Hematology 425 Megakaryopoiesis

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Hematology 425 Megakaryopoiesis

• Russ Morrison
• October 13, 2006

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Megakaryopoiesis

• Platelets (thrombocytes) are cytoplasmic
  fragments that are released from a parent cell
  known as a megakaryocyte
• Megakaryocytes are large, 80-150 um
• Megakaryocytes are found predominantly in the BM
  and to a lesser degree in the spleen and lungs
• As with RBCs and WBCs, megakaryocytes develop
  from a PSC influenced by CSFs
• These CSFs are produced by macrophages,
  fibroblasts, T lymphocytes, and stimulated
  endothelial cells

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Megakaryopoiesis

• As with RBCs and WBCs, interleukins play an
  influential role, particularly IL-3, IL-6 and
  IL-11
• Meg-CSF and G-CSF together stimulate production
  of megakaryocyte progenitor cells
• Meg-CSF is thourhg to be produced by
• BM cells in response to the megakaryocytic mass.

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Megakaryopoiesis

• As the number of megakaryocytes decrease, the
  production of Meg-CSF increases
• Thrombopoietin is generated by the kidney with a
  lesser amount produced by the liver and spleen
• Thrmbopoietin binds to its receptor, C-mpl, and
  stiulates megakaryocyte growth and platelet
  production
• Thrombopoietin also stimulates the release of
  platelets from the megakaryocyte

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Megakaryopoiesis

• Role of the spleen in platelet production
• The spleen plays a key role in the regulation of
  platelet numbers
• 30 of the PB platelets are sequestered in the
  spleen at any given time
• Rapid use of platelets through clotting or
  destruction rapidly empties the splenic pool
• When the platelet count decreases, thrombopoietin
  causes maturation of the megakaryocyte to produce
  a marrow response equal to the loss of platelets

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Megakaryopoiesis

• Any increase in thrombopoietin speeds up the
  maturation of megakaryocytes
• Accelerated maturation results in fewer platelets
  produced per megakaryocyte
• Sustained usage/destruction of platelets causes
  the platelet count to fall to a level incapable
  of maintaining normal vascular and hemostatic
  integrity
• This results in a condition called acute
  thrombocytopenia

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Megakaryopoiesis
Platelet production is unique from other
hematopoietic cell production

• Megakaryocytes do not experience complete
  cellular division, but undergo a proces called
  endomitosis

• RBC and WBC precursors usually divide four times
  during maturation, producing 16 mature cells from
  each committed stem cell.

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Megakaryopoiesis

• In endomitosis, normal telophase is missing
  creating a cell with a multilobed nucleus
• Each lobe of the nucleus is diploid, 2N, and
  contains a full complement of 23 pairs of
  chromosomes capable of transcription
• Megakaryocytes are, therefore, polyploid (have
  more than 2 complete sets of chromosomes)

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Megakaryopoiesis

• During endomitotic division of the nucleus, the
  more ploidy there is, the larger the cytoplasmic
  volume will be
• Megakaryocytes may achieve 16N, or 16 chromosome
  pairs and develop as many as 16 lobes, 32N
• When the platelet turnover is in equilibrium, the
  average megakaryocyte which is 8N or 16N,
  produces 2000 to 4000 platelets

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Megakaryopoiesis

• The maturation process of the Megakaryocyte is
  from PSC to megakaryoblast to basophilic
  cytoplasmic fragments which are later release as
  platelets
• The first in the maturation sequence is called
  the megakaryoblast or MK1 cell

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Megakaryopoiesis

• Megakaryoblast (MK1)
• 10-15 um in size
• High nuclear to cytoplasmic ratio
• Single nucleus with 2-6 nucleoli
• Cytoplasm is minimal, blue, and contains not
  granules
• Not distinguishable bo microscopy alone
• At this stage may reach 50 um in size

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Megakaryopoiesis

• Promegakaryocyte (MK2)
• Reaches 80 um in size
• Develops three kinds of granules formed in the
  Golgi apparatus
• Granules are termed dense, alpha and lysosomal
  and are dispersed throught the cytpolasm

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Megakaryopoiesis

• Basophilic Megakaryocyte (MK3)
• Final divisions of the nucleus occur
• Distinct granulation patterns develop
• Cytoplasmic separation lines begin to be seen,
  outlining individual cytoplasmic fragments which
  will later be released as platelets
• Each demarcated area consists of a membrane,
  cytoskeleton, system of microtubules, canals and
  a portion of cytoplasmic granules and also has a
  store of glycogen to sustain the platelet for
  9-11 days

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Megakaryopoiesis

• Basophilic Megakaryocyte (MK3)
• The cytoplasmic fragments further develop a
  membrane with several types of glycoprotein
  receptors
• These receptors which develop during this stage
  allow activation, adherence, aggregation, and
  cross-linking of the platelet

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Megakaryopoiesis

• Megakaryocyte (MK4)
• Final stage of cell line maturation
• The mature megakaryocyte releases cytoplasmic
  fragments through marrow sinusoid fenestrations
  in a process called budding or shedding of the
  platelets
• When all platelets are released into the blood
  stream, the naked nucleus that remains is
  phagocytized by marrow histiocytes

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Megakaryopoiesis

• Megakaryocyte (MK4)
• Since thousands of platelets are shed from each
  mature megakaryocyte, fewer progenitor cells are
  needed compared to the other cell lines
• Megakaryocytes, because of their size, are
  quickly evident when present on a BM smear
• Presence of megakaryocytes in the BM without
  searching, indicates adequate production

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Megakaryopoiesis

• Megakaryocyte (MK4)
• Megakaryocytic hyperplasia indicates normal
  response to increased demand or autonomous
  proliferation as is seen in myeloproliferative
  disease
• Clustering of megakaryocytes is usually seen in
  myeloproliferative diseases

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Platelet Structure

• Platelets entering the peripheral blood have an
  average diameter of 2.5 um
• Unstimulated platelets are lentiform discs with
  smooth margins
• Platelet structure can be divided into
  peripheral, sol-gel, organelle and membrane zones
• EM work has defined the zones as well as the
  structures contained in each zone

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Platelet Structure

• Peripheral Zone
• Includes the platelet membrane which contains
  membrane-bound glycoproteins, an exterior coat
  called the glycocalyx and a cytoskeleton packed
  with actin and myosin fibrils and microtubules
• The glycoproteins of the platelet membrane
  function similarly to those of the RBC

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Platelet Structure

• Peripheral Zone
• The glycocalyx is a coat of side chains
  protruding beyond the membrane surface and
  connected to the glycoproteins
• The glycocalyx possesses platelet ABO and HLA
  antigens which act as receptors for thrombin,
  vWF, epinephrine, ADP and platelet-activating
  factor (PAF)

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Platelet Structure

• Peripheral Zone
• The activated platelet changes shape from smooth
  margined discs to spiny projections
• The microtubules and the cytoskeleton represent
  an extension of the platelet membrane winding
  inwardly throughout the platelet interior

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Platelet Structure

• Sol-Gel
• During activation and constriction of platelets,
  the open canalicular system (OCS) delivers the
  granular contents to the surface
• Multiple pores of the OCS connect internal
  contents of the platelet with the surface
• As the platelet circulates through the blood
  stream, the pores also allow plasma to enter the
  microtubules facilitating absorption of plasma
  coagulation factors

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Platelet Structure

• Sol-Gel
• Alpha granules concentrate coagulation factors,
  especially factor I (fibrinogen) and factor V
  (labile factor)
• Megakaryocytes do not synthesize plasma clotting
  factors, but platelets acquire them circulating
  through the blood
• Other blood clotting factors are collected on the
  surface of the platelet and held their by surface
  tension
• When platelets become activated, they have
  clotting factors already available by these
  mechanisms

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Platelet Structure

• Sol-Gel
• The sol-gel zone also includes a dense tubular
  system which is the primary site of sequestration
  of calcium ions
• The calcium becomes the source to drive
  calcium-dependent reactions of coagulation

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Platelet Structure

• Organelle Zone
• The interior of the platelet contains
  mitochondria, lysosomes, dense granules and alpha
  granules that are released during platelet
  activity
• The mitochondria provide oxidative
  phosphorylation of ATP energy through their
  glycolytic and citric acid cycles (EM and Krebs,
  respectively)
• Breakdown of glycogen is necessary to produce the
  ATP needed to change shape or reverse activation
  of the platelet

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Platelet Structure

• Organelle Zone
• Platelet granules are filled with constituents
  released in various phases of hemostasis
• Shape change and calcium mobilization during
  platelet activation assist the dense granules in
  releasing their contents
• The alpha granules release their contents next

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Platelet Structure

• Organelle Zone
• Alpha granules platelet-derived growth factor
  (PDGF) that causes proliferation of endothelial,
  smooth muscle, and fibroblast cells for the
  inflammation and repair process
• Alpha granules also release thrombospondin, a
  large (450-kD) glycoprotein that appears to
  enhance platelet adherence and aggregation by
  attaching to corresponding platelet receptors
• Thrombospondin is instrumental in facilitating
  cell-to-cell interactions by use of its receptor
  for platelet attachment

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Platelet Structure

• Organelle Zone
• Other compounds released from alpha granules
  inhibit heparin released by mast cells and
  basophils
• Those compounds are platelet factor 4 (PF4) and
  beta-thromboglobulin (BTG)
• Both prevent heparin neutralization of thrombin
  and other clotting enzymes
• Alpha granules also release an inhibitor called
  plasminogen activator inhibitor-a that
  neutralizes tissue plasminogen activator released
  by traumatized endothelial cells

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Platelet Structure

• Surface marker
• CMP-140 is an acquired membrane marker of
  platelets that is found on active, but not
  resting platelets
• CMP-140 is thought to be remnants of alpha
  granule membrane bound to the platelet surface
• Flow cytometry can identify this marker and
  differentiate resting and active platelets

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Platelet Structure

• Receptors
• Several glycoprotein receptors have been
  identified on the platelet membrane and internal
  surface
• The membrane receptors initiate the complex
  communication system of signal transduction
• Laboratory evaluation of platelet agonists is
  done with one stimulus at a time and is
  demonstrated in platelet aggregation studies

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Platelet Structure

• Glycoprotein receptors of the platelet include
  ADP, thrombin, vWF, collagen, fibrinogen, fibrin,
  fibrinectin, epinephrine, PAF, thrombospondin,
  and others
• One of the receptor complexes (GPIIb/IIIa) is the
  receptor for fibrinogen and fibronectin, with vWF
  helping platelets adhere and cross-link together
  in a stabilized plug with plasminogen

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Platelet Structure

• If you want to read ahead and begin to connect
  platelets and clotting factors and their
  interrelationships in hemostasis, begin with
  Chapter 42