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Chapter  13  –  Platelet  Production,  Structure   Megakaryocyte  Progenitor  
and  Function    
  Megakaryocyte-­‐Erythrocyte  
Megakaryocytopoiesis   progenitor  
 Platelets   are   anucleated   blood   cells   that  
circulate  in  amounts  of  150  -­‐  400X  109/L  
 Mean   platelet   counts   is   slightly   higher   in   TPO  (thrombopoietin),  
women  than  in  men   IL-­‐3,  IL-­‐6,  IL-­‐11  
 On   a   blood   smear   platelets   are  
distributed   throughout   the   RBC  
monolayer  at  7  to  21  per  100  X  field   BFU-­‐Meg   CFU-­‐Meg   LD-­‐CFU-­‐Meg  
 Platelets  have  a  diameter  of  2.5  𝜇m      
 Platelets   have   a   mean   platelet   volume    
(MPV)   of   8   to   10   fL   in   an   isotonic    BFU-­‐Meg,   CFU-­‐Meg   are   diploid   and  
solution   participate   in   normal   mitosis,  
 Platelets   arise   from   unique   bone   maintaining   pool   of   megakaryocyte  
marrow  cells  called  megakaryocytes     progenitor  
 Megakaryocytes:    LD-­‐CFU-­‐Meg   has   little   proliferative  
a) Large,  polyploid   capacity   and   produces   few   cells,   but  
b) 30-­‐50  μm   begins   the   progress   through  
c) Multilobulated  nucleus   endomitosis   to   reach   increased   nuclear  
d) Abundant  granular  cytoplasm   ploidy  
 Megakaryocytes   cluster   in   the    At   the   LD-­‐CFU-­‐Meg   point   of  
extravascular   compartment   adjacent   to   development,   megakaryocytes  
the   albuminal   membrane   of   venous   progenitors   enter   terminal  
sinusoid  endothelial  cells   differentiation,   as   they   lose   their   ability  
 Myelocytic   and   erythrocytic   precursor   to   undergo   normal   mitosis   but   continue  
cells,   which   locate   further   from   the   with  endomitosis  
endothelial   cells,   may   cross   the    In   specialty   laboratory,   immunologic  
megakaryocyte   cytoplasm   to   reach   probes   and   flow   cytometry   are  
sinusoid   lumen,   a   faux   phagocytosis   employed   to   identify   megakaryocyte  
known  as  emperopolesis   progenitors  
 In   a   bone   marrow   aspirate,   two   to   four    Flow  cytometric  progenitor  marker:  
megakaryocytes   may   be   identified   per   a) Stem  cell  markers  
10X  low  power  field   i) CD34   (Cluster   of  
  Differentiation)  
Endomitosis   ii) HLA-­‐DR   (Human  
 Megakaryocyte   maturation   lacks   Leukocyte  Antigen)  
telophase   and   cytokinesis   (separation   b) Platelet   glycoprotein   IIIa   (GP  
into  daughter  cells)  is  called  endomitosis   IIb/IILa,  CD41)  
 DNA   replication   up   to   32N   with   no   cell    Platelet   peroxidase,   localized   in   the   ER  
division   of  progenitors  and  Megakaryoblast,  also  
 128N   (5x   replication)   –   hematological   may   be   identified   by   cytochemical   stain  
disorder   in  TEM  
 Megakaryocytes   employ   their   copious    Identical   peroxides   activity   is   localized  
DNA  to  synthesis  of  abundant  cytoplasm,   to   the   dense   tubular   system   of   mature  
which  differentiates  to  platelets   platelets  
 2,000-­‐4,000  platelets  are  produced  from    
one  megakaryocyte    
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Terminal  Megakaryocyte  Differentiation   Features  of  the  Three  Terminal  Megakaryocyte  
 Morphologists   call   the   least   Differentiation  Stages  
differentiated   megakaryocyte   precursor     MK-­‐I   MK-­‐II   MK-­‐III  
the  MK-­‐I  stage  or  megakaryoblast   %Precursor   20   25   55  
Diameter   14-­‐18𝜇m   15-­‐40𝜇m   30-­‐50𝜇m  
 Morphologist   may   see   plasma   Nucleus   Round     Indented   Multilobed  
membrane  blebs,  blunt  projections  from   Nucleoli   2-­‐6   Variable   Not  visible  
the  margin  that  resemble  platelet   Chromatin   Homogenous   Condensed   Deeply  but  
 At   this   stage   megakaryocyte   begins   to   variably  
develop   most   of   its   cytoplasmic   condensed  
Nucleus   to   3:1   1:2   1:4  
ultrastructure,   including   procoagulant-­‐
Cytoplasmic  
laden   α-­‐granules,   δ-­‐granules   (dense   ratio  
bodies,  and  demarcation  system  (DMS)   Mitosis   Absent   Absent   Absent  
 Immunological   probes   or   cytochemical   Endomitosis   Present   Ends   Absent  
markers:   Cytoplasm   Basophilic   Basophilic   Eosinophilic  
a) CD42   and   and  
granular   granular  
Ø VWF  (Von  Willebrand  Factor)   α-­‐granules   Present   Present   Present  
adhesion  receptor   δ-­‐granules   Present   Present   Present  
b) mpl     DMS   Present   Present   Present  
Ø Fibrinogen  receptor    
c) PF4   Megakaryoblast  (Stage  I)  
Ø Flow  cytometry    19%  
d) VWF    14-­‐18  um  
Ø Immunostaining      Cytoplasm  
  ♣ Varying  shades  of  blue  
Maturation  of  Megakaryoblast   ♣ Small,  blunt  pseudopodia  
 Unique  characteristics   ♣ Small  to  moderate  amount  
♣ Youngest  cell  smaller  than  adult    Narrow  band  around  nucleus  
♣ Nucleus  become  lobulated   ♣ α  and  δ  granules  
♣ Cytoplasm   increases   in   amount   and   ♣ DMS  
becomes  more  granular    Nucleus  
♣ Asynchronization   ♣ Round,  oval,  or  kidney-­‐shaped  
 Endomitosis     ♣ Homogenous    chromatin  pattern  
 Nucleus  becomes  polyploid    
♣ 2-­‐6  nucleoli  
♣ Functional   end   products   are  
♣ 3:1  
cytoplasmic  fragments  
 
 
Promegakaryocyte  (Stage  II)  
Four  Stages  in  Megakaryopoiesis  
 25%  
 Megakaryoblast  (Stage  I  or  MK  I)  
 15-­‐40  um  
 Promegakaryocyte  (Stage  II  or  MK  II)  
 Cytoplasm  
 Granular   megakaryocyte   (Stage   III   or  
♣ More  abundant  
MK  III)  
♣ Less  basophilic  and  granular  
 Mature   megakaryocyte   (Stage   IV   or   MK  
IV)   ♣ α  and  δ  granules  
Ø Thrombocyte  or  platelet   ♣ DMS  
   Nucleus    
  ♣ Coarse  and  condensed  chromatin  
  ♣ Visible  multiple  nucleoli  
  ♣ Irregularly-­‐shaped  
 
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 Show   slight   lobulation   or   Biology  of  Megakaryocyte  
indentation    Golgi  region  
♣ 1:2   ♣ Packing  of  specific  granules  
  ♣ Distribution   throughout   the  
Granular  Megakaryocyte  (Stage  III)   cytoplasm   except   in   the   peripheral  
 56%   borders  
 30-­‐50  um    Polyribosomes  and  RER  
 Cytoplasm   ♣ Present  at  the  onset  of  maturation  
♣ Abundant    Maturation    
♣ Pinkish  blue   ♣ 4-­‐5  days  in  BM  
♣ Fine  and  diffusely  granular    
♣ Irregular  peripheral  boarder   Pathophysiology  of  Megakaryocyte  
♣ α  and  δ  granules    Escape  from  BM    
♣ DMS    Seen  in  peripheral  blood  occasionally  
 Nucleus      Seen   frequently   on   buffy   coat  
♣ Small  in  comparison  to  cell   preparation    
♣ Multiple   nuclei   or   nucleus   show    Seen  in  these  conditions    
multilobulation   ♣ CML  
♣ Coarser  chromatin   ♣ Different  forms  of  cancers  
♣ Variable  nucleoli   ♣ Myelofibrosis  
♣ 1:4     ♣ Polycythemia  vera  
  ♣ Hodgskin’s  disease  
Mature  Megakaryocyte  (Stage  IV)   ♣ Leukocytosis  following  infection  
 Cytoplasm   ♣ Post  surgery  
♣ Coarse  clumped  granules    
 Aggregate  to  bundles   Dwarf  or  Micro  Megakaryocyte  
 Bud   from   periphery   to   become    Abnormal  production  
platelet    Seen  in  cases  of    
 Nucleus     ♣ Myeloproliferative  disorder  
♣ Multiple  nuclei   ♣ Myelodysplastic  syndrome  
♣ No  visible  nucleoli    Single  lobed  nucleus  
♣ 1:1    Size  of  lymphocyte  
   Smudged  appearance  
Platelet  or  Thrombocyte    Pale  blue  and  foamy  cytoplasm    
 Size   ♣ Granular  or  agranular  
♣ 1-­‐4  um    
 Cytoplasm   Platelet  Production  or  Thrombopoiesis  
♣ Light  blue  to  purple    Cytoplasmic  granules  form  cluster  
♣ Two  parts    Network   of   tubules   develop   that   open  
 Chromomere   externally  
“ Granular   ♣ Invagination  of  membrane  
“ Centrally  located   ♣ Demarcation   membrane   system  
 Hyalomere       (DMS)  
“ Surrounds  chromomere    Fusion   of   tubules   to   form   fissures,   then  
“ Nongranular     margins,   and   plasma   membrane   of  
“ Clear  to  light  blue   individual  platelets  
  ♣ Porplatelet  process  extension  
  ♣ Pierce  through  endothelial  cells  
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 Release   of   platelets   into   peripheral    Reticulated  or  stress  platelets  
blood   ♣ 6  um  
 Nucleus  degenerate  (MPS)   ♣ Cylindrical  and  beaded  (citrate)  
 Low  ploidy    
♣ Larger  platelets   Plasma  Membrane  Receptors  
♣ Denser  and  more  functionally  active    Adhesion    
  ♣ Cellular  adhesion  molecule  
Increase  Platelet  Production    Activation    
 Number   of   megakaryocyte   in   the   BM   ♣ Thromboxane  A2  
increase    
 Size  of  the  megakaryocyte  increase   Platelet  Structure  
 Decrease   in   the   maturation   time   of    Four  anatomical  areas  
megakaryocyte   Ø Peripheral  zone  
  Ø Sol-­‐gel  zone  
Platelet  Life  Span   Ø Organelle  zone  
 9-­‐12  days  in  the  peripheral  blood   Ø Membranous  system    
 Young  platelets    
♣ Larger  and  denser   Peripheral  Zone  
 2/3  in  peripheral  blood    Plasma  membrane    
 1/3  in  the  spleen   ♣ Glycoproteins  and  proteoglycans  
 Damaged  and  nonfunctioning  platelets    Arachidonic   acid   (major   integral  
♣ Removed   by   macrophages   in   the   protein)  
spleen    Glycoprotein   Ib   –   von   Willebrand  
 Turn  over  rate   factor  
♣ 35,000  +/-­‐  4,300  per  ul  each  day      Glucoprotein   IIb   and   IIIa   –  
  fibrinogen  and  VWF  
Hormones   and   Cytokines   of    Glycocalyx  
Megakaryocytopoiesis    Albumin,  fibrinogen  other  plasma  
 TPO   proteins    
♣ Liver      
 Interleukins     Sol-­‐Gel  Zone  
♣ IL3    Microfilaments  
♣ IL6   ♣ Actin   –   anchors   glycoprotein   and  
proteoglycan  
♣ IL11  
 20-­‐30%  of  platelet  proteins  
 Stem  cell  factor  
 Globular   and   amorphous  
♣ Kit  ligand   (rest)and   filamentous   and  
♣ Mast  cell  growth  factor   contractile  (active)  
  ♣ Desmin  and  vimentin    
Megakaryocyte  Growth  Inhibitors  
 Connect  with  actin  and  tubules  
 PF4,  β-­‐thromboglobulin   ♣ Actomyosin  or  thrombasthenin    
 Neutrophil-­‐activating  peptide  2,  IL8    Contractile  protein  important  in  
  clot  retraction    
Platelet  
 Microtubules    
 Granular   cytoplasm   with   nuclear   ♣ Tubulin    
material    
 Disassemble   at   refrigerated  
 Biconvex  when  resting   temperature  (round)  
♣ Circular  to  irregular  (EDTA)  
 
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 Return   to   disc   shape   when   Platelet  Function  
warmed    Hemostasis  
♣ Contract   to   express   ά   granule    Maintaining  capillary  activity  
content      Platelet  Count  
  ♣ Rees-­‐Ecker  
Organelle  Zone   ♣ Unopette  
 Mitochondria   ♣ Automatic  count  
 Alpha  granules    
♣ PF4,   ß   thromboglobulin,   PDGF,   Reasons  Why  Platelets  are  Hard  to  Count  
thrombospondin,   VWF,   fibrinogen,   1. Easily  disintegrated  
fibronectin,  Factor  V     2. Hard  to  distinguish  from  debris  
 Delta  granules  or  dense  bodies   3. Has   the   tendency   to   adhere   to   glass  
♣ ADP,     ATP,     calcium,   serotonin,   and   surfaces  and  foreign  bodies  
magnesium   4. Unevenly  distributed  in  the  blood  
♣ Platelet  aggregation    
 Lysosomal  type  granule   Sources  of  Error  in  Platelet  Count  
♣ Aryl   sulfatase,   β-­‐glucoronidase,   acid   1. Improper  blood  collection  
phosphatase,  and  catalase   2. Inadequate  mixing  of  diluted  blood  
  3. Dirt  in  material  and  diluting  fluid  used  
Membranous  System   4. EDTA   concentration   is   greater   than   2  
 Dense  tubular  system   mg/ml,   platelet   swell   and   fragments  
♣ Derived  from  SER   results  to  increase  count    
♣ Holds  calcium  for  platelet  activation    
♣ Synthesizes  prostaglandin   Platelet  Pipetting    
 Surface   connected   canalicular   system    Less  than  50  platelets  counted,  use  WBC  
(open  canalicular  system)   pipet  (1:20  dilution)  
♣ Store   hemeostatic   proteins   in    Greater   than   500   platelets   counted,   use  
glycocalyx   RBC  pipet  (1:200  dilution)  
♣ Route  for  endocytosis      Percentage   error   in   manual   platelet  
♣ Canal   for   release   of   granule   count  is  +10-­‐22%  
constituent    
  Clinical  Significance  of  Platelet  Count  
Platelet  Composition    Low  
 Protein  –  60%   a) Thrombocytopenia  
 Lipid  –  30%   b) Acute  leukemia  
c) Anemia  
 Carbohydrate  –  8%  
d) Patients   undergoing   chemotherapy  
 Minerals,  water,  and  nucleotides  
and  radiologic  treatment  
 Enzymes  –  9%   e) Patients   with   prolonged   bleeding  
 Other  proteins   time  
♣ Glycoproteins    High  
♣ Coagulation  factors   a) Thrombocytosis  
  b) Rheumatic  fever  
  c) Asphyxia  
  d) Post  surgery  
  e) Patients   undergoing   drug   therapy  
  (leukemic  patients)  
   
   
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Platelet  Disorders  
 Bernard-­‐Soulier  Syndrome  
♣ Inherited  disorder  
♣ Thrombocytopenia,  giant  platelets  
♣ Decreased   response   to   platelet  
activation  by  thrombin  
 
 Wiskott-­‐Aldrich  Syndrome  
♣ X-­‐chromosome   linked   inherited  
disorder  
♣ Thrombocytopenia,  small  platelets  
 
 Gianzmann’s  Disease  
♣ Inherited  hemorrhagic  disorder  
♣ Severely   reduced   or   absent   platelet  
aggregation