Megakaryopoiesis and Platelet Formation

• Focus is on megakaryopoiesis and platelets
• Hemostasis maintains blood balance, stopping bleeding while maintaining fluidity within blood vessels.
• Platelets are the most significant formed elements in the blood for hemostasis.
• Studying the structural components of platelets helps understand their critical role in hemostasis.

Megakaryopoiesis

• Is the maturation process of a hematological cell committed to platelet production
• Hematopoiesis is a controlled process of producing cells, only making what the body requires
• According to theories, all blood cells originate from a single Pluripotential Hematopoietic Stem Cell, giving rise to Myeloid and Lymphoid cells.
• Megakaryopoiesis specifically refers to the production of megakaryocytes
• Responds to Thrombopoietin as a growth factor
• Thrombopoietin is essential for stimulating stem cells to produce megakaryocytes
• Platelets are cytoplasmic fragments of megakaryocytes; without megakaryocytes, there will be no platelets
• Megakaryocyte production is unique, as it sheds platelets later on
• Megakaryocytes are the largest cells in the bone marrow
• During megakaryopoiesis, the cell size increases as the cell matures.

Megakaryocyte Lineage Progenitors

• Recognizable stages can be observed and differentiated under a microscope in a bone marrow specimen
• There are progenitors before these stages
• Endomitosis is unique to megakaryopoiesis, involving DNA replication and cytoplasm maturation without cell division, the nucleus divides up to 32 times
• More nuclei in a megakaryocyte result in more platelets
• The most mature stage in megakaryopoiesis is megakaryocytes
• All mature cells should be present in the blood, except megakaryocytes, because the cytoplasmic fragments (platelets) come out of the bone marrow.
• Megakaryocytes exist in the lungs but are more abundant in bone marrow.
• Cells enlarge during endomitosis to facilitate platelet production.
• More nuclei mean a higher chance of platelet production from the cytoplasm
• Proliferative phase: from BFU-Meg to CFU-Meg, involves mitosis
• Terminal Differentiation phase is LD-CFU-Meg, the cell no longer divides.

Characteristics of Megakaryopoiesis

• Cell size increases as it matures

• The nucleus-to-cytoplasm (N:C) ratio decreases

• The nucleus divides and becomes smaller, while the cytoplasm will stretch to be larger accommodating it

• The number of nuclei increases

• Maturation takes about 7 days from megakaryoblast stage to produce megakaryocytes

• Approximately 1-4 out of 1000 bone marrow cells are megakaryocytes capable of releasing roughly 1000 to 4000 platelets each if there are at least 4 nuclei.

• CFU-GEMM becomes BFU-Meg (Burst Forming Unit Megakaryocyte) and can undergo mitosis.

• BFU-Meg becomes CFU-Meg (Colony Forming Unit Megakaryocyte) and can undergo mitosis.

• CFU-Meg becomes LD-CFU-Meg (Light Density Colony Forming Unit Megakaryocyte) and will undergo endomitosis.

• LD-CFU-Meg loses the capacity to divide and will no longer undergo mitosis

• All colonies look morphologically the same, differentiation can be determined through flow cytometry.

• BFU-Meg and CFU-Meg ensure enough megakaryocyte supply in bone marrow through the proliferative phase.

• LD-CFU-Meg stops mitosis and undergoes continuous nuclear maturation via endomitosis

• There is no telophase in endomitosis

• After the proliferative phase, the cells differentiate, recognizable by their characteristics in the bone marrow.

Stages of Megakaryopoiesis

• Recognizable stages are:

Megakaryoblast (MK I)

• Least differentiated megakaryocyte precursor

• Features plasma membrane blebs

• Has a diameter of 15-20 um

• Granules are absent

• Round or oval kidney shaped at times

• Chromatin pattern is fine

• Earliest recognizable stage under light microscope

• Check for the Demarcating Membrane System (DMS) to know if platelets are there or not (network system of platelets)

• MK I, MK II, and MK III have DMS

• DMS not yet visible in MK I/Megakaryoblast due to the large nucleus.

• Platelets are not yet released at this stage

• Look for blebs/cytoplasmic tags/cytoplasmic protrusions to identify

• These blebs will disappear as the cell matures

Promegakaryocyte (MK II)

• Nuclear lobulation becomes visible or apparent
• DMS becomes clearly visible
• First cell that clearly shows the DMS
• Has a diameter to 20-80 um
• Evidence of visible endomitosis as nuclear lobulation begins

Mekaryocyte (MK III)

• Easily recognized at 10X magnification

• Mature/Immature stage

• Platelet shedding proceeds at full maturation

• Shedding is the release of platelets from the cytoplasm

• Has a diameter of 30-100 um

• Most abundant stage in megakaryopoiesis

• Megakaryocytes can be immature (not releasing) or mature (releasing)

• Platelets start to release if there are at least 4 nuclei in the megakaryocytes

• In each megakaryocyte, 2000-4000 platelets can be released, with about 70% circulating in the blood and the other 30% sequestered in the spleen circulating approximately 7-10 days

• The spleen holds this supply in case there is cases of bleeding

• Once platelets are released, megakaryocytes are "naked megakaryocytes" and phagocytized

Megakaryocyte Membrane Receptors and Markers

• MPL (Murine Myeloproliferative Leukemia)
• Is a TPO receptor site
• Named for VMPL
• If negative in MPL, platelet count will decrease
• CD34
• Stem cell and common myeloid progenitor marker, disappearing in terminal differentiation phase.
• Should not be used to test of megakaryoblast, as not expressing is normal
• Gp IIb/IIIa
• First appears on megakaryocyte progenitors and remains throughout maturation.
• Other markers: CD36, CD42, CD61, CD62
• Other substances:
• Factor VIII
• VWF (Von Willebrand Factor)
• Fibrinogen - seen on megakaryocyte and platelets

Platelet Shedding (Thrombocytopoiesis)

• DMS dilates and create tubules for platelet release in bone marrow through endothelial walls.
• About 70% of the produced platelets circulate in the blood, with the other 30% held in the spleen.

Hormones and Cytokines of Megakaryopoiesis

• Thrombopoietin
• Circulates as a hormone in plasma
• Plasma concentration is inversely proportional to platelet and megakaryocyte mass
• Could be derived from liver, kidneys, or smooth muscles at times, serving as the regulator
• More TPO in plasma = less platelets and megakaryocyte mass and vice versa
• Synthetic thrombopoietin can be used to elevate platelet count
• IL-3 acts in synergy with ΤΡΟ to induce early stem cell differentiation
• IL-6 and IL-11 enhance condomitosis and maturation, acting with thrombopoietin to further enhance endomitosis.
• Other growth factors include: Kit-ligand, mast cell growth factor, GM-CSF, G-CSF, acetylcholinesterase-derived megakaryocyte growth stimulating peptide.
• Inhibitors include PF4, B-TG, neutrophil-activating peptide 2, II-8 can stop production, preventing excess platelet formation.

Platelets Basics

• 2-4 um in diameter
• Volume: 8-10 fL
• Described as an anucleated cell (without golgi bodies and rough endoplasmic reticulum, but with mitochondria and granules)
• Circular to irregular granular purple dots on blood films; 7-21 per 100x field
• has a Life span: 7-10 days (in the blood)
• Normal Value: 150-400 x 10^9/L
• Ensures only ample amount of platelets should be present within the body.
• Platelet description;
• Known as stress platelets, appearing in compensation or as a reaction to thrombocytopenia and appears to be larger than the usual platelets
• In citrated blood, cylindrical and beaded, resembling fragments of megakaryocyte proplatelet processes can be used to further identify: nucleic acid dyes (e.g. thiazole orange)

Platelet Ultra Structure

• Has an outer later

• Peripheral Zone transmits; anything entering or leaving platelets through it.

• Platelets can collect substances (through glycocalyx) for clot formation, releasing them when needed.

• Platelet membrane - phospholipids, cholesterol (phospholipids make the basic structure of platelets and makes membrane selectively permeable)

• Phospholipid bilayer acts as a selective permeable membrane

• Platelets circulate inactively until needed for clot formation during injury or bleeding.

• Phospholipids:

• Phosphatidyl choline, Sphingomyelin, Phosphatidyl inositol, Phosphatidyl serine

• Cholesterol stabilizes the cell membrane and maintains fluidity

• Glycocalyx adheres and responds to hemostatic demands

• Platelets absorbs different components within the granules; such as Calcium needed for clot formation, maintains a negative change, serves as the immunologic site of platelets

• Surface Connected Canalicular System enhances platelet interaction with its environment (OCS)

• Substances pass through it to facilitate intracellular and extracellular environment

• Submembrane region interacts with contractile proteins to modulate platelet adhesion and clot retraction

• Sol-Gel Zone is the cytoskeletal region

• Microtubules keep discoid shape of inactive platelets

• Microfilaments: actin-myosin helps change platelet shape when platelets are activated

• Activated platelets change shape, releasing granules to form a clot.

• Thrombostenin releases granules, acting as platelets contractile proteins.

• Organelle Zone is metabolic region

• Mitochondria produces and gives energy

• Dense Tubular System sequesters calcium, supports platelet activation, main center for platelet activation.

• Granules are essential clotting:

• Alpha, dense, lysosomes, unique to platelets

• Lysosomes digest vessel wall matrix

• Dense granules migrate to plasma post-activation and facilitates platelet activation and clotting (ADP helps platelets to aggregate)

• Alpha granules are stored in alpha granules:

• PDGF (Platelet Derived Growth Factor)

• BTG (Beta Thromboglobulin)

• PF4 (Platelet Factor 4)

• Thrombospondin

• PDGF and BTG are essential in healing the injured vessel

Overall Functions of the Platelets

• Maintains Vascular Integrity, Repairing injured vessels, Formation of Platelet Plug, the Part of the platelet job is to maintain integrity of vessels
• PF4 and Thrombospondin also facilitates the formation of clots
• Platelets aggregate to form a plug in primary hemostasis, using collected granules to stabilize and form a clot.
• Stabilize the plug by activating the fibrin formation or clot formation

Explore megakaryopoiesis, the maturation of cells committed to platelet production, crucial for hemostasis. Understand how thrombopoietin stimulates megakaryocyte development, which are essential for creating platelets. Learn about the structural components of platelets and their significance in maintaining blood balance.