Megakaryocytes and Platelet Formation

The Platelets: Megakaryocytes

• The common myeloid progenitor cell, or CFU-GEMM, differentiates into megakaryocytes and platelets.
• GATA1 and FOG1 transcription factors differentiate CFU-GEMM into CFU-Meg and CFU-E.
• The progenitor cell CFU-MegE differentiates from CFU-GEMM before this step.
• Cytokines and interleukins play a role in differentiation:
  • TPO (thrombopoietin)
  • Meg-CSF (megakaryocyte colony stimulating factor)
  • IL-3
• MYB, another transcription gene, can suppress megakaryocyte differentiation.
  • CFU-MegE differentiates into CFU-E when transcription gene MYB activates.
• GATA1 and FOG1 are counterparts:
  • Under activation, the CFU-GEMM differentiates into the CFU-Meg.
• GATA1 and FOG1's function is opposite to MYB's function.
• Includes at least 3 megakaryocyte lineage committed progenitor stages:
  • BFU-Meg (burst-forming unit megakaryocyte), counterpart of the megakaryocyte lineage from erythrocyte lineage
  • CFU-Meg (colony forming unit megakaryocyte)
  • LD-CFU-Meg (light density colony forming unit megakaryocyte)
• Undergo cytoplasmic division after BFU-Meg and CFU-Meg.
• The BFU-Meg contains more daughter cells than in in the CFU-Meg progenitor cell.
• BFU-Meg has proliferative properties for the formation of hundreds of colonies and CFU-Meg produces dozens of new colonies.
• LD-CFU-Meg undergoes endoreduplication or endomitosis for unique type of mitosis.
• Under lineage of common myeloid progenitor cells
• After the pluripotential stem cell, it will differentiate into common myeloid and common lymphoid progenitor cell.

Cell Structure

• 30 to 50 um in size
• < 0.5% of all BM cells
  • Largest, it is one of the fewest cells in the BM
• Multilobulated nucleus
  • Varies depending on the maturation of the megakaryocyte
• Abundant granular cytoplasm
• One mature one produces 2,000 to 4,000 platelets
  • Platelets form as the cytoplasm sheds and dissolves as a result
• Largest cells in the bone marrow and possess multiple chromosome copies (polyploid)
• Wright-stained BM aspirate smear contains 2-4 megakaryocytes per 10x lpf

Endomitosis & RUNX1

• Mitosis occurring within the nucleus without cytoplasmic division
• Transcription control gene RUNXI is activated as GATA1 and FOG1 slow down
• RUNXI mediates the switch from mitosis to endomitosis
• A form of mitosis that lacks telophase and cytokinesis, it is not capable of dividing into daughter cells
• Suppresses the rho/rock signaling pathway, which is needed for cytokinesis
• Inadequate levels of actin and myosin in the cytoplasm due to its suppressed rho/rock signal
• The LD-CFU-Meg converts to megakaryocyte stages or terminal megakaryocytes upon the function/influence of TPO and IL-11
• There are three stages of differentiation of terminal megakaryocytes
• Transcription gene factors influences there maturation and differentiation: - Transcription factor NF-E2 and DNA replication happen inside the nucleus proceeds to the production of 8N and 16N or even 32N ploidy resulting to the duplication further of the chromosome - Aside from 32N ploidy it could reach until 128 but the problem with the increase in duplication of chromosome, it could lead to hemorrhagic condition. - Synthesizes abundant cytoplasm with their multiple DNA copies which ultimately differentiates into platelets

Megakaryocytopoiesis

• Cytoplasm sheds primarily before becoming platelets to increase its size, first becoming proplatelet process and eventually platelet shedding
• Only found in the BM environment, not in the peripheral blood
• Next stage will be the terminal megakaryocyte differentiation from MK-I, MK-II to MK-III which is facilitated by another transcriptional gene, which is NF-E2.

Megakaryoblast - MK I

• Makes up 20% of precursors
• Diameter of 14-18 um
• Nucleus is round and there are 2-6 nucleoli
• Homogenous Chromatin
• N:C ratio of 3:1
• Mitosis absent and Endomitosis present
• Basophilic Cytoplasm
• Alpha and Dense- granules present
• Has a Demarcation system

Megakaryocyte-MK III

• Makes up 55% of precursors
• Diameter of 30-50 um or 15-40 um
• Nucleus is Multilobed and Nucleoli are not visible
• Deeply & variably condensed Chromatin with a N:C ratio of 1:4
• Mitosis and Endomitosis are absent
• Cytoplasm is Azurophilic & granular
• Alpha and Dense- granules present
• Has a Demarcation system
• Is in the last stage of terminal differentiation
• Ready to form proplatelet processes to shed off into individual platelets
• Recognizable characteristic is formation of more granules

MK II

• Makes up 25% of precursors
• Diameter of 15-40 um
• Indented Nucleus
• Variable Nucleoli
• Moderately condensed Nucleoli
• N:C ratio if 1:2
• Mitosis absent and Endomitosis ends
• Cytoplasm is Basophilic & granular
• Alpha and Dense- granules present
• Has a Demarcation system
• Formation of demarcation membrane, which will eventually grow inward and divide into making individual platelets

BM Environment

• Characteristics of the megakaryocytes is the increase in the formation of lobulation
• To recognize the formation of the lobules could be stained with the use of propidium iodide
• Cytoplasm is usually azurophilic or lavender and granular
• Undergoes full maturation with the demarcation membrane present and granules, it is ready to shed off into platelets
• With the endothelial lining composed of endothelial cells that lines the venous sinus
• Differenciated and mature megakaryocytes localize to the abluminal surface of sinusoid-lining endothelial cells in preparation for movement into the blood stream
• Proplatelets are shed off between endothelial cells and will come out as individual platelets into the cicrulation

Platelet Shedding

• Megakaryocytes extends a proplatelet process through or between the endothelial cells into the vascular sinus
• Starts as MK-I, MK-II, and MK-III with the formation of more blebs or pseudopods in MK-III for proplatelet release and eventually individual platelets
• Cytoplasm, elongation of proplatelets will starts the thrombocytopoiesis through microtubule disarrangement

The Markers

• Consists of maturation of platelets during Membrane Receptors and Markers
• Includes:
  • MPL
    • TPO receptor site present at all maturation stages

Thrombopoietin (TPO)

• 70,000 Dalton
• Possesses 23% homology with the red blood cell-producing hormone erythropoietin.
• Found in the kidney, liver, stromal cells, and smooth muscle cells.
• Functions include inducing stem cells to differentiate into megakaryocyte progenitors as a ligand hormone that circulates as a megakaryocyte and MPL.

Hormones

• Include IL-3, IL-6, IL-11, with a variety of uses to affect the differentiation of Progenitors, Megakaryocytes, and Thrombocytopiesis
• Diminish megakaryocytopoiesis at the progenitor, endomitotic, and terminal maturation phases
• Reduction in the transcription factors FOG1, GATA-1, and NF-E2 is inhibited in vitro
• Other cytokines and hormones that participate synergistically with TPO:
  • Stem cell factor/kit ligand/mast cell growth factor
  • Granulocyte-macrophage colony stimulating factor (GM-CSF)
  • Granulocyte colony-stimulating factor (G-CSF)
  • Acetylcholinesterase-derived megakaryocyte growth stimulating peptide
• IL-6 and IL-11:
  • Work with TPO to enhance endomitosis, megakaryocyte maturation, and thrombocytopoiesis
• IL-3 is a hormone
• Acts in synergy with TPO to induce the early differentiation of stem cells

Plate Characteristics

• 2-4um
• 8-10fL Mean Platelet Volume (MPV)
• 8-9 or 7-10 days lifespan
• 150-400 x10⁹/L or 150-4509/L Reference values in adults, but it requires higher demand for older women
• Men: 122-350 x10⁹/L is higher demand for older women
• Women: 140-379 x10⁹/L is higher demand for older women

Distribution of Plate Attributes

• 2/3 in circulation
  • Causes decrease of platelets when present in Splenomegaly, hypersplenism, or Big Spleen Syndrome
• 1/3 in spleen
  • During lifespan, there can be 14,000 trips which may retain up to at least 1/3 of all platelets in the spleen
• Throbocytosis(More)/throbocytopenia(Less)
• Is maintained for 5 days in the BM before they can get reloesed into PB, with 2 days in the spleen before apoptosis

Miscellaneous Facts

• 7-21 platelets per 100x magnification
• Approx. 1 trillion platelets in a single average adult
• Small anucleated cytoplasmic fragments of megakaryocytes
• Gray-blue with purple granules in Wright's stain

Giant Platelets Overview

• Premature release and has an increased demand of premature release and increased demand that is forced from the BM
• Can be viewed in the PB; abnormal Megakaryocytes are known as acute megakaryocytic leukemia (M7)
• Can be circulated, resting Platelets due to its Biconvex features, but if this is not present, use an EDTA chemical reagent to round and activate the molecule
• Smooth flow in arteries, veins, and capillaries during movement
• Clusters near the center of the blood vessel

Activated & Releasing - Platelets Overview

• They form pseudopodia in their filopodia states, which are ready to release granulated structures.
• The microtubules is sabay form sa filopodia
• Microtubules provides rigitidy to the activating platelet, but offers rigidity when structure is made aware