Megakaryocyte contribution to bone marrow fibrosis: many arrows in the quiver
Balduini

Megakaryocyte Contribution to Bone Marrow Fibrosis: many Arrows in the Quiver

Alessandro Malara1-2, Vittorio Abbonante1-2, Maria Zingariello3, Annarita Migliaccio4 and Alessandra Balduini1,2,5.

1 Department of Molecular Medicine, University of Pavia, Pavia, Italy.
2 Biotechnology Research Laboratories, IRCCS San Matteo Foundation, Pavia, Italy.
3 Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, University Campus Bio-Medico, Rome, Italy
4 Department of Biomedical and Neuromotorial Sciences, Alma Mater University, Bologna, Italy.
5 Department of Biomedical Engineering, Tufts University, Medford, MA, USA

Correspondence to: Prof. Alessandra Balduini, MD, Department of Molecular Medicine, University of Pavia, Pavia, Italy. Tel: +39 0382 502968. E-mail: alessandra.balduini@unipv.it; Prof. Annarita Migliaccio, PhD, Department of Biomedical and Neuromotorial Sciences, Alma Mater University, Bologna, Italy. Tel: +39 051 2091547. E-mail: annarita.migliaccio@unibo.it

Published: November 1, 2018
Received: August 15, 2018
Accepted: October 23, 2018
Mediterr J Hematol Infect Dis 2018, 10(1): e2018068 DOI 10.4084/MJHID.2018.068

This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(
https://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In Primary Myelofibrosis (PMF), megakaryocyte dysplasia/hyperplasia determines the release of inflammatory cytokines that, in turn, stimulate stromal cells and induce bone marrow fibrosis. The pathogenic mechanism and the cells responsible for progression to bone marrow fibrosis in PMF are not completely understood. This review article aims to provide an overview of the crucial role of megakaryocytes in myelofibrosis by discussing the role and the altered secretion of megakaryocyte-derived soluble factors, enzymes and extracellular matrices that are known to induce bone marrow fibrosis.



Introduction

Bone marrow (BM) fibrosis is characterized by increased deposition of reticulin fibres and in some cases collagen fibres.[1] There are a number of hematologic and non-hematologic disorders that are associated with increased BM fibrosis. In particular, reticulin fibres are composed by type III collagen and may be evident in many benign situations, including autoimmune and granulomatous diseases, and different tumors, such as lymphoid neoplasms, myelodysplastic syndromes, and acute myeloid leukemia. On the contrary, collagen fibres, are composed of type I collagen and appear to be characteristic of the advanced phases of myeloproliferative neoplasms (MPN), such as primary myelofibrosis (PMF) or secondary myelofibrosis (MF) arising from a pre-existing diagnosis of polycythemia vera (PV) or essential thrombocythemia (ET).[1] The content of BM fibres in routine sections of trephine biopsies of patients is usually demonstrated by histochemical staining using silver impregnation for reticulin fibres or by trichrome stains for collagen fibres.
Recent evidence has shown that the amount of BM reticulin often exhibits no correlation to disease severity, while the presence of collagen fibres is often associated with more severe disease and a poorer prognosis.[2] The exact pathogenesis of BM fibrosis is not fully understood. Aberrant tyrosine kinase signaling is a common hallmark in MPNs and has been shown to represent a key driver of the disease. The three currently recognized driver mutations in PMF are JAK2 (Janus kinase 2; located on chromosome 9p24), CALR (calreticulin; located on chromosome 19p13.2), and MPL (myeloproliferative leukemia virus oncogene; located on chromosome 1p34). The JAK2 V617F substitution of a valine for a phenylalanine destabilizes the JH2 domain of JAK2 and causes loss of the auto-inhibitory activity of this domain.[3] The most common MPL mutations, W515L (tryptophan-to-leucine substitution) and W515K (tryptophan-to-lysine substitution), cause both cytokine-independent growth and hyper-TPO sensitivity.[4] JAK2V617F and MPL 515 mutations are present in about 50% and 5% of PMF cases respectively, resulting in permanent activation of the JAK/STAT signaling pathways and conferring in vitro altered response of mutated clones to thrombopoietin (TPO) and other cytokines.[3,5] In 2013 type 1 mutations (52-bp deletion) and type 2 mutations (5-bp insertion) were discovered in the Calreticulin gene. These mutations determine a constitutive activation of the MPL receptor through abnormal interaction with mutated calreticulin.[6,7]
Of the myeloproliferative disorders, PMF has the worst overall prognosis and morbidity.[8] Despite many significant advances in the treatment of this disease, many aspects of its origin and progression remain poorly understood. While recent understanding, on the pathogenic mechanisms in hematopoietic stem cells (HSCs), provides an explanation for myeloproliferation, several pieces of evidence clearly demonstrate that other processes are involved in this disease than the simple uncontrolled growth of mutant cells. In addition to BM fibrosis, the malignant stem cells exit from the BM as the disease progresses, and relocate in other hematopoietic organs, mostly the spleen and the liver.[9] This leads to the enlargement of the spleen and liver that is characteristic of this disease, causing significant morbidity.[10] In PMF the pathogenesis of myelofibrosis appears to be intimately linked with megakaryocyte (Mk) proliferation and differentiation.[11] Mks have the primarily function to generate and release platelets in close proximity of BM vasculature,[12] but they have also been shown to be involved in the control of BM homeostasis through the generation of signals that regulate HSC self-renewal and quiescence,[13,14] or differentiation of others BM cell niche, such as plasma cells[15] or osteoblasts.[16]
In PMF clusters of immature and necrotic Mks, surrounded by fibrotic areas in the BM, suggests that improper or premature release of their neat cargo of intracellular proteins unleash the uncontrolled and disseminated fibrotic reaction driven by BM stromal cells.[17] Noteworthy, Mks are very rich in cytokines, growth factors, cross-linking enzymes and extracellular matrix proteins (ECM) that are known to directly cause tissue fibrosis by stimulating stromal cells to produce collagen or that physically participate to ECM remodeling and BM scarring. Thus, here we will review what is known about the potential contribution of Mks to the onset and progression of BM fibrosis.

Megakaryocyte cargo in physiological thrombopoiesis

Mks are unique, polyploid hematopoietic cells that are found only in mammals, responsible for everyday production and release of millions of platelets into the bloodstream.[18]
Megakaryopoiesis is mainly driven by TPO, although this cytokine may be dispensable for terminal megakaryocyte maturation in vitro.[19] During the early stages of their differentiation, Mks become polyploid through repeated DNA replication and endomitotic cycles without cytokinesis.[20] At the end of maturation, the Mk cytoplasm becomes very specialized with the development of a complex system of membranes, called the demarcation membrane system (DMS), and three different types of granules including lysosomes, dense granules and 
α-granules.[20] Proteins contained in the α-granules (specialized secretory granules) can be synthesized or endocytosed.[21] As in other cells, the cell-specific proteins are synthesized by ribosomes on the rough endoplasmic reticulum and then packaged via the Golgi apparatus into nascent granules.[22] Mk α-granules are the most abundant secretory organelles and contain a large variety of adhesive proteins, such as β-thromboglobulin, CXCL4 (platelet factor 4, PF4), thrombospondin, fibronectin (FN), von Willebrand factor (vWF) and P-selectin. In addition, anti-angiogenic factors, endostatin and angiostatin, and pro-angiogenic factors such as VEGF and SDF-1α, are involved in the regulation of lymphatic system development and vascular integrity promoted by α-granule.[23,24] Mk α-granules contain additional growth factors for vascular repair, such as Platelet Derived Growth Factor (PDGF), Transforming Growth Factor-β1 (TGFβ1), Epidermal Growth Factor (EGF) and Insulin Growth Factor (IGF).[25] Furthermore, members of the metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) family, which are important factors in angiogenesis and tissue remodeling, are also stored in Mk cytoplasm.[26] In addition, these granules have been shown to contain several plasma proteins, such as fibrinogen,[27] Factor V, albumin and immunoglobulin,[28] which are not synthesized by the cell and are, therefore, endocytosed. Lastly, the pro-coagulant factors II, V, XI and XIII, high molecular weight kininogens, the anti-fibrinolytic factors plasminogen activator inhibitor-1, α2-antiplasmin and carboxypeptidase B2, anti-coagulant factors, for example, antithrombin, protein S, C1-inhibitor, TFPI and protease nexin 2 and pro-fibrinolytic proteins such as plasminogen and plasmin have been all localized in α-granules.[25]
Differently from 
α-granules, secretory lysosomes contain distinct acid hydrolases, such as beta- hexosaminidase, heparanase, elastase, and cathepsin D and E.[29] Finally, Mk dense granules store small bioactive molecules, while α-granules are predominantly protein-packed. The cargo comprises nucleotides (e.g., ADP, ATP), polyphosphates, Ca2+ and Mg2+ cations, but also neurotransmitters and hormones, such as serotonin and histamine.[30]

How do megakaryocytes target and deliver their protein cargo within bone marrow milieu? 

The constitutive in vitro secretion of both α- and dense granules-derived bioactive molecules by developing Mks has been previously demonstrated.[31,32] These functional experiments revealed that secretion of intracellular Mk products governs autocrine mechanisms that sustain cell development and platelet release.[33-36] On the contrary, the physiological in vivo relevance of these autocrine loops has been less explored. However, recent data shed new light on the in vivo involvement of Mks in maintaining BM homeostasis though the controlled release of targeted stimuli.[37] To this regard, conditional ablation of Mks in mice resulted in increased BM HSC frequency and cycling, suggesting that Mks normally restrain HSC proliferation through the production of CXCL4 and TGF-β.[13,14] Conversely, Mk production of fibroblast growth factor-1 (FGF-1) is thought to play a key role in supporting HSC and osteoblast expansion recovery following myeloablative therapy.[16] Therefore, these studies raise the fundamental question: how is Mk compound exocytosis regulated during physiologically or pathologically BM functions? Differently from Mks, platelet activation is at the heart of the control of vascular integrity.[38] During circulation, platelets are reactive to various stimuli and release the materials stored in their specific granules.[39] The extrusion of storage granules' content to the platelet's environment occurs according to regulated secretion events: movements of granules, apposition and fusion of granules and plasma membranes.[40] This 'release reaction' is a key step of primary hemostasis, but it participates also in inflammation, atherosclerosis, antimicrobial host defense, wound healing, angiogenesis, and malignancy.[41] Our current understanding of Mk/platelet secretion at the molecular level is still insufficient to explain how the careful balance between all the bioactive molecules released from granules, under certain activation conditions, is achieved. One of the hypotheses is that Mk/platelet granules are not uniform and may be differentially packaged and thus released in a segregated manner following specific stimuli.[42]
To this regard, Ma et al. observed that platelet stimulation with specific protease-activated receptor- 1 (PAR-1) or PAR-4 agonist resulted in the preferential release of VEGF or endostatin (anti- and pro-angiogenic factors, respectively).[24,43] More recently, a super-resolution immunofluorescence co-localization analysis of 15 platelet α-granule cargoes failed to confirm any functional co- clustering of these proteins.[44] Moreover, Zingariello et al. demonstrated by immunoelectron microscopy that P-selectin and von Willebrand factor (vWF) are co-localized within the same intracellular α-granules in immature Mks. The two proteins, however, are not co-localized in α- granules of mature Mks after wild type mice treatment with TPO.[45] These results suggested that P- selectin and vWF are associated in the Mk cytoplasm at early stages of maturation but that they are routed into separate anti-angiogenic or pro-angiogenic α-granule subtypes as these cells mature. Thus, the differential association of vWF and P-selectin with anti-angiogenic or pro-angiogenic factors suggest that a mechanism regulating the sequential release of different α-granule subtypes may be involved in Mk/platelet function during tissue repair.[45] Interestingly, Mks derived from the GATA-1low mice, which harbors a hypomorphic mutation that blocks Mk maturation and displays BM fibrosis, showed reduced levels of expression of vWF and displaced P-selectin on the demarcation membrane system.
Further, the loss of alpha granules within BM Mks in a mouse model of Gray Platelet Syndrome (GPS) induced a myelofibrotic phenotype.[46] In PMF patients, ultrastructural abnormalities and variety in Mk-granules were reported by Thiele et al. more than 25 years ago.[47] Platelets derived from MPN patients showed several qualitative abnormalities, including decreased alpha granules and mitochondria and also alterations of the dense and tubular canalicular system.[48] Reduced levels of ADT, ATP and serotonin content in dense granules and lower content of beta-thromboglobulin (BTG) and platelet factor 4 (PF4) in alpha granules were also described.[49,50] Thus, aberrant assembly and secretion of Mk granules represent a potential mechanism of BM fibrosis progression (Figure 1).


Figure 1 Figure 1. Schematic representation of potential mechanisms for aberrant release of Mk content in bone marrow fibrosis. 

A second intriguingly hypothesis is that intracellular Mk/platelet products may be delivered in the surrounding space by extracellular vesicles (EVs). EVs are membrane-enclosed structures of varying size (50-10,000 nm) released from cells to mediate both local and distant intercellular communication.[51] EVs of various shapes and sizes have been demonstrated in several body fluids, with substantial variation in their structure, content and function.[52] There are three main types: exosomes (50-100 nm), microparticles (200 nm-1 µm) and apoptotic bodies. Protein, lipid and RNA components contribute to cell-cell crosstalk at a short distance, in a paracrine or endocrine manner via the bloodstream. In addition, they may transfer surface receptors from one cell to another and deliver proteins, mRNA, bioactive lipids, and even whole organelles (e.g., mitochondria) into target cells.[53] It has been reported that two mechanisms used by target cells to integrate EVs are cell endocytosis and membrane fusion.[53] Recent studies of EVs in the BM have shown that these vesicles serve to regulate hematopoiesis, participate in immune cell activation, and hemostasis.[54,55] Several lines of evidence suggest that EVs are involved in regulating BM function during homeostasis and in response to injury, but also that hematological malignancies such as leukemia, multiple myeloma or viral infections can exploit EVs trafficking to reinforce tumor growth, chemotherapeutic resistance, invasion and metastasis.[56,57] Platelet-derived vesicles were first identified by electron microscopy over 50 years ago, but the definition of their features and activities have only become a major focus of interest in recent years.[58] Platelet-derived microparticles (PMPs) are released from the platelet surface and are distinguished from platelet exosomes, which are derived from endocytosis and released from multivesicular endosomes.[59] PMPs may directly stimulate other cells (e.g., hematopoietic cells, lymphocytes and endothelium),[60] transfer platelet expressed receptors (e.g., CD41 or CXCR4) to the surface of other cells,[61] and, in some situations, transfer mRNA, proteins, and even infectious particles to the target cells. Interestingly, in healthy donors, the majority of circulating CD41+ PMPs do not express surface activation marker CD62P, suggesting that they do not originate from activated platelets.[62] In a very elegant study, Flaumenhaft et al. report that a significant number of circulating CD41+ MPs in healthy individuals are derived directly from Mks.[63] Authors first demonstrated via electron microscopy of spontaneous formation of Mk-derived MPs (MkMPs) from cultured murine Mk and that these MkMPs were different from PMPs. However, a functional role for MkMPs was not revealed until a recent study, which documented a novel biological role of MkMPs that are able to induce HSC differentiation towards the Mk lineage without exogenous TPO stimulation.[64] In this paper, Jiang et al. demonstrated that MkMPs, which are distinct from Mk exosomes, target HSC with high specificity since they have no effect on other BM-resident cells, such as mesenchymal stem cells, endothelial cells or granulocytes. They showed that both endocytosis and membrane fusion were responsible for the delivery of MkMP cargo to HSCs, and that MkMPs attached to and entered HSCs preferentially through their uropods, with CD54, CD11b, CD18 and CD43 being involved in target-cell recognition.[64] Aside from the role of EVs in the Mk-HSC crosstalk in the BM under physiological conditions, we can speculate that EV trafficking may also play a distinct role in deregulated hematopoiesis during fibrotic progression (Figure 1). Interestingly, increased MP generation under high shear stress has been reported in platelets in the presence of TPO,[65] a main trigger of BM fibrosis in human and mice.

Megakaryocyte-derived pro-fibrotic cytokines in bone marrow fibrosis

In MPNs different mutations lead to myelofibrosis. The most frequent driver mutation in MPNs, JAK2 V617F, is found in 50-60% of PMF as well as 50-60% of ET, but in almost all cases of PV.[66] It is now clear that the clinical phenotype of myelofibrosis is a consequence both of primary clonal myeloproliferation and secondary inflammation, characterized by profound changes to BM stromal compartment and an atypical cytokine storm.[67] Several evidences argue for an impaired microenvironment in association with inflammation rather than one single genetic trigger: 1) MPNs (PV, ET, and PMF) are all characterized by a significant change in the cytokine production mirrored by increased plasma levels of several inflammatory cytokines (e.g., IL1, IL2, IL6, IL8, IL12, TNFα, and IFNγ), growth factors (e.g., GM-CSF, G-CSF, HGF, PDGF, and EGF), and  angiogenic factors (e.g., VEGF);[68] 2) clinical evidences that chronic inflammation is responsible of the constitutional symptoms which negatively affect the quality of life of MPN patients;[69] 3) clinical use of JAK inhibitors has confirmed that functional symptoms and splenomegaly in patients were concomitant  with a significant increase in the plasma levels of many cytokines.[70]
In PMF, Mks and monocytes are supposed to be the main source of reactive cytokines that force fibroblast proliferation, fibrotic evolution, neoangiogenesis, and osteosclerosis.[11] Several lines of evidence obtained both from studies of patients or murine models are in favor of a crucial role of Mk in myelofibrosis induction: 1) all driver mutations in MPNs result in overproduction of abnormal Mks by hyperactive Jak2/Stat signaling;[71] 2) mice bearing a human JAK2V617F gene restricted exclusively to the Mk lineage develop many of the features of MPNs;[72] 3) patients and rats treated with TPO mimetics show BM fibrosis (usually reversible);[73] 4) high and persistent TPO production by transduced hematopoietic cells in mice results in a fatal myeloproliferative disorder that has a number of features in common with human PMF;[74] 5) impaired expression of the transcription factor GATA-1, involved in erythroid and megakaryocytic differentiation, results in the development of myelofibrosis;[75] 6) patients with GPS, a rare macro- thrombocytopenia with agranular Mk/platelets, manifest myelofibrosis and splenomegaly.[76] On the contrary, contribution of monocytes is less clear. Monocytes from patients with PMF were reported to be spontaneously activated and to secrete abnormally TGF-β1,[77] however, TPO overexpression inducing BM fibrosis in NOD/SCID mice, which harbor impaired mononuclear phagocyte functions, led to controversial data.[78,79] Thus, based on the potential role of inflammation during fibrotic progression, we below describe the involvement and features of individual main Mk- derived cytokines/chemokines in the context of myelofibrosis (Table 1).

Table 1 Table 1. List of Mk-derived cytokines implicated in bone marrow fibrosis progression.

Platelet-Derived Growth Factor (PDGF). PDGF is one of the first growth factors that has been implicated in the role of Mk in development of BM fibrosis.[80] PDGF is produced by Mks in the BM and is physiologically carried to the circulation in the α-granules of the platelets to act at the site of tissue injury as a mediator of tissue repair.[81] PDGF receptors (PDGFRs) are members of the membrane tyrosin-kinase family, composed of the two subunits PDGFRα and PDGFRβ, which form homo- or heterodimers. In the context of tissue repair, the PDGF/PDGFR axis not only enhances the replication, survival, and migration of myofibroblasts but also modulates the production and release of several pro- and anti-inflammatory mediators in fibrotic diseases.[82] Interestingly, PDGF has been reported to increase the expression of the collagen cross-linking enzyme, Lysil Oxidase (LOX), which in turn, oxidizes the PDGF receptor on smooth muscle cells, fibroblasts, and Mks, enhancing the proliferation signaling from this cytokine.[83,84] Ultimately, this loop has the potential to further boost the fibrotic phenotype.[85]
Increased levels of PDGF in plasma and urine from patients with MPNs have been reported.[86,87] Further, Mks and erythroid precursors contained increased levels of immunohistochemically detectable PDGF in BM biopsies of PMF patients.[88] The expression of members of the PDGF system in BM cells derived from PMF patients has been also investigated by real-time RT-PCR.
Increased expression of PDGFs could be demonstrated to be a feature of advanced fibrosis in PMF that is not demonstrable in the pre-fibrotic phase of the disease.[88]
Differently from their ligands, up-regulation of both PDGFRs during fibrotic progression is more controversial. In normal BM PDGFRα appeared in endothelial and endosteal cells in addition to strong labeling in Mks and platelets. In contrast, PDGFRβ subunit marked perisinusoidal stromal cells and adventitial fibrocytes of the larger vessels. However, in PMF patients, Bedekovics et al. found that PDGFRβ expression closely correlates with the grade of MF, while this was not evident for PDGFRα.[90] On the contrary, Bock et al. reported a strong up-regulation of the PDGFR
α in patients with advanced myelofibrosis.[89]
Recently, the involvement of the PDGF/PDGFR axis in BM fibrosis has been definitively proven. Conditional deletion of the PDGFR-α gene and inhibition of PDGFRα by imatinib in leptin receptor+ stromal cells was shown to suppress their expansion and to ameliorate BM fibrosis in mice.[91]
Transforming Growth Factor-
β (TGF-β). Among the abnormally expressed cytokines in PMF, TGF-β1 has received attention due to its critical role in inducing fibrosis not only in BM, but also in other organs.[92] TGF-β occurs in 3 isoforms: TGFβ1, TGFβ2 and TGFβ3. TGFβ1 is the most abundant of all these isoforms and platelets, Mks and monocytes cells are sources of TGF-β production.[93] TGFβ1 is secreted as latent protein and is stored in the extracellular matrix. Reactive oxygen species, proteases, integrins and thrombospondin-1 (TSP-1), convert the inactive latent complexes to the active forms.
Once activated, TGFβ-1 induces BM fibrosis on one hand, by increasing the synthesis of types I, III and IV collagen, FN, proteoglycans and tenascin;[94] while on the other hand, by decreasing matrix degradation through down-regulation of metalloproteinases (MMPs), particularly MMP3, and up- regulation of tissue inhibitors of metalloproteinase (TIMP), particularly TIMP-1.[95] However, effects of TGF-
β1 are not restricted to the stromal compartment and TGF-β1-mediated changes to the BM niche remain to be fully elucidated. It is well known that TGF-β has direct effects on hematopoietic cells by negatively regulating granulocyte, erythroid, Mk and macrophage progenitor proliferation.[96] Further, Erba et al. showed that release and activation of TGF-β1 by Mks and platelets, forced endothelial cells from the BM microvasculature of PMF patients, and mouse model of PMF, to acquire a mesenchymal phenotype through Endothelial Mesenchymal Transition (EndMT), during the development of fibrosis.[97]
Not surprisingly, several groups reported on quantitative alterations of TGF-β and its receptors in Mk, platelets, and CD34+ progenitor cells from MPN patients and concluded that TGF-β was involved in myelofibrosis and myeloproliferation.[31,98-100] In addition to quantitative alterations, Ciaffoni et al., recently demonstrated abnormalities in TGF-β1 signaling genes in the marrow and spleen of PMF patients.[101] These alterations included genes of TGF-β1 signaling, cell cycling, Hedgehog and p53 signaling and suggested a non-canonical TGF-β1 signaling in marrow identifying, for the first time, autoimmunity as a possible cause of BM fibrosis in PMF.[101] Among the genes that predict the activation of the non-canonical TGF-β signaling, expression level of the Jun gene was increased in BM of PMF patients. Interestingly, over-expression of Jun was sufficient to induce myelofibrosis, severe fibrosis in multiple organs and steatohepatosis in mice.[102]
Moreover, the involvement of TGF-
β in in vivo mouse model has been deeply investigated. TGF-β was significantly increased in the extracellular fluid of the BM, plasma and platelet extracts in two widely MF studied mouse models, which include the TPOhigh and the GATA-1low mice.[103,104] To test directly the impact of TGF-β1 in the pathogenesis of MF, BM stem cells from homozygous TGF-β1 null (TGF-β1(-/-)) and wild-type littermates were infected with a retrovirus encoding the murine TPO protein and engrafted into lethally irradiated wild type hosts for long-term reconstitution. Differently from wild type mice, none of the mice repopulated with TGF-β1(-/-) cells showed deposition of reticulin fibres at any time during the follow-up.[105] Consistently with patient data, alterations of TGF-β1, Hedgehog, and p53 signaling pathways were identified in the BM of GATA- 1low mice model.[106] Inhibition of TGF-β1 signaling in these mice by an inhibitor of the tyrosine kinase activity of TGF-β1 receptor type I, led to restoration of normal Mk development, reduced fibrosis, neoangiogenesis, and osteogenesis in the BM.[106] Based on these consistent observations, TGF-β inhibition has become a potential therapeutic strategy to decrease BM fibrosis in MPNs and is also being investigated in several clinical and experimental scenarios.[107]
Cxcl-4 (Pf-4). Cxcl-4, (C-X-C motif) ligand 4 (CXCL4) (also known as platelet factor 4 [PF4]), is one of the most abundant protein in the α-granules of Mk/platelets (estimated micromolar concentration), together with CXCL7.[108] This 70 a.a., cationic, lysine-rich, 7.8-kDa chemokine, is mainly synthesized by Mks, and comprises 2%-3% of the releasate from agonist-activated platelets. Once secreted, CXCL4 avidly binds to glycosaminoglycans, but only a splice-variant of the human chemokine receptor CXCR3 (CXCR3B), which is not present in mice, and LDLR90 have been identified as high-affinity receptors.[109] In contrast, the specific receptor for CXCL4 has not yet been identified in mice. However, in some circumstances, CXCL4 can interact with other chemokines (e.g. CCL5) and thereby modulate their effects on target cells. A central role of platelet-derived CXCL4 was demonstrated in solid organs. In vivo, mice lacking CXCL4 are significantly protected from severe liver fibrosis, demonstrating the pro-fibrotic phenotype of this chemokine and that its effects in mice are indeed mediated by other receptors than CXCR3.[110] In addition, it was shown that CXCL4 is secreted not only by activated platelets, but also by plasmacytoid dendritic cells and fibroblasts in systemic sclerosis.[111] Thus, these studies further involve Mks/platelets to pro- inflammatory and pro-fibrotic programmes in fibrosis. Interestingly, more than 30 years ago, Burstein et al. linked CXCL4 to myelofibrosis, by suggesting that abnormal Mks stimulate the proliferation of fibrosis-driving fibroblasts though the release of CXCL4.[112] However, no correlation was seen between BM fibrosis and plasma levels or the platelet content of CXCL4 in the same study.[112] Recently, Schneider et al. using a mouse model with genetic fate tracing in vivo, provided evidence that Gli1+ cells are key players in the initiation and progression of BM fibrosis and that Mk-derived CXCL4 was necessary and sufficient to induce the migration of Gli1+ stromal cells and their myofibroblastic differentiation.[113] In these experiments, CXCL4 was shown to induced myofibroblast differentiation of Gli1+ cells comparable to induction with TGF-β, a known stimulus for differentiation of MSCs into myofibroblasts.[113]
Other cytokines. Oncostatin M (OSM), is a pleiotropic cytokine belonging to the interleukin-6 (IL-6) family.[114] Produced mainly by activated T cells and monocytes, OSM can elicit different biological effects, depending on the cell type. OSM acts through two types of receptors. The type I OSM receptor is composed of gp130 and the leukemia inhibitory factor (LIF) receptor β-subunit (LIFR), and the type II OSM receptor is composed of gp130 and the OSM-specific receptor β- subunit (OSMR).[115] OSM has emerged as an important cytokine in the control of hematopoiesis. Transplantation experiments with OSM-deficient mice have shown that OSM stimulates stromal cells as well as hematopoietic progenitors and is required for the proper generation and maintenance of microenvironment in the BM.[116,117] Noteworthy, BM Mks express substantial amounts of OSM,[118] and OSM has been reported to behave as a megakaryocytic maturation factor in vitro and to augment platelet production in vivo.[119] Within the context of myelofibrosis, JAK2 V617F mutation promotes expression of OSM in neoplastic myeloid cells and, consequently, OSM mRNA levels are increased in the BM of patients with MPNs compared to control patients.[120] Mechanistically, OSM secreted by JAK2V617F+ cells stimulated growth of fibroblasts and endothelial cells by sustaining the production of angiogenic and pro-fibrogenic cytokines.[120]
Aberrant packaging of 
α-granule-specific proteins is supposed to trigger myelofibrosis in patients with GPS. Using a Nbeal2-/- murine model of GPS, Guerrero et al. demonstrated that BM Mks from these mice were enriched in a restricted set of chemokines transcripts, namely CCL3 and CCL4, which encode macrophage inflammatory protein (MIP) 1α and 1β, respectively, well-known pro- inflammatory chemokines increased in PMF.[46] A peculiar role for MIP 1α in sustaining osteoblasts proliferation in MPN mice model has been also proposed.[121]
Interleukin 8 (IL-8) is a member of the family of chemokines related by a CXC motif. It binds to CXC chemokine receptor 1 (CXCR1) and 2 (CXCR2).[122] It is produced by several cell types, including Mks[123] and exhibits many biological functions in inflammation, HSC proliferation and mobilization and neo-angiogenesis. Increased levels of IL-8 were found in serum[124] and plasma[68] of patients with PMF. Additionally, IL-8 and its receptors were reported to be involved in PMF-altered Mk growth.[124] Finally, rodents lack a direct homologue of IL-8, but the chemokines CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX are regarded as functional homologues of IL-8.[125]
Finally,  Mks were repeatedly identified as the main cellular source of an increasing list of cytokines, which show higher plasma levels in PMF patients, and that are individually involved in the promotion of myelofibrosis. This list further includes bone morphogenic proteins (e.g. BMP- 1[126], BMP-2, -4, and -6[127]), Lipocalin-2 (LCN-2),[128] Fibroblasts Growth Factor (FGF),[129] Vascular Endothelial Growth Factor (VEGF),[130] Secreted Phospho Protein-1 (SPP1)[131] and Thrombospondin- 1 (TSP-1).[132,133]

Megakaryocyte expression of extracellular matrices and cross-linking enzymes in bone marrow fibrosis

Deregulated extracellular matrix (ECM) dynamics in terms of amount, composition and topography is a hallmark of BM fibrosis.[1] This in turn potentiates the oncogenic effects of growth factor signaling pathways and alters cell behaviors during fibrosis progression. ECM components are not solely expressed by stromal cells, several evidences suggest that Mks may directly influence the biochemical properties and architecture of BM ECM both in physiological and pathological conditions.[134] It is known that Mks can secrete various ECM components which are supposed to sustain Mk maturation and platelet release by creating a regulatory niche within the BM environment.[135] Mks express different collagen types (e.g., III, IV), glycoproteins (e.g., Fibronectin and Thrombospondin) and proteoglycans. Interestingly, TPO has been recently recognized as a pivotal regulator of this new Mk function, by inducing TGF-β1 release and consequent activation of TGF-β downstream signaling pathways, both in vitro and in vivo.[136] This activation led to a dose dependent increase of ECM component synthesis by Mks, which was reverted upon incubation with JAK and TGF-β1 receptor specific inhibitors.[136]
In parallel with ECM secretion, Mks express several modifiers of ECM structure. Factor XIII-A is synthesized by Mks and both protein and mRNA are packaged into the cytoplasm of forming platelets.[137] Factor XIII-A belongs to transglutaminases, a class of calcium ion-dependent enzymes that catalyze an acyl transfer reaction in which y-carboxamide groups of peptide-bound glutaminyl residues are acyl donors and primary amine including the δ-amino group of peptide-bound lysyl residues, are acyl acceptors. By this reaction, transglutaminases catalyze the formation of δ-(y- glutamy1) lysine linkages between proteins. Thus, based on these properties, the potential role of FXIII-A in the BM environment may consist in the cross-link of extracellular fibrillar FN matrix with collagen.[138,139]
LOX is a copper-dependent amine oxidase that catalyzes oxidative deamination of lysine and hydroxylysine residues on collagen and elastin, leading to cross-linking within these proteins and changes in ECM elasticity. Eliades et al., detected LOX expression in diploid-tetraploid Mks, but scarce traces in polyploid Mks and identified a peculiar role for this enzyme in BM fibrosis.[84] They found that in the GATA-1low mouse model, which is characterized by increased frequency of low ploidy Mks and extensive matrix of fibres, LOX was abundantly expressed by low ploidy Mks. More importantly, administration of β-aminopropionitrile (a LOX inhibitor) to the GATA-1low mice inhibited the progression of myelofibrosis. Consistently, human platelets and Mks from patients with PMF overexpress LOX and show higher adhesion to collagen that is dependent on LOX activity.[140]
In addition to cross-linking enzymes, also ECM degradation directly impacts cell behavior and migration. Metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases and function in remodeling the ECM by its ability to degrade and cleave ECM components with wide substrate specificities.[141] Once activated, the MMPs are subject to inhibition by the tissue inhibitors of metalloproteinases (TIMPs) that bind MMPs non-covalently and counteract their proteolytic activity.
Mks synthetize several MMPs, particularly gelatinases MMP-2 and MMP-9.[142] Moreover, transcripts for MMP-1, 11, 14, 15, 17, 19, 24 and 25 have also been identified.[26] Conversely, biosynthesis of TIMPs 1-4 in Mks/platelets intervenes in excessive tissue remodeling.[143] It is suggested that BM fibrosis in PMF results from enhanced TIMP and decreased MMP activities. In particular, TIMP-1 (both the total, complex and the free form) is significantly increased in MPNs, while MMP-3 is significantly decreased, and levels of MMP-2 and MMP-9 are not different from control values.[95,144] Further, membrane type 1-MMP (MMP-14) was found overexpressed by up to 80-fold in advanced stages of fibrosis, and Mks and endothelial cells were unmasked as the major cellular source.[145] By contrast, a significantly higher expression of neutrophil collagenase (MMP-8) was encountered in the pre-fibrotic stages of PMF. Although the JAK-STAT signaling pathway is directly involved in the regulation of genes encoding MMPs, the altered expression of MMPs seem not influenced by the JAK2 mutation status but predominantly related to the stage of disease.[145]

Direct Megakaryocyte-cell interactions in the context of bone marrow fibrosis

In addition to secretory events, one more pathophysiological mechanism operating in the development of myelofibrosis is the abnormal interaction of Mks with cell components of the BM (Figure 1). Selectins (CD62L, CD62P) and Mk glycoproteins (CD41a, CD42b) were demonstrated to mediate Mk-fibroblast interactions in human BM and to increase fibroblast growth.[146]
Abnormalities in mesenchymal stem cells derived from PMF patients were reported to alter the ability of these cells to support Mk differentiation in vitro.[147-149] Further, a pathological interaction, between polymorphonuclear (PMN) leukocytes and Mk, correlated with MF development, has been also proposed.[17] Emperipolesis is the random passage of the different types of BM cells through Mk intracellular space. The phenomenon is strongly increased in BM of patients with MPN disorders.[150] Schmitt et al., first showed both in the BM of patients with PMF, and in the TPOhigh murine model, abnormal subcellular P-selectin distribution, which appeared to correlate with excessive and pathological emperipolesis of PMN leukocytes within Mk.[150] This abnormal interaction was considered the main cause of the destruction of Mk storage organelles and leakage of α-granular contents into the BM microenvironment.[151] As in patients, a similar pathologic neutrophil emperipolesis was detected in the GATA-1low mouse model of myelofibrosis.[152] In BM Mk of these mice, P-selectin, although normally expressed, was found frequently associated with the demarcation membrane system (DMS) instead of within granules. In addition, pathologic Mks were surrounded by myeloperoxidase-positive neutrophils, some of which appeared in the process to establish contact with Mks by fusing their membrane with those of the DMS. Quantification of this process revealed that 34% (in BM) of GATA-1(low) Mks contained 1 to 3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1(low) Mks displayed morphologic features compatible with those of cells dying from para-apoptosis, confirming the hypothesis that emperipolesis sustains myelofibrosis by driving the release of fibrogenic Mk cytokines and neutrophil proteases in the BM microenvironment.[152] Moreover, abnormal localization of P-selectin in Mks and platelets, induced by the GATA-1(low) mutation, was further involved in the pathological interactions of circulating platelets with leucocytes, responsible for the increased presence of thrombosis seen in these mice,[153] as well as, in the promotion of extramedullary hematopoiesis.[154] Consistently, high rate of emperipolesis is detectable in BM biopsies of patients with GPS, a rare inherited bleeding disorder characterized by deficiency of platelet α-granules, macrothrombocytopenia and marrow fibrosis.[155]

Is the pro-fibrotic role of Megakaryocytes/ platelets restricted to the bone marrow? 

New discoveries in the field of thrombopoiesis and platelet roles have revealed unprecedented features of the Mk/Platelet lineage that open new avenues in the study of these cells, particularly in diseased conditions. Bioactive mediators, stored in platelets, have been implicated in fibrotic conditions that target solid organs, rather than BM (Table 2).

Table 2 Table 2. Megakaryocytes/Platelets contribution to organ fibrosis.

A large amount of experimental evidence implies that platelets participate in the liver fibrotic process mainly by releasing pro- fibrotic mediators. Using mice carrying a Mk/platelet-specific targeted conditional deletion of the TGF-β1 gene (PF4CreTgfβ1f/f), Ghafoory et al. demonstrated that platelet TGF-β1 deficiency decreases liver fibrosis in a mouse model of carbon tetrachloride (CCl4)-induced liver injury.[156] However, there is also evidence that platelets under certain circumstances may have a protective role against liver fibrosis. To this regard, thrombocytopenic mice, with selective disruption of the anti-apoptotic gene Bcl-xL, were shown to be more prone to liver fibrosis by bile duct ligation compared to their wild type counterparts.[157] The authors, suggested that the anti-fibrotic Hepatocyte Growth Factor (HGF) released from activated platelets in liver, attenuated the expression of collagen in hepatic stellate cells, the key cell type in liver fibrosis.[157] Additionally, Mk-specific disruption of the TGF-β1 gene resulted in mice protection from cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction, suggesting that platelet profibrotic behavior is not solely restricted to the liver.[158] Similarly, evidence has been accumulated implicating platelets in the pathogenesis of interstitial lung fibrosis in several animal models. Piguet et al. found that trapping of platelets in contact with the alveolar endothelium of the lungs after bleomycin injection was increased and correlated with the deposition of collagen.[159] The authors suggested that this could represent not only a simple correlation but also a potential pathological mechanism that links platelets and pulmonary fibrosis. Interestingly, in a recent study platelets were shown to promote acute lung injury through the massive release of the Wnt/β-catenin inhibitor Dickkopf-1 (Dkk-1) from their α-granules, leading to increased expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on the surface of alveolar epithelial cells (AECs) and abnormal macrophage/neutrophils interaction with AECs.[160] In addition to the direct involvement of platelets in fibrosis of solid organs, the potential contribution of Mks to organ fibrosis still needs to be uncovered. It is becoming increasingly clear that Mks transfer unique genetic codes to platelets, and that environmental changes can alter transcriptional, translational, and post-translational processes in Mks, affecting the genetic code of platelets in circulation.[161] Change in Mk-platelet transcriptional axis is a dynamic process, especially in disease situations, and can rapidly affect the platelet repertoire of messenger RNAs (mRNAs), microRNAs (miRNAs), and proteins that contribute to their primary and alternative functions. Freishtat et al., revealed for the first time, that a de novo transcriptome is imparted to platelets by BM residing Mks during sepsis.[162] Septic Mks produce platelets with acutely altered mRNA profiles, and these platelets mediate lymphotoxicity via the potent cytotoxic serine protease, granzyme B.[162] Similarly, in the context of cancer, Mks of tumor-bearing mice endocytose circulating thrombospondin-1 (TSP-1) and increase its synthesis to produce platelets with elevated levels of TSP-1, one of the most potent angiogenesis inhibitors. These TSP-1-enriched platelets were shown to adhere to tumors and to act as potent inhibitors of angiogenesis and cancer growth.[163] Thus, similar changes may occur in fibrotic conditions, but this has not been demonstrated yet.

Conclusions

In this review, we summarized the involvement of the Mk lineage in the development of BM fibrosis. We now know that, in addition to genetic triggers, BM fibrosis is sustained by the intramedullary release of cytokines that are responsible for the abnormal activation of stromal cells, resulting in extensive deposits of reticulin and collagens. Mks are supposed to constitute the main source of these reactive cytokines. Abnormal Mk differentiation, apoptosis and emperipolesis were all proposed as major mechanisms for the enhanced release of cytokines with a fibrogenic potential. Unfortunately, mechanisms underlying Mk secretion, their relationships with other BM lineages and their functional activities in physiological conditions as well as during myelofibrosis progression, are not well understood to date. The first attempt to directly target the Mk lineage was shown, recently, to revert the disease in both Jak2V617F and MPLW515L mice models. Using a small molecule, the AURKA inhibitor MLN8237, that induce Mk polyploidization, differentiation, and subsequent apoptosis, the Crispino’s group demonstrated that the pharmacological induction of Mk maturation was beneficial in terms of reduced burden of immature Mks and amelioration of PMF features, including BM fibrosis.[164] Thus, developing drugs able to re-establish Mk normal function may represent a new strategy to treat the disease and, at the same time, to understand its pathogenic mechanisms (Figure  2).

Figure 2 Figure 2. Schematic representation of potential novel mechanisms for MPN pathogenesis.


Acknowledgments

This paper was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC IG 2016 18700, AIRC; Milano, Italy).
 
 

References   

  1. Kuter DJ, Bain B, Mufti G, Bagg A, Hasserjian RP. Bone marrow fibrosis: pathophysiology and clinical significance of increased bone marrow stromal fibres. Br J Haematol. 2007;139(3):351-362. https://doi.org/10.1111/j.1365-2141.2007.06807.x PMid:17910625
  2. Gianelli U, Fiori S, Cattaneo D, et al. Prognostic significance of a comprehensive histological evaluation of reticulin fibrosis, collagen deposition and osteosclerosis in primary myelofibrosis patients. Histopathology. 2017;71(6):897-908. https://doi.org/10.1111/his.13309 PMid:28710830
  3. James C, Ugo V, Le Couédic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434(7037):1144-1148. https://doi.org/10.1038/nature03546 PMid:15793561
  4. Pikman Y, Lee BH, Mercher T, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 2006;3(7):e270. https://doi.org/10.1371/journal.pmed.0030270 PMid:16834459 PMCid:PMC1502153
  5. Pardanani AD, Levine RL, Lasho T, et al. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood. 2006;108(10):3472-3476. https://doi.org/10.1182/blood-2006-04-018879 PMid:16868251  
  6. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369(25):2379-2390. https://doi.org/10.1056/NEJMoa1311347 PMid:24325356
  7. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013;369(25):2391-2405. https://doi.org/10.1056/NEJMoa1312542 PMid:24325359 PMCid:PMC3966280
  8. Vannucchi AM, Guglielmelli P, Tefferi A. Advances in understanding and management of myeloproliferative neoplasms. CA Cancer J Clin. 2009;59(3):171-191. https://doi.org/10.3322/caac.20009 PMid:19369682
  9. Mesa RA, Verstovsek S, Cervantes F, et al. Primary myelofibrosis (PMF), post polycythemia vera myelofibrosis (post-PV MF), post essential thrombocythemia myelofibrosis (post-ET MF), blast phase PMF (PMF-BP): Consensus on terminology by the international working group for myelofibrosis research and treatment (IWG-MRT). Leuk Res. 2007;31(6):737-740. https://doi.org/10.1016/j.leukres.2006.12.002 PMid:17210175
  10. Cervantes F, Dupriez B, Pereira A, et al. New prognostic scoring system for primary myelofibrosis based on a study of the International Working Group for Myelofibrosis Research and Treatment. Blood. 2009;113(13):2895-2901. https://doi.org/10.1182/blood-2008-07-170449 PMid:18988864
  11. Ciurea SO, Merchant D, Mahmud N, et al. Pivotal contributions of megakaryocytes to the biology of idiopathic myelofibrosis. Blood. 2007;110(3):986-993. https://doi.org/10.1182/blood-2006-12-064626  PMid:17473062 PMCid:PMC1924766
  12. Avecilla ST, Hattori K, Heissig B, et al. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med. 2004;10(1):64-71. https://doi.org/10.1038/nm973  PMid:14702636
  13. Bruns I, Lucas D, Pinho S, et al. Megakaryocytes regulate hematopoietic stem cell quiescence through CXCL4 secretion. Nat Med. 2014;20(11):1315-1320. https://doi.org/10.1038/nm.3707 PMid:25326802 PMCid:PMC4258871
  14. Zhao M, Perry JM, Marshall H, et al. Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells. Nat Med. 2014;20(11):1321-1326. https://doi.org/10.1038/nm.3706  PMid:25326798
  15. Winter O, Moser K, Mohr E, et al. Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow. Blood. 2010;116(11):1867-1875. https://doi.org/10.1182/blood-2009-12-259457 PMid:20538807
  16. Olson TS, Caselli A, Otsuru S, et al. Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning. Blood. 2013;121(26):5238-5249. https://doi.org/10.1182/blood-2012-10-463414 PMid:23667055 PMCid:PMC3695366
  17. Schmitt A, Jouault H, Guichard J, Wendling F, Drouin A, Cramer EM. Pathologic interaction between megakaryocytes and polymorphonuclear leukocytes in myelofibrosis. Blood. 2000;96(4):1342-1347. PMid:10942376
  18. Kaushansky K. The molecular mechanisms that control thrombopoiesis. J Clin Invest. 2005;115(12):3339-3347. https://doi.org/10.1172/JCI26674 PMid:16322778 PMCid:PMC1297257
  19. Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, et al. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell. 1995;81(5):695-704. https://doi.org/10.1016/0092-8674(95)90531-6  
  20. Patel SR, Hartwig JH, Italiano JE. The biogenesis of platelets from megakaryocyte proplatelets. J Clin Invest. 2005;115(12):3348-3354. https://doi.org/10.1172/JCI26891 PMid:16322779 PMCid:PMC1297261
  21. Blair P, Flaumenhaft R. Platelet alpha-granules: basic biology and clinical correlates. Blood Rev. 2009;23(4):177-189. https://doi.org/10.1016/j.blre.2009.04.001 PMid:19450911 PMCid:PMC2720568
  22. Maynard DM, Heijnen HF, Gahl WA, Gunay-Aygun M. The α-granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome. J Thromb Haemost. 2010;8(8):1786-1796. https://doi.org/10.1111/j.1538-7836.2010.03932.x PMid:20524979 PMCid:PMC2953603
  23. Stellos K, Langer H, Daub K, et al. Platelet-derived stromal cell-derived factor-1 regulates adhesion and promotes differentiation of human CD34+ cells to endothelial progenitor cells. Circulation. 2008;117(2):206-215. https://doi.org/10.1161/CIRCULATIONAHA.107.714691 PMid:18086932
  24. Battinelli EM, Markens BA, Italiano JE. Release of angiogenesis regulatory proteins from platelet alpha granules: modulation of physiologic and pathologic angiogenesis. Blood. 2011;118(5):1359-1369. https://doi.org/10.1182/blood-2011-02-334524 PMid:21680800 PMCid:PMC3152500
  25. Coppinger JA, Cagney G, Toomey S, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood. 2004;103(6):2096-2104. https://doi.org/10.1182/blood-2003-08-2804 PMid:14630798
  26. Cecchetti L, Tolley ND, Michetti N, Bury L, Weyrich AS, Gresele P. Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events. Blood. 2011;118(7):1903-1911. https://doi.org/10.1182/blood-2010-12-324517 PMid:21628401 PMCid:PMC3158719
  27. Handagama P, Scarborough RM, Shuman MA, Bainton DF. Endocytosis of fibrinogen into megakaryocyte and platelet alpha-granules is mediated by alpha IIb beta 3 (glycoprotein IIb-IIIa). Blood. 1993;82(1):135-138. PMid:8391871
  28. George JN. Platelet immunoglobulin G: its significance for the evaluation of thrombocytopenia and for understanding the origin of alpha-granule proteins. Blood. 1990;76(5):859-870. PMid:2203482
  29. Ciferri S, Emiliani C, Guglielmini G, Orlacchio A, Nenci GG, Gresele P. Platelets release their lysosomal content in vivo in humans upon activation. Thromb Haemost. 2000;83(1):157-164. https://doi.org/10.1055/s-0037-1613772  PMid:10669170
  30. McNicol A, Israels SJ. Platelet dense granules: structure, function and implications for haemostasis. Thromb Res. 1999;95(1):1-18. https://doi.org/10.1016/S0049-3848(99)00015-8  
  31. Badalucco S, Di Buduo CA, Campanelli R, et al. Involvement of TGFβ1 in autocrine regulation of proplatelet formation in healthy subjects and patients with primary myelofibrosis. Haematologica. 2013;98(4):514-517. https://doi.org/10.3324/haematol.2012.076752 PMid:23403314 PMCid:PMC3659980
  32. Balduini A, Di Buduo CA, Malara A, et al. Constitutively released adenosine diphosphate regulates proplatelet formation by human megakaryocytes. Haematologica. 2012;97(11):1657- 1665. https://doi.org/10.3324/haematol.2011.059212 PMid:22689668 PMCid:PMC3487437
  33. Saulle E, Guerriero R, Petronelli A, et al. Autocrine role of angiopoietins during megakaryocytic differentiation. PLoS One. 2012;7(7):e39796. https://doi.org/10.1371/journal.pone.0039796 PMid:22792187 PMCid:PMC3391299
  34. Lambert MP, Rauova L, Bailey M, Sola-Visner MC, Kowalska MA, Poncz M. Platelet factor 4 is a negative autocrine in vivo regulator of megakaryopoiesis: clinical and therapeutic implications. Blood. 2007;110(4):1153-1160. https://doi.org/10.1182/blood-2007-01-067116 PMid:17495129 PMCid:PMC1976471
  35. Nagata Y, Yoshikawa J, Hashimoto A, Yamamoto M, Payne AH, Todokoro K. Proplatelet formation of megakaryocytes is triggered by autocrine-synthesized estradiol. Genes Dev. 2003;17(23):2864-2869. https://doi.org/10.1101/gad.1128003 PMid:14665668 PMCid:PMC289146
  36. Casella I, Feccia T, Chelucci C, et al. Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor. Blood. 2003;101(4):1316-1323. https://doi.org/10.1182/blood-2002-07-2184 PMid:12406876
  37. Malara A, Abbonante V, Di Buduo CA, Tozzi L, Currao M, Balduini A. The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control. Cell Mol Life Sci. 2015;72(8):1517-1536. https://doi.org/10.1007/s00018-014-1813-y PMid:25572292 PMCid:PMC4369169
  38. Clemetson KJ. Platelets and primary haemostasis. Thromb Res. 2012;129(3):220-224. https://doi.org/10.1016/j.thromres.2011.11.036 PMid:22178577
  39. Eckly A, Rinckel JY, Proamer F, et al. Respective contributions of single and compound granule fusion to secretion by activated platelets. Blood. 2016;128(21):2538-2549. https://doi.org/10.1182/blood-2016-03-705681 PMid:27625359
  40. Rendu F, Brohard-Bohn B. The platelet release reaction: granules' constituents, secretion and functions. Platelets. 2001;12(5):261-273. https://doi.org/10.1080/09537100120068170 PMid:11487378
  41. Golebiewska EM, Poole AW. Platelet secretion: From haemostasis to wound healing and beyond. Blood Rev. 2015;29(3):153-162. https://doi.org/10.1016/j.blre.2014.10.003 PMid:25468720 PMCid:PMC4452143
  42. Italiano JE, Richardson JL, Patel-Hett S, et al. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood. 2008;111(3):1227-1233. https://doi.org/10.1182/blood-2007-09-113837 PMid:17962514 PMCid:PMC2214735
  43. Ma L, Perini R, McKnight W, et al. Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets. Proc Natl Acad Sci U S A. 2005;102(1):216- 220. https://doi.org/10.1073/pnas.0406682102 PMid:15615851 PMCid:PMC544057
  44. Kamykowski J, Carlton P, Sehgal S, Storrie B. Quantitative immunofluorescence mapping reveals little functional coclustering of proteins within platelet α-granules. Blood. 2011;118(5):1370-1373. https://doi.org/10.1182/blood-2011-01-330910  PMid:21622648
  45. Zingariello M, Fabucci ME, Bosco D, et al. Differential localization of P-selectin and von Willebrand factor during megakaryocyte maturation. Biotech Histochem. 2010;85(3):157-170. https://doi.org/10.3109/10520290903149612 PMid:20426698 PMCid:PMC3700322
  46. Guerrero JA, Bennett C, van der Weyden L, et al. Gray platelet syndrome: proinflammatory megakaryocytes and α-granule loss cause myelofibrosis and confer metastasis resistance in mice. Blood. 2014;124(24):3624-3635. https://doi.org/10.1182/blood-2014-04-566760 PMid:25258341
  47. Thiele J, Kuemmel T, Sander C, Fischer R. Ultrastructure of bone marrow tissue in so-called primary (idiopathic) myelofibrosis-osteomyelosclerosis (agnogenic myeloid metaplasia). I. Abnormalities of megakaryopoiesis and thrombocytes. J Submicrosc Cytol Pathol. 1991;23(1):93- 107. PMid:2036630
  48. Raman BK, Van Slyck EJ, Riddle J, Sawdyk MA, Abraham JP, Saeed SM. Platelet function and structure in myeloproliferative disease, myelodysplastic syndrome, and secondary thrombocytosis. Am J Clin Pathol. 1989;91(6):647-655. https://doi.org/10.1093/ajcp/91.6.647 PMid:2524965
  49. Sacchi S, Curci G, Piccinini L, et al. Platelet alpha-granule release in chronic myeloproliferative disorders with thrombocytosis. Scand J Clin Lab Invest. 1986;46(2):163-166. https://doi.org/10.3109/00365518609083653 PMid:2424075
  50. Holme S, Murphy S. Platelet abnormalities in myeloproliferative disorders. Clin Lab Med. 1990;10(4):873-888. PMid:2272179
  51. Muralidharan-Chari V, Clancy JW, Sedgwick A, D'Souza-Schorey C. Microvesicles: mediators of extracellular communication during cancer progression. J Cell Sci. 2010;123(Pt 10):1603-1611. https://doi.org/10.1242/jcs.064386 PMid:20445011 PMCid:PMC2864708
  52. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373-383. https://doi.org/10.1083/jcb.201211138 PMid:23420871 PMCid:PMC3575529
  53. Costa Verdera H, Gitz-Francois JJ, Schiffelers RM, Vader P. Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis. J Control Release. 2017;266:100-108. https://doi.org/10.1016/j.jconrel.2017.09.019  PMid:28919558
  54. Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat Commun. 2011;2:282. https://doi.org/10.1038/ncomms1285 PMid:21505438 PMCid:PMC3104548
  55. Goloviznina NA, Verghese SC, Yoon YM, Taratula O, Marks DL, Kurre P. Mesenchymal Stromal Cell-derived Extracellular Vesicles Promote Myeloid-biased Multipotent Hematopoietic Progenitor Expansion via Toll-Like Receptor Engagement. J Biol Chem. 2016;291(47):24607- 24617. https://doi.org/10.1074/jbc.M116.745653 PMid:27758863 PMCid:PMC5114412
  56. Crompot E, Van Damme M, Pieters K, et al. Extracellular vesicles of bone marrow stromal cells rescue chronic lymphocytic leukemia B cells from apoptosis, enhance their migration and induce gene expression modifications. Haematologica. 2017;102(9):1594-1604. https://doi.org/10.3324/haematol.2016.163337 PMid:28596280 PMCid:PMC5685228
  57. Lin LY, Du LM, Cao K, et al. Tumour cell-derived exosomes endow mesenchymal stromal cells with tumour-promotion capabilities. Oncogene. 2016;35(46):6038-6042. https://doi.org/10.1038/onc.2016.131 PMid:27132512 PMCid:PMC5116561
  58. Wolf P. The nature and significance of platelet products in human plasma. Br J Haematol. 1967;13(3):269-288. https://doi.org/10.1111/j.1365-2141.1967.tb08741.x PMid:6025241
  59. Boilard E, Duchez AC, Brisson A. The diversity of platelet microparticles. Curr Opin Hematol. 2015;22(5):437-444. https://doi.org/10.1097/MOH.0000000000000166 PMid:26214207
  60. Baj-Krzyworzeka M, Majka M, Pratico D, et al. Platelet-derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells. Exp Hematol. 2002;30(5):450-459. https://doi.org/10.1016/S0301-472X(02)00791-9  
  61. Rozmyslowicz T, Majka M, Kijowski J, et al. Platelet- and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV. AIDS. 2003;17(1):33-42. https://doi.org/10.1097/00002030-200301030-00006 PMid:12478067
  62. Ratajczak MZ. Megakaryocyte-derived microvesicles, please stand up! Blood. 2009;113(5):981-982. https://doi.org/10.1182/blood-2008-10-182964 PMid:19179472
  63. Flaumenhaft R, Dilks JR, Richardson J, et al. Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles. Blood. 2009;113(5):1112-1121. https://doi.org/10.1182/blood-2008-06-163832 PMid:18802008 PMCid:PMC2635076
  64. Jiang J, Kao CY, Papoutsakis ET. How do megakaryocytic microparticles target and deliver cargo to alter the fate of hematopoietic stem cells? J Control Release. 2017;247:1-18. https://doi.org/10.1016/j.jconrel.2016.12.021 PMid:28024915 PMCid:PMC5804484
  65. Nomura S, Nakamura T, Cone J, Tandon NN, Kambayashi J. Cytometric analysis of high shear-induced platelet microparticles and effect of cytokines on microparticle generation. Cytometry. 2000;40(3):173-181. https://doi.org/10.1002/1097-0320(20000701)40:3<173::AID-CYTO1>3.0.CO;2-L  
  66. Vainchenker W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017;129(6):667-679. https://doi.org/10.1182/blood-2016-10-695940 PMid:28028029
  67. Desterke C, Martinaud C, Ruzehaji N, Le Bousse-Kerdilès MC. Inflammation as a Keystone of Bone Marrow Stroma Alterations in Primary Myelofibrosis. Mediators Inflamm. 2015;2015:415024. https://doi.org/10.1155/2015/415024 PMid:26640324 PMCid:PMC4660030
  68. Tefferi A, Vaidya R, Caramazza D, Finke C, Lasho T, Pardanani A. Circulating interleukin (IL)-8, IL-2R, IL-12, and IL-15 levels are independently prognostic in primary myelofibrosis: a comprehensive cytokine profiling study. J Clin Oncol. 2011;29(10):1356-1363. https://doi.org/10.1200/JCO.2010.32.9490 PMid:21300928
  69. Hasselbalch HC. Perspectives on chronic inflammation in essential thrombocythemia, polycythemia vera, and myelofibrosis: is chronic inflammation a trigger and driver of clonal evolution and development of accelerated atherosclerosis and second cancer? Blood. 2012;119(14):3219-3225. https://doi.org/10.1182/blood-2011-11-394775 PMid:22318201
  70. Al-Ali HK, Griesshammer M, le Coutre P, et al. Safety and efficacy of ruxolitinib in an open-label, multicenter, single-arm phase 3b expanded-access study in patients with myelofibrosis: a snapshot of 1144 patients in the JUMP trial. Haematologica. 2016;101(9):1065-1073. https://doi.org/10.3324/haematol.2016.143677 PMid:27247324 PMCid:PMC5060023
  71. Skoda RC, Duek A, Grisouard J. Pathogenesis of myeloproliferative neoplasms. Exp Hematol. 2015;43(8):599-608. https://doi.org/10.1016/j.exphem.2015.06.007 PMid:26209551
  72. Zhan H, Ma Y, Lin CH, Kaushansky K. JAK2. Leukemia. 2016;30(12):2332-2341. https://doi.org/10.1038/leu.2016.114 PMid:27133820 PMCid:PMC5158308
  73. Kuter DJ, Mufti GJ, Bain BJ, Hasserjian RP, Davis W, Rutstein M. Evaluation of bone marrow reticulin formation in chronic immune thrombocytopenia patients treated with romiplostim. Blood. 2009;114(18):3748-3756. https://doi.org/10.1182/blood-2009-05-224766  PMid:19671919
  74. Villeval JL, Cohen-Solal K, Tulliez M, et al. High thrombopoietin production by hematopoietic cells induces a fatal myeloproliferative syndrome in mice. Blood. 1997;90(11):4369- 4383. PMid:9373248
  75. Vannucchi AM, Bianchi L, Cellai C, et al. Development of myelofibrosis in mice genetically impaired for GATA-1 expression (GATA-1(low) mice). Blood. 2002;100(4):1123-1132. https://doi.org/10.1182/blood-2002-06-1913  PMid:12149188
  76. Jantunen E, Hänninen A, Naukkarinen A, Vornanen M, Lahtinen R. Gray platelet syndrome with splenomegaly and signs of extramedullary hematopoiesis: a case report with review of the literature. Am J Hematol. 1994;46(3):218-224. https://doi.org/10.1002/ajh.2830460311  PMid:8192152
  77. Rameshwar P, Narayanan R, Qian J, Denny TN, Colon C, Gascon P. NF-kappa B as a central mediator in the induction of TGF-beta in monocytes from patients with idiopathic myelofibrosis: an inflammatory response beyond the realm of homeostasis. J Immunol. 2000;165(4):2271-2277. https://doi.org/10.4049/jimmunol.165.4.2271  PMid:10925316
  78. Frey BM, Rafii S, Teterson M, Eaton D, Crystal RG, Moore MA. Adenovector-mediated expression of human thrombopoietin cDNA in immune-compromised mice: insights into the pathophysiology of osteomyelofibrosis. J Immunol. 1998;160(2):691-699. PMid:9551904
  79. Wagner-Ballon O, Chagraoui H, Prina E, et al. Monocyte/macrophage dysfunctions do not impair the promotion of myelofibrosis by high levels of thrombopoietin. J Immunol. 2006;176(11):6425-6433. https://doi.org/10.4049/jimmunol.176.11.6425 PMid:16709799
  80. Castro-Malaspina H, Jhanwar SC. Properties of myelofibrosis-derived fibroblasts. Prog Clin Biol Res. 1984;154:307-322. PMid:6382300
  81. Bowen-Pope DF, Raines EW. History of discovery: platelet-derived growth factor. Arterioscler Thromb Vasc Biol. 2011;31(11):2397-2401. https://doi.org/10.1161/ATVBAHA.108.179556 PMid:22011752 PMCid:PMC3209478
  82. Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev. 2004;15(4):255-273. https://doi.org/10.1016/j.cytogfr.2004.03.006 PMid:15207816
  83. Lucero HA, Ravid K, Grimsby JL, et al. Lysyl oxidase oxidizes cell membrane proteins and enhances the chemotactic response of vascular smooth muscle cells. J Biol Chem. 2008;283(35):24103-24117. https://doi.org/10.1074/jbc.M709897200 PMid:18586678 PMCid:PMC2527118
  84. Eliades A, Papadantonakis N, Bhupatiraju A, et al. Control of megakaryocyte expansion and bone marrow fibrosis by lysyl oxidase. J Biol Chem. 2011;286(31):27630-27638. https://doi.org/10.1074/jbc.M111.243113 PMid:21665949 PMCid:PMC3149354
  85. Papadantonakis N, Matsuura S, Ravid K. Megakaryocyte pathology and bone marrow fibrosis: the lysyl oxidase connection. Blood. 2012;120(9):1774-1781. https://doi.org/10.1182/blood-2012-02-402594 PMid:22767499 PMCid:PMC3433087
  86. Gersuk GM, Carmel R, Pattengale PK. Platelet-derived growth factor concentrations in platelet-poor plasma and urine from patients with myeloproliferative disorders. Blood. 1989;74(7):2330-2334. PMid:2804368
  87. Lev PR, Marta RF, Vassallu P, Molinas FC. Variation of PDGF, TGFbeta, and bFGF levels in essential thrombocythemia patients treated with anagrelide. Am J Hematol. 2002;70(2):85-91. https://doi.org/10.1002/ajh.10091 PMid:12111780
  88. Yoon SY, Tefferi A, Li CY. Cellular distribution of platelet-derived growth factor, transforming growth factor-beta, basic fibroblast growth factor, and their receptors in normal bone marrow. Acta Haematol. 2000;104(4):151-157. https://doi.org/10.1159/000046507 PMid:11279303
  89. Bock O, Loch G, Büsche G, von Wasielewski R, Schlué J, Kreipe H. Aberrant expression of platelet-derived growth factor (PDGF) and PDGF receptor-alpha is associated with advanced bone marrow fibrosis in idiopathic myelofibrosis. Haematologica. 2005;90(1):133-134. PMid:15642683
  90. Bedekovics J, Kiss A, Beke L, Károlyi K, Méhes G. Platelet derived growth factor receptor- beta (PDGFRβ) expression is limited to activated stromal cells in the bone marrow and shows a strong correlation with the grade of myelofibrosis. Virchows Arch. 2013;463(1):57-65. https://doi.org/10.1007/s00428-013-1434-0 PMid:23748876
  91. Decker M, Martinez-Morentin L, Wang G, et al. Leptin-receptor-expressing bone marrow stromal cells are myofibroblasts in primary myelofibrosis. Nat Cell Biol. 2017;19(6):677-688. https://doi.org/10.1038/ncb3530 PMid:28481328 PMCid:PMC5801040
  92. Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J. 2004;18(7):816-827. https://doi.org/10.1096/fj.03-1273rev PMid:15117886
  93. Martyré MC, Magdelenat H, Bryckaert MC, Laine-Bidron C, Calvo F. Increased intraplatelet levels of platelet-derived growth factor and transforming growth factor-beta in patients with myelofibrosis with myeloid metaplasia. Br J Haematol. 1991;77(1):80-86. https://doi.org/10.1111/j.1365-2141.1991.tb07952.x PMid:1998600
  94. Le Bousse-Kerdilès MC, Martyré MC. Dual implication of fibrogenic cytokines in the pathogenesis of fibrosis and myeloproliferation in myeloid metaplasia with myelofibrosis. Ann Hematol. 1999;78(10):437-444. https://doi.org/10.1007/s002770050595 PMid:10550553
  95. Wang JC, Novetsky A, Chen C, Novetsky AD. Plasma matrix metalloproteinase and tissue inhibitor of metalloproteinase in patients with agnogenic myeloid metaplasia or idiopathic primary myelofibrosis. Br J Haematol. 2002;119(3):709-712. https://doi.org/10.1046/j.1365-2141.2002.03874.x PMid:12437648
  96. Blank U, Karlsson S. The role of Smad signaling in hematopoiesis and translational hematology. Leukemia. 2011;25(9):1379-1388. https://doi.org/10.1038/leu.2011.95 PMid:21566654
  97. Erba BG, Gruppi C, Corada M, et al. Endothelial-to-Mesenchymal Transition in Bone Marrow and Spleen of Primary Myelofibrosis. Am J Pathol. 2017;187(8):1879-1892. https://doi.org/10.1016/j.ajpath.2017.04.006 PMid:28728747
  98. Le Bousse-Kerdilès MC, Chevillard S, Charpentier A, et al. Differential expression of transforming growth factor-beta, basic fibroblast growth factor, and their receptors in CD34+ hematopoietic progenitor cells from patients with myelofibrosis and myeloid metaplasia. Blood. 1996;88(12):4534-4546. PMid:8977245
  99. Le Bousse-Kerdilès MC, Martyré MC, Myelofibrosis FIrnoI. Involvement of the fibrogenic cytokines, TGF-beta and bFGF, in the pathogenesis of idiopathic myelofibrosis. Pathol Biol (Paris). 2001;49(2):153-157. https://doi.org/10.1016/S0369-8114(00)00021-3  
  100. Campanelli R, Rosti V, Villani L, et al. Evaluation of the bioactive and total transforming growth factor β1 levels in primary myelofibrosis. Cytokine. 2011;53(1):100-106. https://doi.org/10.1016/j.cyto.2010.07.427 PMid:20801055
  101. Ciaffoni F, Cassella E, Varricchio L, Massa M, Barosi G, Migliaccio AR. Activation of non- canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis. Blood Cells Mol Dis. 2015;54(3):234-241. https://doi.org/10.1016/j.bcmd.2014.12.005 PMid:25703685 PMCid:PMC4338409
  102. Wernig G, Chen SY, Cui L, et al. Unifying mechanism for different fibrotic diseases. Proc Natl Acad Sci U S A. 2017;114(18):4757-4762. https://doi.org/10.1073/pnas.1621375114 PMid:28424250 PMCid:PMC5422830
  103. Vannucchi AM, Bianchi L, Paoletti F, et al. A pathobiologic pathway linking thrombopoietin, GATA-1, and TGF-beta1 in the development of myelofibrosis. Blood. 2005;105(9):3493-3501. https://doi.org/10.1182/blood-2004-04-1320 PMid:15665119
  104. Yanagida M, Ide Y, Imai A, et al. The role of transforming growth factor-beta in PEG- rHuMGDF-induced reversible myelofibrosis in rats. Br J Haematol. 1997;99(4):739-745. https://doi.org/10.1046/j.1365-2141.1997.4843288.x PMid:9432016
  105. Chagraoui H, Komura E, Tulliez M, Giraudier S, Vainchenker W, Wendling F. Prominent role of TGF-beta 1 in thrombopoietin-induced myelofibrosis in mice. Blood. 2002;100(10):3495- 3503. https://doi.org/10.1182/blood-2002-04-1133 PMid:12393681
  106. Zingariello M, Martelli F, Ciaffoni F, et al. Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis. Blood. 2013;121(17):3345-3363. https://doi.org/10.1182/blood-2012-06-439661 PMid:23462118 PMCid:PMC3637011
  107. Ceglia I, Dueck AC, Masiello F, et al. Preclinical rationale for TGF-β inhibition as a therapeutic target for the treatment of myelofibrosis. Exp Hematol. 2016;44(12):1138-1155.e1134. https://doi.org/10.1016/j.exphem.2016.08.007 PMid:27592389 PMCid:PMC5778911
  108. Gleissner CA, von Hundelshausen P, Ley K. Platelet chemokines in vascular disease. Arterioscler Thromb Vasc Biol. 2008;28(11):1920-1927. https://doi.org/10.1161/ATVBAHA.108.169417 PMid:18723831 PMCid:PMC2657037
  109. Lasagni L, Francalanci M, Annunziato F, et al. An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4. J Exp Med. 2003;197(11):1537-1549. https://doi.org/10.1084/jem.20021897 PMid:12782716 PMCid:PMC2193908
  110. Zaldivar MM, Pauels K, von Hundelshausen P, et al. CXC chemokine ligand 4 (Cxcl4) is a platelet-derived mediator of experimental liver fibrosis. Hepatology. 2010;51(4):1345-1353. https://doi.org/10.1002/hep.23435 PMid:20162727
  111. van Bon L, Affandi AJ, Broen J, et al. Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis. N Engl J Med. 2014;370(5):433-443. https://doi.org/10.1056/NEJMoa1114576 PMid:24350901 PMCid:PMC4040466
  112. Burstein SA, Malpass TW, Yee E, et al. Platelet factor-4 excretion in myeloproliferative disease: implications for the aetiology of myelofibrosis. Br J Haematol. 1984;57(3):383-392. https://doi.org/10.1111/j.1365-2141.1984.tb02912.x PMid:6743563
  113. Schneider RK, Mullally A, Dugourd A, et al. Gli1. Cell Stem Cell. 2017;20(6):785- 800.e788. https://doi.org/10.1016/j.stem.2017.03.008 PMid:28457748
  114. Gómez-Lechón MJ. Oncostatin M: signal transduction and biological activity. Life Sci. 1999;65(20):2019-2030. https://doi.org/10.1016/S0024-3205(99)00296-9  
  115. Mosley B, De Imus C, Friend D, et al. Dual oncostatin M (OSM) receptors. Cloning and characterization of an alternative signaling subunit conferring OSM-specific receptor activation. J Biol Chem. 1996;271(51):32635-32643. https://doi.org/10.1074/jbc.271.51.32635 PMid:8999038
  116. Tanaka M, Hirabayashi Y, Sekiguchi T, Inoue T, Katsuki M, Miyajima A. Targeted disruption of oncostatin M receptor results in altered hematopoiesis. Blood. 2003;102(9):3154- 3162. https://doi.org/10.1182/blood-2003-02-0367 PMid:12855584
  117. Minehata K, Takeuchi M, Hirabayashi Y, et al. Oncostatin m maintains the hematopoietic microenvironment and retains hematopoietic progenitors in the bone marrow. Int J Hematol. 2006;84(4):319-327. https://doi.org/10.1532/IJH97.06090 PMid:17118758
  118. Hoermann G, Cerny-Reiterer S, Perné A, et al. Identification of oncostatin M as a STAT5- dependent mediator of bone marrow remodeling in KIT D816V-positive systemic mastocytosis. Am J Pathol. 2011;178(5):2344-2356. https://doi.org/10.1016/j.ajpath.2011.01.020 PMid:21457934 PMCid:PMC3081146
  119. Wallace PM, MacMaster JF, Rillema JR, Peng J, Burstein SA, Shoyab M. Thrombocytopoietic properties of oncostatin M. Blood. 1995;86(4):1310-1315 PMid:7632937
  120. Hoermann G, Cerny-Reiterer S, Herrmann H, et al. Identification of oncostatin M as a JAK2 V617F-dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms. FASEB J. 2012;26(2):894-906. https://doi.org/10.1096/fj.11-193078 PMid:22051730
  121. Schepers K, Pietras EM, Reynaud D, et al. Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche. Cell Stem Cell. 2013;13(3):285-299. https://doi.org/10.1016/j.stem.2013.06.009 PMid:23850243 PMCid:PMC3769504
  122. Murphy PM. Neutrophil receptors for interleukin-8 and related CXC chemokines. Semin Hematol. 1997;34(4):311-318. PMid:9347581
  123. Takeuchi K, Higuchi T, Yamashita T, Koike K. Chemokine production by human megakaryocytes derived from CD34-positive cord blood cells. Cytokine. 1999;11(6):424-434. https://doi.org/10.1006/cyto.1998.0455 PMid:10346982
  124. Emadi S, Clay D, Desterke C, et al. IL-8 and its CXCR1 and CXCR2 receptors participate in the control of megakaryocytic proliferation, differentiation, and ploidy in myeloid metaplasia with myelofibrosis. Blood. 2005;105(2):464-473.m https://doi.org/10.1182/blood-2003-12-4415 PMid:15454487
  125. Hol J, Wilhelmsen L, Haraldsen G. The murine IL-8 homologues KC, MIP-2, and LIX are found in endothelial cytoplasmic granules but not in Weibel-Palade bodies. J Leukoc Biol. 2010;87(3):501-508. https://doi.org/10.1189/jlb.0809532  PMid:20007247
  126. Bock O, Höftmann J, Theophile K, et al. Bone morphogenetic proteins are overexpressed in the bone marrow of primary myelofibrosis and are apparently induced by fibrogenic cytokines. Am J Pathol. 2008;172(4):951-960. https://doi.org/10.2353/ajpath.2008.071030 PMid:18349123 PMCid:PMC2276425
  127. Garimella R, Kacena MA, Tague SE, Wang J, Horowitz MC, Anderson HC. Expression of bone morphogenetic proteins and their receptors in the bone marrow megakaryocytes of GATA- 1(low) mice: a possible role in osteosclerosis. J Histochem Cytochem. 2007;55(7):745-752. https://doi.org/10.1369/jhc.6A7164.2007 PMid:17371937
  128. Lu M, Xia L, Liu YC, et al. Lipocalin produced by myelofibrosis cells affects the fate of both hematopoietic and marrow microenvironmental cells. Blood. 2015;126(8):972-982. https://doi.org/10.1182/blood-2014-12-618595 PMid:26022238 PMCid:PMC4543230
  129. Martyré MC, Le Bousse-Kerdiles MC, Romquin N, et al. Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis. Br J Haematol. 1997;97(2):441-448. https://doi.org/10.1046/j.1365-2141.1997.292671.x PMid:9163611
  130. Boiocchi L, Vener C, Savi F, et al. Increased expression of vascular endothelial growth factor receptor 1 correlates with VEGF and microvessel density in Philadelphia chromosome- negative myeloproliferative neoplasms. J Clin Pathol. 2011;64(3):226-231. https://doi.org/10.1136/jcp.2010.083386 PMid:21217153
  131. Ruberti S, Bianchi E, Guglielmelli P, et al. Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients. Leukemia. 2018;32(2):438-449. https://doi.org/10.1038/leu.2017.220 PMid:28745329 PMCid:PMC5808097
  132. Evrard S, Bluteau O, Tulliez M, et al. Thrombospondin-1 is not the major activator of TGF- β1 in thrombopoietin-induced myelofibrosis. Blood. 2011;117(1):246-249. https://doi.org/10.1182/blood-2010-07-294447 PMid:20944070
  133. Muth M, Engelhardt BM, Kröger N, et al. Thrombospondin-1 (TSP-1) in primary myelofibrosis (PMF) - a megakaryocyte-derived biomarker which largely discriminates PMF from essential thrombocythemia. Ann Hematol. 2011;90(1):33-40. https://doi.org/10.1007/s00277-010-1024-z PMid:20625903
  134. Abbonante V, Di Buduo CA, Gruppi C, et al. A new path to platelet production through matrix sensing. Haematologica. 2017. https://doi.org/10.3324/haematol.2016.161562 PMCid:PMC5566016
  135. Malara A, Currao M, Gruppi C, et al. Megakaryocytes contribute to the bone marrow-matrix environment by expressing fibronectin, type IV collagen, and laminin. Stem Cells. 2014;32(4):926- 937. https://doi.org/10.1002/stem.1626 PMid:24357118 PMCid:PMC4096110
  136. Abbonante V, Di Buduo CA, Gruppi C, et al. Thrombopoietin/TGF-β1 Loop Regulates Megakaryocyte Extracellular Matrix Component Synthesis. Stem Cells. 2016;34(4):1123-1133. https://doi.org/10.1002/stem.2285  PMid:26748484
  137. Malara A, Gruppi C, Rebuzzini P, et al. Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A. Blood. 2011;117(8):2476-2483. https://doi.org/10.1182/blood-2010-06-288795 PMid:21131589
  138. Malara A, Gruppi C, Rebuzzini P, et al. Megakaryocyte-matrix interaction within bone marrow: New roles for fibronectin and factor XIII-A. Blood. 2011;117(8):2476-2483. https://doi.org/10.1182/blood-2010-06-288795 PMid:21131589
  139. Mosher DF, Schad PE, Vann JM. Cross-linking of collagen and fibronectin by factor XIIIa. Localization of participating glutaminyl residues to a tryptic fragment of fibronectin. J Biol Chem. 1980;255(3):1181-1188. PMid:7356656
  140. Abbonante V, Chitalia V, Rosti V, et al. Upregulation of lysyl oxidase and adhesion to collagen of human megakaryocytes and platelets in primary myelofibrosis. Blood. 2017;130(6):829- 831. https://doi.org/10.1182/blood-2017-04-777417 PMid:28592432 PMCid:PMC5553577
  141. Rundhaug JE. Matrix metalloproteinases and angiogenesis. J Cell Mol Med. 2005;9(2):267- 285. https://doi.org/10.1111/j.1582-4934.2005.tb00355.x PMid:15963249
  142. Lane WJ, Dias S, Hattori K, et al. Stromal-derived factor 1-induced megakaryocyte migration and platelet production is dependent on matrix metalloproteinases. Blood. 2000;96(13):4152-4159. PMid:11110686
  143. Villeneuve J, Block A, Le Bousse-Kerdilès MC, et al. Tissue inhibitors of matrix metalloproteinases in platelets and megakaryocytes: a novel organization for these secreted proteins. Exp Hematol. 2009;37(7):849-856. https://doi.org/10.1016/j.exphem.2009.03.009 PMid:19410025  
  144. Wang JC. Importance of plasma matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinase (TIMP) in development of fibrosis in agnogenic myeloid metaplasia. Leuk Lymphoma. 2005;46(9):1261-1268. https://doi.org/10.1080/10428190500126463 PMid:16109602
  145. Bock O, Neuse J, Hussein K, et al. Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status. Am J Pathol. 2006;169(2):471-481.https://doi.org/10.2353/ajpath.2006.060110 PMid:16877349 PMCid:PMC1780160
  146. Wickenhauser C, Schmitz B, Baldus SE, et al. Selectins (CD62L, CD62P) and megakaryocytic glycoproteins (CD41a, CD42b) mediate megakaryocyte-fibroblast interactions in human bone marrow. Leuk Res. 2000;24(12):1013-1021. https://doi.org/10.1016/S0145-2126(00)00063-1  
  147. Abbonante V, Gruppi C, Catarsi P, et al. Altered fibronectin expression and deposition by myeloproliferative neoplasm-derived mesenchymal stromal cells. Br J Haematol. 2016;172(1):140- 144. https://doi.org/10.1111/bjh.13471 PMid:25940987
  148. Avanzini MA, Abbonante V, Catarsi P, et al. The spleen of patients with myelofibrosis harbors defective mesenchymal stromal cells. Am J Hematol. 2018. https://doi.org/10.1002/ajh.25047 PMid:29359451
  149. Schneider RK, Ziegler S, Leisten I, et al. Activated fibronectin-secretory phenotype of mesenchymal stromal cells in pre-fibrotic myeloproliferative neoplasms. J Hematol Oncol. 2014;7:92. https://doi.org/10.1186/s13045-014-0092-2 PMid:25498831 PMCid:PMC4271470
  150. Cashell AW, Buss DH. The frequency and significance of megakaryocytic emperipolesis in myeloproliferative and reactive states. Ann Hematol. 1992;64(6):273-276. https://doi.org/10.1007/BF01695470  
  151. Schmitt A, Drouin A, Massé JM, Guichard J, Shagraoui H, Cramer EM. Polymorphonuclear neutrophil and megakaryocyte mutual involvement in myelofibrosis pathogenesis. Leuk Lymphoma. 2002;43(4):719-724. https://doi.org/10.1080/10428190290016809 PMid:12153156
  152. Centurione L, Di Baldassarre A, Zingariello M, et al. Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA- 1(low) mice. Blood. 2004;104(12):3573-3580. https://doi.org/10.1182/blood-2004-01-0193 PMid:15292068
  153. Zetterberg E, Verrucci M, Martelli F, et al. Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis. Platelets. 2014;25(7):539-547. https://doi.org/10.3109/09537104.2013.840720 PMid:24176039 PMCid:PMC4045657
  154. Spangrude GJ, Lewandowski D, Martelli F, et al. P-Selectin Sustains Extramedullary Hematopoiesis in the Gata1 low Model of Myelofibrosis. Stem Cells. 2016;34(1):67-82. https://doi.org/10.1002/stem.2229 PMid:26439305
  155. Di Buduo CA, Alberelli MA, Glembostky AC, et al. Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients. Sci Rep. 2016;6:23213. https://doi.org/10.1038/srep23213 PMid:26987485 PMCid:PMC4796794
  156. Ghafoory S, Varshney R, Robison T, et al. Platelet TGF-β1 deficiency decreases liver fibrosis in a mouse model of liver injury. Blood Adv. 2018;2(5):470-480. https://doi.org/10.1182/bloodadvances.2017010868 PMid:29490978 PMCid:PMC5851416
  157. Kodama T, Takehara T, Hikita H, et al. Thrombocytopenia exacerbates cholestasis-induced liver fibrosis in mice. Gastroenterology. 2010;138(7):2487-2498, 2498.e2481-2487.
  158. Meyer A, Wang W, Qu J, et al. Platelet TGF-β1 contributions to plasma TGF-β1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload. Blood. 2012;119(4):1064- 1074. https://doi.org/10.1182/blood-2011-09-377648 PMid:22134166 PMCid:PMC3271718
  159. Piguet PF, Vesin C. Pulmonary platelet trapping induced by bleomycin: correlation with fibrosis and involvement of the beta 2 integrins. Int J Exp Pathol. 1994;75(5):321-328. PMid:7528044 PMCid:PMC2001873
  160. Guo Y, Mishra A, Howland E, et al. Platelet-derived Wnt antagonist Dickkopf-1 is implicated in ICAM-1/VCAM-1-mediated neutrophilic acute lung inflammation. Blood. 2015;126(19):2220-2229. https://doi.org/10.1182/blood-2015-02-622233 PMid:26351298 PMCid:PMC4635118
  161. Rondina MT, Weyrich AS. Regulation of the genetic code in megakaryocytes and platelets. J Thromb Haemost. 2015;13 Suppl 1:S26-32. https://doi.org/10.1111/jth.12965 PMid:26149034 PMCid:PMC4498409
  162. Freishtat RJ, Natale J, Benton AS, et al. Sepsis alters the megakaryocyte-platelet transcriptional axis resulting in granzyme B-mediated lymphotoxicity. Am J Respir Crit Care Med. 2009;179(6):467-473. https://doi.org/10.1164/rccm.200807-1085OC PMid:19136373 PMCid:PMC2654976
  163. Zaslavsky A, Baek KH, Lynch RC, et al. Platelet-derived thrombospondin-1 is a critical negative regulator and potential biomarker of angiogenesis. Blood. 2010;115(22):4605-4613. https://doi.org/10.1182/blood-2009-09-242065 PMid:20086246 PMCid:PMC2881490
  164. Wen QJ, Yang Q, Goldenson B, et al. Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition. Nat Med. 2015;21(12):1473-1480. https://doi.org/10.1038/nm.3995 PMid:26569382 PMCid:PMC4674320

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