia can be stimulated with alternative thrombopoietic agents

Norma E Fox 1,1, Jihyang Lim 1,1, Rose Chen 1, Amy E Geddis 1,*
PMCID: PMC2862996  NIHMSID: NIHMS182070  PMID: 20188141

Abstract

Objective

To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists.

Methods

We created cell line models of type II c-Mpl mutations identified in CAMT. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing WT or F104S c-Mpl in response to thrombopoetin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD 4665) domains of the receptor by MTT assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species.

Results

Cells expressing F104S c-Mpl proliferated in response to LGD 4665 but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD 4665 stimulates signaling with delayed kinetics in both WT and F104S c-Mpl expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen bonding interactions that are critical for binding to thrombopoietin.

Conclusions

These findings suggest that a small subset of patients with CAMT might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin.

Introduction

CAMT is an inherited bone marrow failure syndrome caused by mutations in the receptor for thrombopoietin, c-Mpl ([1,2]. Affected children typically present with thrombocytopenia at birth, and evaluation of the bone marrow reveals severely reduced or absent megakaryocytes. In most cases, isolated thrombocytopenia progresses to pancytopenia due to trilineage bone marrow failure within the first decade of life and these children ultimately require stem cell transplantation. Due to the lack of receptor-mediated receptor uptake, high plasma thrombopoietin levels are characteristic of this disease [1].

Mutations have been described throughout the c-Mpl receptor, although mutations in exons 2 and 3 are the most frequent [3]. Mutations of c-Mpl have been classified as either type I, in which the receptor has lost all activity, or type II, in which the receptor retains some degree of function [1]. Clinically, type II patients have a slightly delayed onset of bone marrow failure (mean age 48 mo) compared to type I patients (22 mo) [3].

Thrombopoietin receptor agonists have been developed and are currently approved for the treatment of chronic ITP in adults [4–6]. The role of these agents in the treatment of inherited thrombocytopenia is not defined. Two types of thrombopoietin receptor agonists are currently approved for clinical use: a peptibody that interacts with the extracellular domain of the receptor (Romiplostim, Amgen)[5] and a small molecule that binds to the transmembrane region of the receptor (Eltrombopag, GlaxoSmithKline) [6]. Additional transmembrane domain binding agents are currently in development, including LGD 4665 from Ligand Pharmaceuticals [7]. None are structurally related to thrombopoietin (reviewed in [8]).

We have hypothesized that although endogenous thrombopoietin levels are already highly elevated in CAMT, there may exist type II c-Mpl mutations involving the extracellular domain of the receptor that could be stimulated by a thrombopoietic agent that interacts with the receptor differently than does native thrombopoietin. Previously, we determined that the most common type II mutation, R102P c-Mpl, cannot be stimulated by either extracellular or transmembrane domain-binding receptor agonists in a cell line model. Although R102P c-Mpl is synthesized and stable within the cell, it is not normally glycosyolated and does not reach the cell surface and is therefore inaccessible to external stimulation [9]. Here we describe a type II c-Mpl mutation, F104S c-Mpl, which is expressed on the cell surface and can be stimulated by a thrombopoietin receptor agonist. Intriguingly, cells expressing F104S do not respond to stimulation by thrombopoietin or by m-AMP4, an extracellular domain binding receptor agonist, but they do respond to LGD 4665, which binds within the transmembrane domain. This work suggests that a small subset of patients with CAMT and type II c-Mpl mutations might be responsive to thrombopoietin receptor agonists, but that drugs that bind to the extracellular and transmembrane domain of the receptor are not interchangeable.

Methods

Materials and DNA constructs

The cDNA for human c-Mpl was cloned into pMx-Puro (the gift of Toshio Kitamura [10]); F104S, W154R, R257C and R257L mutations were introduced into the human c-Mpl cDNA using Quikchange XL-Sited Directed Mutagenesis kit (Stratagene, Cedar Creek, TX) according to the manufacturer's protocol and verified by DNA sequencing. Recombinant human thrombopoietin (rhTPO) was the gift of Zymogenetics (Seattle, WA). m-AMP4 was a gift of Amgen (Thousand Oaks, CA). LGD 4665 was a gift of Ligand pharmaceuticals (La Jolla, CA).

Cell lines

BaF3 cells are an IL-3 dependent murine lymphoblastic cell line that does not express c-Mpl. Cells were maintained in RPMI (Gibco, Carlsbad, CA) containing 10% fetal bovine serum (FBS), penicillin, streptomycin, glycine, and 2 µl/ml murine IL-3 supernatant purified from transfected baby hamster kidney cells. WT or mutant c-Mpl was inserted into the vector pMx-puro and introduced into BaF3 cells by retroviral transduction; expressing cells were selected with puromycin and clonal cell lines were derived by single cell dilution. All clones were assayed for c-Mpl expression by western blotting, using polyclonal rabbit anti-Mpl (Millipore, Billerica, MA). BaF3 cells expressing R102P c-Mpl have been reported previously [9].

Total and surface expression of c-Mpl

Western blotting

BaF3 cells expressing WT or mutant c-Mpl and growing in IL-3 were lysed in NP-40 buffer (50mmM Tris HCl PH 7.4,150mM NaCl, 1 mM EDTA, 1% NP-40) containing a protease and phosphatase inhibitors (Complete Protease Inhibitor, (Roche, Basel, Switzerland), 2 mM Na3VO4 and 5 mM NaF), according to standard protocols. Protein concentrations were determined using the Protein/DC assay (Bio-Rad, Hercules, CA). For each sample, 30 µg of total protein was denatured by boiling for 10 min in loading buffer containing 30 µl/ml β-mercaptoethanol and separated on a 4–15% gradient polyacrylamide gel (Bio-Rad). Proteins were electrophoretically transferred to a PDVF membrane (Bio-Rad) which was then blocked for 1 h at room temperature with Tris-buffered saline (pH 8.0) containing 0.1% Tween-20 (TBST) and 5% nonfat dry milk. The membrane was incubated overnight at 4 °C in TBST with 5% milk and anti-c-Mpl antibody (1:3000). After washing, the membrane was incubated in TBST with 5% milk containing a 1:3000 dilution of secondary antibody conjugated to horseradish peroxidase (goat anti-rabbit, Santa Cruz Biotechnologies, Santa Cruz, CA). Secondary detection was performed with ECL-plus (Amersham, Piscataway, NJ) and blots were exposed to autoradiographic film.

Flow cytometry

Parental BaF3 cells and BaF3 cells expressing WT or mutant c-Mpl growing in IL-3 were washed in PBS/0.5% bovine serum albumin (BSA) then incubated with a 1:100 dilution of mouse monoclonal antibody specific for the extracellular domain of c-Mpl (gift from Amgen) in PBS/0.5% BSA for 30 min at 4°C. After washing, cells were incubated with secondary antibody conjugated to Alexafluor 488 (Invitrogen, Carlsbad, CA) for 30 min at 4°C, then fixed in phosphate buffered saline (PBS) containing 2% paraformaldehyde prior to analysis. Relative fluorescence was assayed by flow cytometry using CellQuest software (Becton Dickinson, Franklin Lakes, NJ). Background antibody staining was taken as that present in parental BaF3 cells. Relative surface expression was measured as the difference in geometric mean intensity of fluorescence compared to that in parental Baf3 cells. Three independent clones were analyzed for surface expression of F104S c-Mpl.

Assays for response to rhTPO and thrombopoietin mimetics

MTT assays

Parental BaF3 cells and BaF3 cells expressing WT or mutant c-Mpl were washed twice with PBS to remove IL-3, then transferred to RPMI containing 10% fetal bovine serum alone or with 5 ng/ml of rhTPO, 3 µM LGD 4665, or 50 ng/ml m-AMP4. Thrombopoietin responsiveness was defined as dose-dependent cell growth at 72 hr and measured by MTT assay as previously described [11]. Cell growth in optimal concentrations of IL-3 was simultaneously determined for comparison.

Phosphorylation of Erk, Akt and Stat5

To detect activation of c-Mpl by LGD 4665 or rhTPO, BaF3 cells expressing WT or mutant c-Mpl were washed twice, starved of serum and cytokines overnight in RPMI containing 0.5% BSA, then stimulated for 0–120 min with 1.5 µM LGD 4665 or 5 ng/ml rhTPO. Cells were lysed and analyzed by western blotting using anti-phospho p42/p44 Erk, anti-phospho-S473Akt and anti-phospho STAT5 antibodies (Cell Signaling Technologies, Beverly, MA). Blots were reprobed for β actin as a loading control.

Internalization of c-Mpl

BaF3 expressing WT c-Mpl cells growing with IL-3 were harvested in log phase, washed and resuspended at 2.5×105 cells/ml in RPMI medium supplemented with 10% FBS. Two ml of cell suspension was plated in wells of 6 well plates and the plates equilibrated at 37 °C, before adding either rhTPO (5 ng/ml)or LGD 4665 (1.5 µM) to the wells. At time points ranging from 0–120 min, cells were washed in ice cold PBS to stop receptor internalization, and pellets were incubated in 100 µl of PBS/0.5% BSA containing 1 µg/ml anti c-Mpl monoclonal antibody followed by secondary detection with 2 µg/ml secondary antibody conjugated to Alexafluor 488. Cells were washed and fixed in 2% paraformaldehyde prior to analysis. Exposed surface receptor was measured by flow cytometry and expressed as a percentage of mean fluorescence intensity detected prior to stimulation.

Thrombopoietin uptake assay

To measure the ability of WT or mutant c-Mpl to bind thrombopoietin, we determined if cells bearing WT or mutant receptors could effectively remove rhTPO from the cell culture medium. Wells were set up containing 0 or 2×107 BaF3 cells expressing WT or mutant c-Mpl and incubated at 37°C in culture medium containing 0.75 ng/ml rhTPO. As a control for non-specific uptake or degradation, additional wells were prepared containing parental BaF3 cells and no cells. After 30 min, samples were placed on ice to stop uptake and then cells were removed by centrifugation and the amount of rhTPO remaining in the medium was determined using TPO ELISA (Quantikine for human TPO, R&D, Minneapolis, MN).

Results

Screening of type II mutants for potential surface expression

We had previously found that a relatively common mutation found in CAMT, R102P c-Mpl, results in a receptor that is not expressed on the cell surface. Therefore, we reasoned that if we screened additional c-Mpl mutants for cell surface expression we could select the best candidates for further testing to determine if any mutation could be stimulated by a thrombopoietin mimetic. We created a panel of cell lines expressing type II c-Mpl mutations as described in the literature and made protein lysates of each for evaluation by western blotting. WT c-Mpl usually migrates on western blotting as two bands, a slowly migrating band around 85 kDa that represents mature, glycosylated receptor and a faster migrating band around 80 kDa that represents immature receptor. The presence of glycosylated receptor correlates with cell surface expression (manuscript in preparation). When we analyzed our panel of type II c-Mpl mutants, only F104S c-Mpl showed a glycosylated band and therefore this was suggestive of surface expression and the potential for response to our thrombopoietin receptor agonists (Figure 1).

Figure 1.

Figure 1

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BaF3 cells expressing F104S c-Mpl make a glycosylated receptor. BaF3 cells were transfected with WT, F104S, R257C, R257L, W154R or R102P c-Mpl and selected with puromycin. Whole cell lysates were probed for expression of c-Mpl by immunoblotting. F104S is the only mutant that demonstrates the WT-pattern of receptor glycosylation with expression of the 85 kD form. The WT receptor on this blot is shown at a longer exposure to make up for differences in expression levels between cell lines.

To confirm that F104S c-Mpl is expressed on the cell surface, we evaluated receptor expression by flow cytometry in parental BaF3 cells and BaF3 cells expressing WT c-Mpl or F104S c-Mpl. For these experiments, we used a monoclonal antibody developed by Amgen that recognizes the extracellular domain of c-Mpl. In our hands, the commercially available antibodies we have tried have not been useful for detection of c-Mpl in flow cytometry. As we would have predicted by its glycosylation pattern, F104S c-Mpl is expressed on the cell surface (Figure 2). Therefore, F104S c-Mpl represents a good candidate to examine for responsiveness to thrombopoietin receptor agonists. We generated clonal cell lines for further studies.

Figure 2.

Figure 2

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BaF3 cells express F104S c-Mpl on the cell surface. BaF3 cells alone or expressing WT or F104S c-Mpl were fixed and incubated with primary antibody to the extracellular domain of c-Mpl, followed by secondary antibody conjugated to Alexafluor 488. Both BaF3 cells expressing WT and F104S c-Mpl show significant reactivity compared to parental BaF3 cells that do not express c-Mpl. Results are representative of 3 different clones.

Cells expressing F104S c-Mpl do not proliferate in response to rhTPO or an extracellular domain-binding receptor agonist but do respond to a transmembrane domain-binding drug

To determine if F104S c-Mpl could be stimulated by thrombopoietin or a thrombopoietin receptor agonist, we treated BaF3 cells expressing either WT or F104S c-Mpl with rhTPO, m-AMP4 or LGD 4665 and analyzed their growth response. We washed cells from 3 F104S c-Mpl clones and 2 WT controls of IL-3, then cultured them with of maximal doses of rhTPO 5 ng/ml), m-AMP4 (50 ng/ml), LGD 4665 (3 µM) or IL-3 and measured cell growth at 72 hr using an MTT assay. Cell growth was normalized to maximal growth in IL-3 to account for intrinsic differences in growth potential for individual cell lines. Although cells expressing the WT receptor grew well in all of the thrombopoietic agents, cells expressing F104S c-Mpl only demonstrated significant growth in LGD 4665 and did not grow in rhTPO or m-AMP4 (Figure 3A). We then performed a dose response curve using LGD 4665 in cell lines expressing either WT or F104S c-Mpl. We found that although there is significant clone-to-clone variability, both WT and F104S c-Mpl demonstrated dose-dependent growth in response to LGD 4665, with maximal response seen at approximately 1.5 µM (Figure 3B). This is comparable to findings in primary cells, in which LGD-4665 stimulated the formation of polyploid, CD41+ megakaryocytes from purified human bone marrow CD34+ progenitor cells with an EC50 ≈ 0.07 µM and a maximum response at 1 µM (personal communication, M. Meglasson, August 2009). As expected, parental BaF3 cells, which do not express c-Mpl, do not respond to LGD 4665, indicating that response requires the thrombopoietin receptor.

Figure 3.

Figure 3

Figure 3

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BaF3 cells expressing F104S c-Mpl grow in response to LGD 4665 but not to rhTPO or m-AMP4. (A) Three independent clones of BaF3 cells expressing F104S c-Mpl were washed of IL-3 and tested for growth in maximal IL-3, 5 ng/ml rhTPO, 50 ng/ml m-AMP4 or 3 µM LGD 4665. After 72 h, cells were lysed and an MTT assay was performed. Results are expressed relative to maximal growth in IL-3. Points were performed in triplicate. Error bars represent SEM. Growth of 2 clones expressing WT c-Mpl under the same conditions is shown for comparison. (B) Dose dependent growth of WT and F104S c-Mpl in response to LGD 4665. Two clones for each receptor type were studied, selecting the clones exhibiting the best response to LGD 4665 from Figure 3A. Cells were washed of IL-3 and cultured with increasing doses of LGD 4665 from 0- 3 µM for 72 hr and growth was measured by MTT assay. Values are expressed as percentage of maximal growth in IL-3. Points were performed in triplicate and error bars indicate SEM. The growth curves shown are representative of 3 independent assays.

To further evaluate the function of the mutant receptor, we starved BaF3 cells expressing WT or F014S c-Mpl of serum and IL-3 overnight, and then stimulated them with rhTPO or LGD 4665 for 0–120 min and probed for phosphorylation of Erk, Akt and Stat5 by immunoblotting. A concentration of 1.5 µM LGD 4665 was chosen as this yielded optimal growth in the MTT assay. As expected, in cells expressing WT receptor rhTPO stimulation resulted in strong phosphorylation and activation of Erk, Akt and Stat5 within 15 min. In comparison, the addition of LGD 4665 resulted in phosphorylation of these pathways with delayed kinetics, with onset only after 60 min and maximal activation at 120 min (Figure 4A). There was no apparent difference in signaling intensity or signaling kinetics in cells expressing F1014S or WT c-Mpl treated with LGD 4665. To further investigate this difference in the onset of signaling, we examined receptor internalization of WT c-Mpl in cells stimulated with either rhTPO or LGD 4665. We have previously shown that c-Mpl is rapidly internalized in response to TPO, reaching maximal internalization within 30 min of stimulation. We found that, despite prolonged incubation times up to 120 min, WT c-Mpl is not internalized after stimulation by LGD 4665 (Figure 4B). These data suggest that the temporal and spatial mechanisms of signaling by TPO and LGD 4665 are different.

Figure 4.

Figure 4

Figure 4

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BaF3 cells expressing WT or F104S c-Mpl demonstrate delayed signaling in response to LGD 4665 compared to WT cells stimulated with rhTPO. (A) BaF3 cells expressing F104S or WT c-Mpl were starved overnight and then stimulated with 1.5 µM LGD 4665 or 5 ng/ml rhTPO for 0–120 min, lysed and assayed for phosphorylation of Erk, Akt and Stat5 by immunoblotting. Blots were reprobed for β actin as a loading control. Blot shown is representative of 3 independent experiments. (B) Internalization of c-Mpl following stimulation with LGD 4665 is delayed compared to cells stimulated with rhTPO. BaF3 cells expressing WT c-Mpl were washed of IL-3 and treated with 5 ng/ml rhTPO or 1.5 µM LGD 4665 for 0–120 minutes, then incubated on ice with anti-c-Mpl monoclonal antibody and Alexafluor 488-conjugated secondary antibody. Residual surface expressed c-Mpl was measured by flow cytometry. Results are expressed as a percentage of mean fluorescence intensity detected prior to stimulation. A representative of 4 experiments is shown.

F104S c-Mpl does not bind thrombopoietin as efficiently as WT c-Mpl

Because rhTPO did not stimulate cells expressing F104S c-Mpl, we asked if the mutant receptor was capable of binding thrombopoietin. When thrombopoietin binds to cells expressing normal c-Mpl, it is internalized with the receptor through the process of endocytosis [12,13] and thrombopoietin is removed from the plasma. We used this phenomenon to compare the binding of rhTPO to WT or mutant c-Mpl in cultured cells by measuring the depletion of rhTPO from the medium. Equal numbers of parental BaF3 cells, BaF3 cells expressing WT receptor or 2 clones of BaF3 cells expressing c-Mpl F104S were cultured with media containing 0.75 ng/ml of rhTPO, and rhTPO levels were measured using an ELISA assay both prior to the addition of cells and following 30 min incubation at 37°C. We found that compared to BaF3 cells expressing WT receptor, which effectively removed rhTPO from the media, cells expressing F104S c-Mpl did not take up any more rhTPO than did parental BaF3 cells (Figure 5). Although these measurements are relative, this result suggests that the mutant receptor is unable to efficiently bind thrombopoietin.

Figure 5.

Figure 5

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F104S c-Mpl does not bind TPO as efficiently as WT c-Mpl. To compare the ability of mutant and WT receptors to bind TPO, we incubated BaF3 parental cells or BaF3 cells expressing WT c-Mpl or F104S c-Mpl in medium containing fixed amounts of rhTPO and measured the ability of the cells to deplete the cytokine from the culture medium. 2×107 cells were washed of IL-3 and cultured with 0.75 ng/ml rhTPO at 37°C for 30 min. TPO levels were measured by ELISA in the input medium as well as after 30 min incubation with cells, and are expressed as percentage of input TPO remaining. Results shown are the mean of three independent experiments performed on 2 separate clones for each receptor type, error bars represent SEM.

Discussion

At the present time, platelet transfusion and bone marrow transplant represents the only treatments for children with CAMT. Although thrombopoietin receptor agonists are approved for the treatment of thrombocytopenia in chronic immune thrombocytopenia, the role for these agents in inherited disease is not defined. Most patients with CAMT do not express a functional thrombopoietin receptor and are therefore unable to respond to thrombopoietin or a thrombopoietin mimetic. However, thrombopoietin receptor agonists that bind to c-Mpl at a site distinct from thrombopoietin could potentially stimulate a receptor with an extracellular domain mutation. Here, we provide proof of concept that a previously described CAMT mutation, F104S c-Mpl, can be stimulated by a thrombopoietin receptor agonist in a cell line model.

In contrast to the extracellular domain mutation we examined in our prior work (R102P c-Mpl), the substitution F104S does not prevent transport of the receptor to the surface of the cell, making it a candidate for stimulation. Nevertheless, cells expressing F104S c-Mpl are unable to bind rhTPO and do not grow in either rhTPO or m-AMP4. They are however stimulated at least partially by LGD 4665. Although LGD 4665 and hrTPO activate similar signaling pathways in our cell line model of BaF3 cells expressing the WT c-Mpl receptor, the kinetics of signaling in response to the two agents are different. Intriguingly, we found that c-Mpl internalization following stimulation with LGD 4665 is also significantly reduced as compared to rhTPO. In fact, in most experiments we saw an increase in surface expressed c-Mpl over time. We speculate that this may be due to trapping of additional receptor as it is transported to the cell surface without subsequent internalization. In separate work, we have created cell line models in which c-Mpl internalization is altered. For example, mutation of intracellular domain tyrosine 591 (Y591) impairs c-Mpl internalization in response to TPO; however, in this case signaling occurs with normal kinetics and is relatively increased compared to the WT receptor [13]. It is possible Y591 has functions in addition to regulation of receptor internalization which confound interpretation of these results. An alternate interpretation of our data is that impaired receptor internalization following stimulation with LGD 4665 is due to a slower onset of signaling; however, this seems unlikely as no c-Mpl internalization is demonstrable even at 120 minutes when signaling with LGD 4665 is similar in intensity to that seen with rhTPO. The reasons behind these differences in signaling kinetics between rhTPO and LGD 4665 and their impact on cellular responses are not known but are the subject of ongoing research.

We postulate that a site important for the interaction of both m-Amp4 and thrombopoietin with c-Mpl is disrupted by the mutation F104S. F104 lies within the conserved erythropoietin receptor ligand binding homology domain of c-Mpl. Alignment of c-Mpl and erythropoietin receptor sequences from multiple species indicates that phenylalanine is highly conserved at this position (Figure 6). Although c-Mpl has not been crystallized, the corresponding phenylalanine in the erythropoietin receptor is predicted to be involved in critical non-polar interactions with its ligand [14], and mutation of this residue of the erythropoietin receptor results in loss of ligand binding activity [15,16]. In F104S c-Mpl, the conformation of the transmembrane domain appears to be retained as the mutant receptor can still be stimulated by LGD 4665.

Figure 6.

Figure 6

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Sequence alignment reveals conservation of F104 in c-Mpl and erythropoietin receptors of multiple species. Alignment of this region within the erythropoietin receptor ligand binding domain of different receptors was performed using the NCBI website. The EpoR ligand binding protein crystallized by Syed et al. [14] served as the reference sequence. The conserved phenylalanines are highlighted in red. Although c-Mpl from zebrafish bears a tyrosine at this position, the hydrophobic nature of this residue is conserved.

We have now created cell line models to examine 5 reported type II c-Mpl mutations and have identified only one that yields a receptor that is expressed on the cell surface. In addition, F104S c-Mpl has been reported in only one patient [17], but collection of patients with clinical findings of CAMT and careful genotyping would help to identify additional patients who might respond to a thrombopoietin receptor agonist. Nevertheless, it is likely that only a small subset of patients with CAMT could be treated in this way. In this particular case, we found that LGD 4665 but not m-AMP4 was effective in stimulating the mutant receptor in our cell line model. We predict that other small molecule agonists that bind to the transmembrane domain of c-Mpl would also be effective in stimulating F104S c-Mpl. However, different receptor mutants may have unique requirements for stimulation. Multiple thrombopoietin receptor agonists that interact at different sites on the receptor have been reported [8,18–20] and it is possible that individual patients may respond to one and not others of these agents. Novel treatment strategies are especially important for patients with CAMT who lack a matched sibling donor, as transplant outcomes remain inferior in this setting.

Acknowledgments

The authors would like to thank Dr. Kenneth Kaushansky for advice and critical review of the manuscript and Monica Gudea for administrative support. This work was supported with funds provided by NIH grant R01 DK049855-15. AEG has received prior research support from Amgen.

Footnotes

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Conflict of interest disclosure:

No financial interest/relationships with financial interest relating to the topic of this article have been declared.

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