Hematology 425 Megakaryopoiesis

1. Hematology 425 Megakaryopoiesis Russ Morrison October 13, 2006

2. 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

3. 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.

4. 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

5. 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

6. 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

7. RBC and WBC precursors usually divide four times during maturation, producing 16 mature cells from each committed stem cell. Megakaryocytes do not experience complete cellular division, but undergo a proces called endomitosis Megakaryopoiesis Platelet production is unique from other hematopoietic cell production:

8. 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)

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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)

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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