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Contents

Introduction

Figure 1. A schematic of the platelet production. [1]

Megakaryocytes (Greek: mega- (enlargement), -karyo- (nucleus) -cyte (cell); /'PHONETIC/), like other blood cell types are derived from the 'Haematopoeitic Stem Cells (HSCs)' found in the bone marrow and belong to the myeloid lineage. They are the largest cells found in the bone marrow and perform their vital functions and metabolic processes only at this site. They have a lobulated nucleus and are more importantly known for their ability to produce platelets which are abundantly found circulating the bloodstream.

Platelets are a distinct class of blood cells that play a vital role in normal blood clotting which is essential for the reparation of small vascular injuries. Megakaryocytes also play a very important role in maintaining 'bone marrow homeostasis' by tightly regulating the activation of HSCs through the secretion of various bioactive molecules. [2] Under normal circumstances, megakaryocytes constitute 1 in every 10,000 bone marrow cells but in the presence of certain diseases, they can proliferate by ten-fold. [3]

History

Year Historical Event
1906 James Wright examined blood smears and describes the cells as ‘plates’. In his 1910 publication, however, he changed the term to ‘platelets’, which is now universally accepted. [4]
1956 Platelets are found to form in bone marrow in a precursor form (megakaryocytes). [5]
1968 Established that the time needed for MK’s to complete polyploidization, mature and release platelets is approximately 5 days in humans, and 2-3 days in rodents. [6]
1969 Proplatelet production observed under light microscope of rodent MKs. [7]
1974 The size and shape of the MK cells are established in the bone marrow. Found to account for ~0.01% of nucleated bone marrow cells. [8]
1976 Studies find endomitosis is a metabolic process driven by TPO and that the MKs achieve polyploidy status through DNA cycle replication with no cell division. [9]
1976 MK production observed via electron microscope scans of proplatelets extending into the blood vessels of bone marrow (in vivo). [10]
1977 Life expectancy of MKs discovered, approximately 7-10 days for humans, and 4-5 days for rodents. 10^11 MKs are produced daily in humans. [11]
1982 MKs found to develop from hematopoietic stem cells (HSCs) that produced and exist in bone marrow for adults, and in the yolk sac, liver and spleen during development. [12] MK tubular system was discovered and analysed. [13]
1994 Identification of the major regulator of MK, promoting growth, development and proliferation of cells from their HSC precursors. The MK-specific receptor c-Mpl and Thrombopoietin (Tpo) revolutionized the field of study of platelet biology. [14][15]
2007 Geddis et al. discovered the reason why MKs can undergo endomitosis - their inability of MKs to form contractile ring and undergo cytokinesis. [16]
2010 New stage of platelet formation identified, known as the preplatelet. These platelets are anucleated discoid particles, 2-10um in diameter. Found that preplatelets can mature into platelets in vitro and after transfusion into mice in vivo. [17]

Structure

Megakaryocytes are solely located in the bone marrow and are large polypoid cells. Their function is to maintain the normal blood platelet count, which is carried out by mature megakaryocytes releasing approximately 20 to 30 × 10^9 platelets/L platelets/L of blood per day. [18]

Figure 2. Megakaryocytes with a large lobulated nucleus in the growing bone of a rabbit.

Megakaryocytes

Megakaryocytes are 10- 15 times bigger than the average red blood cell with a diameter of 50-100μm. Due to endomitosis, megakaryocytes grow in size and duplicate their DNA without cytokinesis allowing for the nucleus to become larger and lobulated, giving the illusion of many nuclei. It has been noticed that the greater the DNA content in a cell, the larger the size of the megakaryocyte. Megakaryocytes are distinctive from other cells because of the density and complexity of their cytoplasm. The cytoplasm of a megakaryocyte is subdivided into the following three zones:[19]

Figure 3. An image showcasing some of the contents in a megakaryocyte.

Innermost (Perinuclear) Zone

The Innermost (Perinuclear) Zone is comprised of a narrow band of cytoplasm that encircles the nucleus. Organelles found in this layer include mitochondria, small vesicles and endoplasmic reticulum. Additionally, the golgi apparatus and centrioles can be found within this margin. [20] Ribosomes also exist within this layer both freely unbound and attached to the endoplasmic reticulum. Platelet granules and membrane demarcations are not present within this region. The endoplasmic reticulum is most commonly found residing in the perinuclear zone along and also in the deeper portions of the intermediate zone.[21]

Middle (Intermediate) Zone

The Middle Zone is separated by an indistinct boundary from the perinuclear zone and is quite large in size. Out of the three different zones, it contains the greatest volume of cytoplasm and contains vast quantities of mitochondria, small vesicles and endoplasmic reticulum. The vesicular stage in the formation of platelets, can be found in this area with vesicles of about 400 A, present as they align in rows. Portions of the intermediate zone showed ribosomes are also present in this layer both free from and attached to the endoplasmic reticulum that resides in the cytoplasm. The mitochondria are small in size and range from 0.15- 0.3μm in their narrow diameter and are round ellipsoid in shape. [22]

Figure 4. An image that summarizes the structure of a megakaryocyte.

Furthermore, the Middle Zone is significant for the presence of the 'demarcation membrane system (DMS)'.This system consists of a large number of small vesicles which appear to line up and merge with one another to form a long, paired membranous profile. It is the precursor of the platelet cell membranes and little is known about its properties in living megakaryocytes. Platelet granules found in the Middle Zone are ovoid and occasionally elongated. They vary in size and their long axis ranges from approximately 0.3- 0.7μm. The Middle Zone is separated from the Marginal Zone at the site where the demarcation membrane ends. [23]

Outer (Marginal) Zone

The Outer (Marginal) Zone is the outermost layer of cytoplasm in the cell of a megakaryocyte. It is a narrow band of cytoplasm that contains fine granules along with few ribosomes and vesicles. It is also known that the marginal zone varies in width and usually lacks organelles as well as demarcation membranes. This region of the cell is more importantly the site at which the platelets are released. [24]

Mature megakaryocytes have large multilobulated polypoid nuclei which are often found towards the periphery of the cell. They have large amounts of cytoplasm that contain platelet specific secretory granules known as alpha granules. Alpha granules are 200-500nm in diameter and consist of a dense centre embedded in a fine granular matrix. [23]

Figure 5. Transmission electron micrographs of MKs, preplatelets, proplatelets, and platelets. [1]

Platelets

Platelets that are unactivated are biconvex discoid (lens-shaped) structures, with a diameter of around 2–3 µm. [19] Platelets bud off from the megakaryocyte's cytoplasm which contain dense bodies and α-granules. The term "alpha granules" is used to describe granules containing several growth factors in platelets.[21] Some of the contents in the platelets include insulin-like growth factor 1, platelet-derived growth factors, TGFβ, platelet factor 4 (which is a heparin-binding chemokine) and other clotting proteins (such as thrombospondin, fibronectin, factor V, and von Willebrand factor). Furthermore, the alpha granules express the adhesion molecule P-selectin and CD63. These are transferred to the membrane after synthesis. The other type of granules within platelets are called dense granules. [22] [21] Dense bodies are comprised of ADP, ATP, 5-HT, calcium and pyrophosphate. These contents are important components of the platelet’s biological activities. Further on the exterior surface of platelets is the presence of polymorphic glycoproteins. Some of the platelet membrane constituents contains a variety of polymorphic glycoprotein molecules in order to interact with ligands to generate the primary hemostatic plug. Some examples of ligands include; coagulation factors, vessel wall components and other molecules.


Structurally the platelet can be divided into four zones, from peripheral to innermost:

Figure 6. Microtubules line the shafts of proplatelets and platelets. [1]

Peripheral zone

This zone is abundant in glycoproteins that are required for platelet function which includes adhesion, activation, and aggregation. some examples are; GPIb/IX/X; GPVI; GPIIb/IIIa. [23][21]

Sol-gel zone

The Sol-gel zone is rich in microtubules and microfilaments, allowing the platelets to preserve their discoid shape. [23][21]

Organelle zone

Ample platelet granules are found in this region of platelets. There are two main types of granules: [24]

  • α-granules: factor V, factor VIII, fibrinogen, fibronectin, platelet-derived growth factor, and chemotactic agents.
  • Dense bodies (delta granules): contain ADP, calcium, serotonin, which are platelet-activating mediators.

Membranous zone

This region includes membranes derived from megakaryocytic smooth endoplasmic reticulum which has been organised into a dense tubular system. It is responsible for thromboxane A2 synthesis. This dense tubular system is connected to the platelet's membrane to assist in thromboxane A2 release. [23][21]

Similarities and Differences

Comparison between megakaryocytes and platelets  
Features Megakaryocyte Platelet
Size 50-100μm 2–3 µm
Nucleus Yes, nucleus is large and lobulated No
Demarcated membrane Yes, present in the intermediate zone and is a portion of the megakaryocyte which is broken off to form platelet areas (proplatelets) No
Mitochondria Yes, located in the perinuclear and intermediate zones Yes, mitochondria here regulates the pro-thrombotic function of platelets through the initiation of apoptosis [25]
Golgi Apparatus Yes, present in both the intermediate zone and the perinuclear zone Yes, present in platelets and functions as a molecular assembly line for which membrane proteins go through post-translational modification
Endoplasmic reticulum Yes, present in the perinuclear and the intermediate zones of a megakaryocyte Yes, as membrane transport also occurs between the endoplasmic reticulum and the Golgi
Secretory organelles Yes, megakaryocyte dense granules contain glycoproteins Ib and IIb-IIIa [26] Yes, the dense granules and the α-granules [27]
Zones Yes; Innermost (Perinuclear) Zone, Middle (Intermediate) Zone,Outer (Marginal) Zone Yes; Peripheral zone, Sol-gel zone, Organelle zone, Membranous zone
Function The most important role of a megakaryocyte is to produce platelets so that blood count is kept at a 'homeostatic level' and within a normal healthy range (150,000-400,000 platelets/μL of blood). Platelets are primary effectors of 'primary haemostasis', which is the process of blood clotting at the site of injury to repair damaged endothelium

Development and Maturation Process

Megakaryopoiesis Stages

Figure 7. Overview of Megakaryopoiesis with level indications of the major factors contributing to the process.

Haematopoietic Stem Cells

Haematopoietic Stem Cells (HSCs) are the precursor cells for all blood cell types, including megakaryocytes which produce platelets. As a type of multipotent progenitor cells (MPP), HSCs have the ability to regenerate themselves and can proliferate and differentiate into other blood cell types. These include red blood cells (erythrocytes), leukocytes (both granulocytes and lymphocytes), macrophages, and megakaryocytes. In the megakaryocyte (MK) lineage, HSCs differentiate into common myeloid progenitors (CMP) under the effects of the cytokine thrombopoietin and GATA1 transcription factor. [28]

Figure 8. Histological image of megakaryoblasts (top) and a mature megakaryocyte containing multiple lobes of nuclei during endomitosis (bottom)

Common Myeloid Progenitor Cells

Common myeloid progenitors (CMPs) are the first differentiation step in myeloid cell production, which includes granulocytes, erythrocytes, and megakaryocytes. Differentiation from HSCs to CMPs occurs when there is significant transcription mediated by GATA1 DNA-binding proteins and when large numbers of c-mpl receptors (thrombopoietin receptor) are activated by thrombopoietin, instead of IL-7R (interleukin 7 receptor) - although other IL growth factors such as IL-3 and IL-6 contribute to its proliferation and growth. Under the effects of IL-7, HSCs will instead be led to produce the CLP (common lymphoid progenitor cells) line which incorporates lymphocytes. [28]

Megakaryocyte-erythroid progenitors

Megakaryocyte-erythroid progenitors (MEP) are derived from CMPs under high levels of GATA1 and erythropoietin. As the name suggests, MEPs will differentiate into either erythrocytes or megakaryocytes. Commitment to enter megakaryocytic lineage can be observed when MEPs are in high levels of thrombopoietin (TPO) which acts on c-mpl receptors, and low erythropoietin levels. [28][29]

Megakaryocyte progenitors (Megakaryoblasts)

After committing to megakaryocytic pathway and still under high levels of TPO, MEPs stop themselves from producing erythrocytes by transforming into megakaryoblasts or megakaryocyte progenitors. Megakaryoblasts can be identified through its large and densely staining nucleus and high platelet peroxidase activity[30] which induces formation of tubular system interconnecting proplatelets during endomitosis. [31]

Mature Megakaryocytes and Endomitosis

Maturation of a megakaryocyte is started when a process called endomitosis has occured. Endomitosis is unique to megakaryocytes, giving the cells their polyploid characteristics, which significantly increases platelet production. Endomitosis requires the progenitor cells' ploidy to be 4N for the process to functionally start. By skipping the second phase of anaphase (formation of central contractile ring) and cytokinesis completely under the effects of RhoA (see subheading below), the cell maintains its integrity while only the nucleus divided into four parts. When compared to normal mitosis, endomitosis has the same cell cycle of G1, S, and G2. However, MKs show a significantly shorter M stage which confirms the lack of some phases compared to normal mitosis. [32] Ultimately, megakaryocytes will start deforming and releasing proplatelets post-endomitosis into the circulation.

Megakaryopoiesis Factors

GATA1 binding protein

GATA1 is a specific protein/transcription factor binding to GATA the section of the chromosomal DNA of haematopoietic stem cells, allowing maturation and differentiation of the cells into various blood cell types (erythrocytes, megakaryocytes, mast cells, and eosinophils specifically). [33] Lack of GATA1 in megakaryocytic HSCs was proven to cause developmental stasis and causing unrestricted proliferation, unlike in proerythroblasts (erythrocyte precursor) which will undergo apoptosis. [34] In addition, GATA1 binding activity locks the lineage to favor megakaryocyte production rather than other myeloid cells and erythrocytes[28], while also maintaining cell cycles of the precursor cells by interacting with E2F1 gene which regulates cell cycle and controlled apoptosis, avoiding various blood conditions such as leukemia. [35]

NF-E2

NF-E2 is another transcription factor vital to the final steps of megakaryocyte maturation, particularly granule and proplatelet formation. p45 is the specific subunit which binds to the promoter region of the genes which regulate the production of the components required during the final stages of megakaryopoiesis. A p45 subunit from NF-E2 knockout experiment on mice by Shivdasani in 1994 showed that the mice without NF-E2 binding will suffere from severe thrombocytopenia, similar to the overexpression of E2F-1 gene from the lack of GATA-1. [36][37]

Thrombopoietin

Thrombopoietin (TPO) and other growth / transcription factors inhibit erythroid growth and enhance megakaryopoiesis, regulating differentiation of haematopoietic stem cells to favor megakaryocyte production in haematopoiesis. [38] As mentioned earlier, thrombopoietin affects the firing rate of c-mpl receptors on HSCs, leading to differentiation to CMPs instead of CLPs. [28] Detailed signalling mechanism of the TPO receptors is described under the signalling section of the page.

Figure 9. Effect of RhoA on maturation of Megakaryocytes[39]

RhoA

RhoA, an intracellular signaling protein, was proved to play important role in the development of megakaryocytes, particularly during cytokinesis stages in endomitosis. RhoA acts using GTP to block contractile ring formation, ultimately stopping cytokinesis, forcing the cell to immediately re-enter G1 phase. In an experiment by Suzuki et al., it was found that RhoA-negative megakaryocytes were larger, contained higher numbers of chromosomes (ploidy), and were rapidly releasing immature, unstable platelets which were immediately discarded by the circulation system. RhoA-positive cells (control) showed normal maturation speed and completed development of platelets before their release. [39]

Interleukins

Interleukin activities are essential in megakaryopoiesis pathway, especially in the early stages. IL-3 and IL-6 are the main growth factors for CMPs, while simulataneously blocking IL-7 activity to inhibit CLP production. [28]

Megakaryocytic Signalling

Signalling within megakaryocytes plays a crucial role as it allows the cell to adapt to changes within the environment. Cell functions are also mediated in this manner in response to ligands that are present and will bind to receptors. One of the main important signalling pathways is the TPO: cMpl pathway. This is an example of a growth factor receptor kinase pathway.

Thrombopoietin receptor

The receptor for the ligand thrombopoietin (TPO) is simply known as the thrombopoietin receptor (TPR), which is a monomer receptor using the receptor kinase pathway. Its other names include the myeloproliferative leukemia protein or cluster of differentiation (CD110). It is encoded by the myeloproliferative leukemia virus (MPL) oncogene and the common name given to the human homologous of this oncogene is c-Mpl belonging to the cytokine receptor superfamily. [40] c-Mpl is abundantly principally located on hematopioetic stem cells and the action of binding to TPO and subsequent signalling cascades allows for the difference of megakaryocytes. c-Mpl is also known to play a role in platelet formation. [41] High levels of circulating TPO drives the differentiation process whereas anti-sense oligodeoxynucleotides of c-mpl inhibits the formation of megakaryocytes, consequently hindering platelet formation. The structure of the CD110 protein encoded by the c-mpl gene is such that there are 635 amino acids in the transmembrane region along with 2 extracellular cytokine receptor regions and 2 intracellular cytokine receptor box motifs. Binding of TPO to the c-Mpl receptor coherently initiates the JAK/STAT cascade.

Figure 10. TPO: c-Mpl signalling pathway.

JAK2/STAT3 signalling

Steps involved:

1. TPO bings to two CD110 proteins attached to JAK2s. [42]

2. JAK2s dimmerise.

3. STAT3 binds to JAK2 and also become phosphorylated.

4. Phosphorylated STAT3 ceases to bind to JAK2s and dimerise.

5. Phosphorylated STAT3s moves into the nucleus and partakes in the "formation of specific DNA-binding complexes" enhancing megakaryopoiesis. [43]

Function and Role

Platelet Production

Figure 11. Overview of the biological phases involved in platelet generation from HSCs and subsequent platelet activation. [44]

The most important role of a megakaryocyte is to produce platelets such that their blood count is kept at a 'homeostatic level' or within a normal healthy range (150,000-400,000 platelets/μL of blood). The main mode of platelet production in megakaryocytes is regulated by the hematopoietic cytokine 'thrombopoietin' which promotes alterations in the membrane of these cells and finally the formation of 'proplatelet processes'. [45] During the process of 'proplatelet formation', megakaryocytes will form "microtubule - dependent extensions of elongated pseudopodal structures" that will eventually break off into platelets. [45] In humans, these platelets will lack a nucleus and appear as small irregularly shaped cells. Platelet generation occurs within the bone marrow where megakaryocytes are solely located and they detach from the proplatelet processes in 'vascular sinusoids' where they enter the blood circulation. [46]

Regulation of Skeletal Homeostasis and Bone Development

Many studies have illustrated that there is a complex interplay between megakaryocytes and bone development. [47] [48] It has been established that only immature and mature megakaryocytes are capable of enhancing the process of osteoblast proliferation whilst reducing the formation of osteoclasts at virtually the same level. [47] As the number of megakaryocytes increase, so does the level of osteoblast proliferation and hence the population of megakaryocytes in the bone marrow is tightly regulated such that there is a balance between osteoclast and osteoblast numbers and activity. [47] It has therefore been suggested that abnormalities in megakaryocyte function may contribute to many metabolic bone and joint disorders such as 'osteoperosis' and 'rheumatoid arthritis'. [49] [48]

Figure 12. Summary of Megakaryocyte Functions.

Maintenance of Haematopoietic Stem Cells in the Quiescent State

Megakaryocytes have been shown to maintain HSC quiescence during homeostasis and are able to promote the repletion of HSCs following chemotherapeutic stress. [50] Many cell types in the bone marrow stroma have been identified as HSC-regulating niche cells. However, whether the progeny of a HSC is recruited as a HSC niche cell is not clearly known and is thought to be regulated by megakaryocyte secretions. [50] It has been demonstrated that megakaryocytes have physical interactions with HSCs in mice bone marrow and that a deficiency of megakaryocytes will lead quiescent HSCs to become activated and thereby proliferate rapidly. [50] In comparison to other stromal niche cells, megakaryocytes were revealed to express higher levels of biologically active transforming growth factor β1 (TGF-β1) through analysing RNA sequencing data. When MKs are removed from the bone marrow, the levels of functional TGF-β1 protein and nuclear-localized phosphorylated SMAD2/3 (pSMAD2/3) is significantly lowered in the resident HSCs. [50] This insinuates that the HSCs are kept in the quiescent state through the TGF-β-SMAD signalling pathway triggered by megakaryocyte secretions. It has also been suggested that when the bone marrow environment is under stress such as during chemotherapy, megakaryocytes release fibroblast growth factor 1 (FGF1) which appear to override the inhibitory signal induced by TGF-β1. This had temporarily enabled the quiescent HSCs to become activated and clonally expand. [50]

Platelet Function

Figure 13. The role of platelets in haemostasis: platelet adhesion, aggregation and formation of a platelet plug.

Haemostasis and Vascular Repair

Platelets are anucleated cellular fragments produced by megakaryocytes and are the primary effectors of 'primary haemostasis'. This is the process of stopping bleeding at the site of injured endothelium and therefore enabling repair of the wounded blood vessel wall. [51] When the endothelial layer of a small blood vessel is damaged, subendothelial collagen is exposed and the platelets will be activated and adhere to the site of injury through the formation of pseudopods.[52] This is mediated by platelet binding to von Willebrand Factor which is released and found lining the subendothelial collagen. [52]

Following platelet adhesion, the activated platelets will release ADP and thromboxane A2 to recruit more circulating platelets to the growing platelet aggregate. [53] Various signalling molecules will be released that will promote vasoconstriction and lead to the generation of a 'platelet plug'.[51] This mass aggregate of activated platelets will prevent the leakage of blood from the wound in the vascular wall. [51]

Simultaneously, a 'coagulation cascade' will occur in which inactive coagulation factors within the plasma will become activated. They will subsequently interact with one another in a complex series of catalytic reactions to ultimately form an insoluble protein called 'fibrin'. This collagen fibre will then deposit on the platelet plug in order to stabilise it and thereby seal the vascular lesion rigidly. [51] Red blood cells will then become trapped in the 'fibrin mesh' embedded on the platelet plug and this strengthens the blood clot. This entire process is known as 'secondary haemostasis'

Eventually, the endothelial cells will undergo cell division and eventually fill in the vascular lesion whilst the basement membrane of the blood vessel will be repaired by fibroblasts. As this process occurs, the blood clot which is a meshwork of platelets, red blood cells and fibrin will become dissolved by an enzyme called 'plasmin'. The final outcome is a completely healed blood vessel. [54]

Modulation of Immune Response

Platelets are small cells of the blood that become activated by an array of stimuli, including infection, inflammation, and injury. When activated, platelets are stimulated to rapidly release a large diversity of bioactive mediators at the site of injury or infection. These molecules are involved in the regulation of innate immune cells, the activation of endothelial cells, and modulating systemic immune responses. [55]

Their unique 'sentinel role' and active involvement in the early signalling stages of the immune response is advantageous due to their vast abundance in the circulation and extreme sensitivity to an array of stimuli. [55]

Furthermore, platelets also contribute to the modulation of the adaptive immune response through the following ways:

  • Modulate dendritic cell activation such that their is an increase in antigen presentation to T cells. [56]
  • Triggers the CD40-dependent pathway in T cells which results in the release of mediators that activates a positive feedback loop, recruiting more T Cells for an enhanced immune response [57]
  • Stimulate B-cells to produce IgG antibodies especially under conditions when there is a limited number of adaptive immune cells. [58] [59]
  • Work in coordination with T cells to accelerate the formation of germinal centres. [59]

Modulation of Inflammatory Process

Platelets may also have a potential role in modulating the inflammatory process at sites of infection or injury through interactions with leukocytes and the secretion of various inflammatory mediators. Their generation is unregulated in response to inflammation and they will be directed to the sites of injury to act as the cellular effectors of haemostasis. [60] An example of their involvement in acute inflammation is their ability to release an eicosanoid called 'dioxolane A3'. This lipid inflammatory mediator has the ability to recruit neutrophils and stimulate their antimicrobial activities in the early stages of tissue infection and injury. [61]

Pathology

Essential Thrombocythaemia

Essential thrombocythemia (ET) or essential thrombocytosis, is a rare chronic disorder that is characterised by an abnormally high production of platelets and megakaryocytes in the bone marrow. This overproduction of platelets in the circulation can progressively lead to the formation of thrombi which can obstruct the blood flow through blood vessels around the body. [62] If left untreated, blood clots may develop in critical arteries such as those supplying the brain and heart (coronary artery) and this can result in life-threatening complications such as strokes and myocardial infarction. [63] Consequently, treatment is generally prophylactic and to reduce the risk of thrombohaemorrhagic events from occurring. [64]

Signs and Symtpoms

These are general descriptions of the signs and symptoms presented in patients with Essential Thrombocytosis:

Signs and Symtpoms Essential Thrombocytosis
More Common
  • Headache
  • Dizziness or lightheadedness
  • Chest pain
  • Weakness
  • Fainting
  • Temporary vision changes
  • Numbness or tingling of the hands and feet
  • Redness, throbbing and burning pain in the hands and feet (erythromelalgia)
  • Mildly enlarged spleen
Less Common
  • Nosebleeds
  • Bruising
  • Bleeding from your mouth or gums
  • Bloody stool

Note: This has not been referenced as it contains general information compiled from organisation websites.

Pathophysiology

The pathophysiology of essential thrombocytosis is not completely understood but is possibly associated with mutations in at least one of the following three genes: Janus kinase 2 (JAK2), Calreticulin (CALR), Myeloproliferative leukemia virus oncogene (MPL).[65] Each of these genes encode for proteins that play a vital role in the TPO:c-Mpl signalling pathway and mutations found in the majority of ET patients lead tocontinual activation of this pathway. [65] These mutations generally lead to the overproduction of platelets and facilitate this through different mechanisms. JAK2 mutations have been shown to turn on the TPO receptor permanently and thereby leading to clonal proliferation of megakaryocytes despite the absence of exogenous TPO. [66] These mutations are found in the blood cells of 50-60% of ET sufferers. [67] The MPL gene encodes for the TPO receptor protein which is central to the growth and proliferation of megakaryocytes in the bone marrow and mutations in this particular gene account for only about 3-5% of ET cases. [66] Mutations in the MPL gene often lead to 'constitutive activation' of the TPO receptor protein which means that it is constantly active to stimulate the growth of megakaryocytes and their release of platelets into the circulation, without the need of TPO binding. [68]

There is an abnormal increase in the production of platelets by megakaryocytes and this is reflected through elevated platelet counts in patients with ET. The cause of this increase in platelet production has not been clearly identified but has been speculated to be due to:

  • Autonomous production
  • Increased sensitivities to cytokines such as IL-3
  • Decreased effect of platelet-inhibiting factors such as TGF-β
  • Defects in the accessory cell microenvironment

Treatment

Treatment options for ET are generally non - invasive and must be provided early in diagnosis to prevent the development of disease complications such as transient ischemic attack due to thrombosis. [69] [70] The main clinical goal of treatment is to reduce thrombohemorrhagic events from occurring and this can be carried out by utilising drugs that prevent platelet aggregation and lower platelet levels to <400 × 10^9/L (platelet concentration in a healthy individual). [62] The table below briefly outlines the effects of the primary drugs used to treat ET:

Treatment Agent Effects of the Drug
Interferon alfa
  • Suppresses the proliferation of hematopoietic progenitors and so it effectively reduces the production of megakaryocytes in the bone marrow. [71]
  • Reduces the level of platelet production and hence decreases the chance of forming thrombi which can lead to stroke. [71]
  • Increases serum lactate dehydrogenase (LDH) which is responsible for energy production in blood cells. [72]
  • Increase the population of myeloid cells in the peripheral circulation. [72]
Hydroxicarbimide
  • Significantly reduces platelet count. [73]
  • Strongly prevents thrombosis which is the major source of morbidity in ET. [73]
  • Reduces reticulin fibrosis in essential thrombocythemia. [73]
  • Inhibits the enzyme, ribonucleoside diphosphate reductase and so blood cells go into G1/S arrest in the cell cycle. [69]
Anagrelide
  • Prevents platelet aggregation by inhibiting cyclic AMP phosphodiesterase. [74]
  • Specific for the megakaryocyte line and thereby does not affect red or white cell progenitor proliferation.
  • Reduces megakaryocyte size, ploidy and maturation.[75]
  • Reduces platelet production by inhibiting megakaryocyte colony development. [75]
  • Speculated to inhibit megakaryocyte proliferation by repressing expression of the transcription factors, GATA-1 and FOG-1.[76]


Video tutorial on Essential Thrombocythemia

Congenital Amegakaryocytic Thrombocytopenia (CAMT)

Congenital amegakaryocytic thrombocytopenia (CAMT) is a a rare autosomal disorder that affects newborns following the first 3 days of life. It is characterised by a defect in the TPO:c-MpL signalling pathway which leads to insufficient production of megakaryocytes from HSCs (megakayocytopoiesis), leading to abnormally low levels of platelets in the bloodstream. [77] In CAMT, mutations can be found in exons 2,3 and 5 of the c-MPL gene which encodes for the thrombopoietin (TPO) receptor, c-MPL. This consequently prevents TPO from binding effectively to the c-MPL receptor, thereby leading to the loss of signalling for megakaryocyte production from HSCs along with their development and maturation. [78] As a result, CAMT patients often display high levels of serum TPO and this is the body's attempt to maintain 'homoeostasis' in the bone marrow. [78] Due to the development of thrombocytopenia in CAMT, excessive bleeding is a primary concern as platelet formation and clotting becomes impaired due to the significant reduction in platelet count.

Clinical Presentation

This table outlines the typical and distinctive features of Congenital Amegakaryocytic Thrombocytopenia in a clinical context:

Clinical Presentation Congenital Amegakaryocytic Thrombocytopenia
Typical Features
  • Thrombocytopenia and megakaryocytopenia, which is characterised by abnormally low numbers of platelets and megakaryocytes respectively.
  • No physical abnormalities are usually noticeable in the structure of platelets or megakarycoytes under microscopic examination. [79]
  • Absence of megakaryocytes in the bone marrow [80]
  • A common pattern is often seen in neonates with CAMT, whereby platelet counts reduce to as low as (50-100)x 10^9 platelets/L at 4-5 days since birth and they return to normal at 7-10 days (150 x 10^9 platelets/L). [81]
  • However,the majority of neonates have late - onset thrombocytopenia which usually occurs after 3 days of life which is characterised by platelet counts rapidly reaching below 50 x 10^9 platelets/L. [81]
  • Excessive Bleeding which can occur in: cutaneous, gastrointestinal, pulmonary, and intracranial haemorrhage. [82]
  • Even though the megakaryocyte count may be within normal range on an initial bone marrow aspirate, CAMT should not be excluded from the differential diagnosis of thrombocytopenia within the first year of life.
  • Development of 'aplastic anaemia' in CAMT patients who have survived beyond birth due to impaired erythrocyte production caused by the lack of MPL signalling in HSCs. [80]
Distinctive Features Since CAMT is a genetic disorder, there are distinctive features that doctors observe in patients with the disease and these include: [83]
  • Familial history of thrombocytopenia, especially parent-child or maternal uncle-nephew.
  • No observable platelet response to autoimmune thrombocytopenia therapies.
  • Blood smears present diagnostic features such as abnormal sized platelets, absence of platelet alpha granules, Dohle-like bodies or microcytosis.
  • Bleeding out of proportion to the platelet count.
  • Onset at birth.
  • Associated clinical features such as renal failure, high tone hearing loss, cataracts absent radii, mental retardation or the development of leukaemia.
  • A persistently low platelet count over several years.
  • Some patients may present with petechial purpura, cranial hematoma or recurrent rectorrhagia.

Pathophysiology

In the majority of patients with CAMT, there is a mutation in the gene for the thrombopoeitin receptor, c-Mpl and this leads to elevated levels of TPO in their blood serum. [84] Effective binding of TPO to the c-Mpl receptor is crucial for the activation of both early and late phases of megakaryocytopoiesis along with increasing structural features of megakaryocytes such as their size, ploidy and also numbers. This binding also plays a central role in promoting the expression of platelet-specific markers which is needed for platelets to carry out their major roles such as adhering to vascular lesions. [51] The TPO:c-Mpl signalling pathway is also vital for the maintainence of the HSC population within the bone marrow and so a dysfunction in this pathway can eventually give rise to both thrombocytopenia and pancytopenia in CAMT patients. Furthermore, this signalling pathway has been observed to reduce brain injury in strokes and improve sensorimotor functions and so abnormalities can lead to impaired cognitive function.[85] It has been been determined that animals with a deficiency in TPO or c-Mpl, have a 90% reduction of megakaryocytic precursor cells and a 60%–80% decrease of both erythroid and myeloid progenitors.[86] As a result, patients often develop persisting thrombocytopenia and 'aplastic anaemia' (low erythrocyte count) which leads to excessive bleeding and lack of sufficient oxygen supply to bodily tissues respectively.

TPO is a cytokine that plays a unique role in the regulation and maintenance of cells found in haematopoietic tissues such as the bone marrow, spleen and the fetal liver. This is because the TPO receptor, c-MPL is restricted to the surface of cells within these tissues. When TPO binds to c-Mpl on the surface of megakaryocytes, it regulates megakaryopeoiesis by influencing the development and maturation of megakaryocytes. It is also the primary signalling molecule required for platelet production from megakaryocytes. This cellular response is triggered by the activation of tyrosine kinase (Tyk2) and Janus kinase (JAK2) family members, which in turn phosphorylate Stat5 and Stat3. [87] These transcription factors then translocate to the nucleus and upregulate the transcription of Stat responsive genes.[87]

CAMT is classified as a bone marrow failure syndrome and manifests itself from birth or is congenital. There is still more to be learnt about the pathophysiology of bone marrow failure in CAMT but current research has illustrated that defects in the activation of TPO:c-Mpl signalling pathway is the most likely cause of this disorder.[84] This is because the stimulation of c-Mpl with TPO is vital for the growth and maintenance of the HSC population and so their ability to differentiate into the various haematopoietic cell lineages and to self-renew is impaired in CAMT. [88] [89] In vivo and in vitro murine models utiling c-Mpl −/− mice, have displayed a similar deficiency in megakaryocytes with a severe reduction in platelet count and high serum TPO levels, as seen in humans affected by CAMT. [89] Although CAMT is often referred to as a platelet disorder, the majority of patients also develop aplastic anemia.within the neonatal period. However, the age at which aplasia manifests is dependent on the level of impairment in the TPO:c-Mpl signalling. The c-Mpl knockout mice also exhibits a global reduction in HSCs and an eventual onset of pancytopenia (deficiency in all 3 cellular components: platelets, RBCs and WBCs) compaired to the c-Mpl + mice and this was partly due to the expression of the inflammatory cytokines, TNF-α and IFN-γ.[88] Hence, it is apparent that a single defect in the TPO:c-Mpl signalling pathway leads to the entire haematopoietic system to become dysfunctional in CAMT. [90]


Figure 14. Simple model of human megakaryopoiesis and erythropoiesis regulated by MPL signalling.

Treatment

CAMT is a genetic disorder and it's clinical manifestations are therefore visible since birth. This disease has a very poor prognosis and is fatal unless a bone marrow (BM) transplantation is surgically provided to the patient. [80] The purpose of performing a BM transplantation is to introduce normally functioning HSCs into the patient's bone marrow. [80] Eventually, the pool of defective HSCs will be replaced by normal ones which are capable of dividing into the different types of haematopoietic cells including erythrocytes and megakaryocytes which are particularly deficient in those afflicted by CAMT. [80] However, before curative BM transplantation is administered to CAMT patients, they are usually provided with repetitive transfusions of erythrocytes and/or platelets. [82] This is because the erythrocyte, platelet and leukocyte counts in the blood of most CAMT patients is abnormally low and this is because each haematopoietic lineage has its own distinct dependency on MPL signalling.[82]

OMIM

The Future of Megakaryocytes

The future of megakaryocytes is still bright as there are many unanswered questions regarding differentiation and maturation just to name a few. Many scientist and laboratories today are looking at ways in which having a better understanding of megakaryocytes will contribute in aiding those suffering from megakaryocytic abnormalities.

Genetic manipulation

Recent laboratory experiments have mimicked platelet function with origins that of murine and human. The ability to create viable platelets and put them to use in humans would be of significance to those suffering from abnormal plate function. A major drawback is that platelets are anucleate and are "therefore not amenable to direct genetic mutations". [91] Megakaryocyte like cell lines have been one of the lineages used in laboratories in order to better understand platelet function. Future approaches may see direct gene manipulation in cell-lineages and aid those with platelet abnormalities.

Sphingosine 1-phosphate on Proplatelet Formation

As not all the processes involving the maturation of megakaryocytes leading to platelet production is known, one way to better understand these processes may lie in looking at the components of blood and its effect on megakaryocytes. The noteworthy study by Zhang et al. (2012) [92] has raised questions in this field as it was discovered that the lipid mediator sphingosine 1-phosphate (S1P) elicits megakaryocitic proplatelet extensions leading to production of platelets into blood circulation. The study was conducted in mice and saw those who lacked the S1P receptor (S1pr1) were prone to thombocytopenia. Further study of this phenomena may lead to the incorporation of sphingosine 1-phosphate when treating patients with thombocytopenia.

Estrogen and Androgen Receptors

The follow-up study by Khetawat et al. (2010) demonstrated that estrogen receptors (ER) along with androgen receptors (AR) located on megakaryocytes affects platelet formation. Platelet formation between females and males was found to be varying in response to differing testosterone levels in test subjects. The availability and abundance of (ERs) and (ARs) in both genders may be the key in developing techniques that could be able to aid in decrease in incidence of thrombotic and atherosclerotic diseases. [93]

Therapies for Bone Disorders

Megakaryocytes are known to play a key role in 'skeletal homeostasis' by increasing the proliferation of osteoblasts through direct cell - cell contact when the activity of osteoclasts is abnormally higher. However, the mechanisms are still being researched and have potential in the development of novel anabolic therapies to treat bone disorders such as osteoperosis where there is significant bone loss. [94]

Glossary

Acronym Expansion Definition
ADP Adenison Diphosphate An important organic compound in metabolism and is essential to the flow of energy in living cells.
AR Androgen Receptor Also known as NR3C4 (Nuclear Receptor, subfamily 3, group C, member 4) is a type of nuclear receptor activated by binding to either of the androgenic hormones - be it testosterone or dihydrotestosterone - in the cytoplasm and then translocating the hormone into the nucleus.
CALR Calreticulin A multifunctional protein that binds with Ca[2+] ions and renders them inactive. It is utilized when misfolded proteins occur in the endoplasmic reticulum and prevents them from being exported, somewhat like a molecular form of quality control.
CAMT Congenntal Amegakaryocytic Thrombocytopenia A rare disorder characterized by isolated thrombocytopenia and megakaryocytopenia in infancy.
CLP Common Lymphoid Progenitor cells Precursor cells which will differentiate into lymphoid cells - lymphocytes.
CMP Common Myeloid Progenitor cells Precursor cells which will differentiate into myeloid cells, including granulocytes (eosinophils, basophils), erythrocytes, and megakaryocytes.
ER Estrogen Receptor A protein found inside and on cells that are activated by estrogen.
ET Essential Thrombocytosis Achronic disorder characterized by the body producing excessive amounts of platelets.
GATA1 Guanine-Adenine-Guanine-Adenine-1 A specific protein/transcription factor binding to 'GATA' section of the chromosomal DNA of hematopoietic stem cells, allowing maturation and differentiation of the cells into various blood cell types.
GPIIIb/IIIa and GPVI Glycoprotein 3b/3a Required for platelet adhesion.
HSC Haematopoeitic Stem Cells The precursor cells for all blood cell types, including megakaryocytes which produce platelets.
JAK2 Janus Activating Kinase 2 A cytoplasmic enzyme responsible for catalyzing the transfer of a phosphate group from a nucleoside triphosphate donor, such as ATP, to tyrosine residues in proteins.
MEP Megakaryocyte-Erythyroid Progenitors Cells after CMP differerentiation and locking into myeloid lineage, will differentiate into either erythrocytes or megakaryocytes.
MK Megakaryocyte The precursor cells to platelets.
MPL Myeloproliferative Leukemia A viral oncogene.
RhoA Ras Homolog gene family, member A A small protein primarily asociated with the cytoskeleton and actomyosin contractility.
TGF-β-SMAD Transforming Growth Factor Plays a role in growth differentiation and development. It is a secreted protein that performs cellular functions such as growth of cells, proliferation differentiation and apoptosis.
TPO Thrombopoietin gene A glycoprotein hormone produced by the liver and kidney which regulates the production of platelets through stimulating production and differentiation of megakaryocytes.
WASp Wiskott-Aldrich Syndrome protein A protein expressed in cells of the hematopoietic system. It is involced in the transduction of signals from receptors on the cell surface to the actin cytoskeleton.


Term Definition
Anaphase A phase of mitosis in which the chromosomes have separated and begun to be pulled to opposite poles of the cell.
Anagrelide An orally administered agent used in the hydrochloride salt form to reduce elevated platelet counts and the risk of thrombosis in treatment of hemorrhagic thrombocythemia.
Anucleate A cell without a nucleus.
Centioles Each of a pair of minute cylindrical organelles near the nucleus in animal cells, involved in the development of spindle fibres in cell division.
Chemokine Is part of a family of small cytokines, or signaling proteins secreted by cells. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines.
Chemotaxis The movement of an organism in response to a chemical stimulus. Somatic cells, bacteria, and other single-cell or multicellular organisms direct their movements according to certain chemicals in their environment.
Cytokinesis The division of two daughter cells cytoplasm following mitotic division of the nucleus. Involves the formation of an actin contractile ring around the equator of the cell after nuclear division.
Cytoskeleton Collective term for the cytosol, organelles and cytoskeleton located outside the nucleus and under the plasma membrane.
Discoid A disc or lens-shaped object.
Effectors An organ or cell that acts in response to a stimulus.
Endomitosis The division of chromosomes that is not followed by nuclear division and that results in an increased number of chromosomes in the cell.
Endoplasmic Reticulum The cytoplasmic organelle forming a single membrane enclosed space continuous with the outer membrane of the nuclear envelope. It has 2 functional domains described by the presence or absence of ribosomes, the rough endoplasmic reticulum and smooth endoplasmic reticulum respectively.
Fibrinogen A soluble protein present in blood plasma, from which fibrin is produced by the action of the enzyme thrombin.
Fibroblasts A cell in connective tissue which produces collagen and other fibres.
Fibronectin A glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Similar to integrins, fibronectin binds extracellular matrix components such as collagen, fibrin, and heparin sulfate proteoglycans.
Glycoproteins A class of proteins which have carbohydrate groups attached to the polypeptide chain.
Golgi apparatus Cytoplasmic organelle consisting of a stack of individual membrane enclosed spaces located near the nucleus. Functions to process and modify proteins for exocytosis and endocytosis pathways.
Granulocytes A group of white blood cells with secretory granules in their cytoplasm, e.g. an eosinophil or a basophil.
Haemostasis The stopping of a flow of blood.
Hematopoietic Another name for stem cells that give rise to all the other blood cells through the process of haematopoiesis. They are derived from mesoderm and located in the red bone marrow, which is contained in the core of most bones.
Hydroxyurea An anti-cancer chemotherapy drug, offically classified as an 'antimetabolite'.
In Vitro A process performed or taking place in a test tube, culture dish, or elsewhere outside a living organism.
In Vivo A process performed or taking place in a living organism.
Isoform Any of two or more functionally similar proteins that have a similar but not identical amino acid sequence and are either encoded by different genes or by RNA transcripts from the same gene which have had different exons removed.
Lobulated A lobulation is an appearance resembling lobules.
Macrophage A large phagocytic cell found in stationary form in the tissues or as a mobile white blood cell, especially at sites of infection.
Megakaryocyte is a large bone marrow cell with a lobulated nucleus responsible for the production of blood thrombocytes (platelets), which are necessary for normal blood clotting.
Microfilaments Cytoskeleton filament system required for cell shape and motility. The name comes from the filaments being the smallest in cross-sectional size of the three filament systems including intermediate filaments and microtubules.
Microtubules Cytoskeleton filament system required for intracellular transport and motility. The name comes from the system filaments forming "tubes", they are also the largest in cross-sectional size of the three filament systems including microfilaments and intermediate filaments.
Oligodeoxynucleotide A short single-stranded synthetic DNA molecule containing a cytosine triphosphate deoxynucleotide followed by a guanine triphosphate deoxynucleotide.
Osteoblast A cell which secretes the substance of bone.
Perinuclear Situated or occurring around the nucleus of a cell.
Plasmin An enzyme, formed in the blood in some circumstances, which destroys blood clots by attacking fibrin.
Ploidy The number of sets of chromosomes in a cell, or in the cells of an organism.
Polyploid Polyploid cells and organisms are those containing more than two paired (homologous) sets of chromosomes. Most species whose cells have nuclei (Eukaryotes) are diploid, meaning they have two sets of chromosomes—one set inherited from each parent.
Pulmonary Relating to the lungs.
Proplatelet Portion of megakaryocyte broken off to form platelet.
Pseudopodal A temporary protrusion of the surface of an amoeboid cell for movement and feeding.
Recombinant The rearrangement of genetic material, especially by crossing over in chromosomes or by the artificial joining of segments of DNA from different organisms.
Ribosomes Cytoplasmic structure formed from RNA and proteins that assembles to synthesise proteins. Ribosomes may be either free in the cytoplasm or attached to the endoplasmic reticulum, forming rough endoplasmic reticulum.
Romiplostim Is a fusion protein and hormone, analogously related to thrombopoietin, that regulates platelet production.
Sol-Gel Zone The Sol-gel zone is rich in microtubules and microfilaments, allowing the platelets to preserve their discoid shape.
Splenomegaly Abnormal enlargement of the spleen.
Stem Cell Term used to describe a cell with the potential to reproduce itself indefinitely, as well as differentiate into any other embryo tissue cell types. There are also a number of different specialised stem cell definitions: totipotential stem cell (as described above), pluripotential stem cell (capable of forming a number of different cell types), embryonic stem cell (derived from the blastocyst), cord blood stem cell (derived from placental cord blood), mesenchymal stem cell and adult stem cell (derived from adult or postnatal tissue).
Stroma The supportive tissue of an epithelial organ, tumour, gonad, etc., consisting of connective tissues and blood vessels.
Thrombocytoppenia Deficiency of platelets in the blood. This causes bleeding into the tissues, bruising, and slow blood clotting after injury.
Thrombospondin One of a family of glycoproteins (carbohydrates complexed with proteins) that are made in cells, secreted by cells, and incorporated into cells including blood platelets (thrombocytes) from which they take their name.
Thromboxane A hormone of the prostacyclin type released from blood platelets, which induces platelet aggregation and arterial constriction.
Translocating In the medical field, 'transloatinng' refers to the moving of a portion of a chromosome to a new position on the same or another chromosome
Transmembrane An event that exists or occurs across a cell membrane.

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