Endomitosis

Megakaryocyte maturation is marked by a mysterious form of mitosis that lacks telophase and cytokinesis (separation into daughter cells) and is called endomitosis. In endomitosis, DNA replication proceeds to the production of 8N, 16N, or 32N ploidy with duplicated sets of chromosomes, but no cell division.⁶ Some megakaryocyte nuclei replicate five times, reaching 128N; this level of ploidy is unusual, however, and may signal hematologic disease. Megakaryocytes employ their copious DNA to synthesize abundant cytoplasm, which differentiates into platelets. A single megakaryocyte may shed 2000 to 4000 platelets. In an average-size healthy human there are 10⁸ megakaryocytes producing 10¹¹ platelets per day. The key to endomitosis is the loss of spindle fiber orientation at the point of telophase, so that chromosomes do not proceed from equatorial plates to polar bodies, but rather duplicate in place. Cytokinesis is arrested, a cell-cycle adaptation found in no other normal human cell.

Megakaryocyte Progenitors

The megakaryocyte-erythrocyte progenitor (see Figure 7-13) differentiates into the megakaryocyte lineage under the influence of the hormone thrombopoietin (TPO) and a series of cytokines (see Table 13-3). There are three megakaryocyte lineage-committed progenitor stages, defined by their culture colony characteristics. In order of differentiation, these are the least mature burst-forming unit (BFU-Meg), the colony-forming unit (CFU-Meg), and the most mature light-density CFU (LD-CFU-Meg).⁷ All three progenitor stages resemble small lymphocytes and cannot be distinguished by Wright-stained light microscopy. The BFU-Meg and CFU-Meg are diploid and participate in normal mitosis, maintaining a pool of megakaryocyte progenitors. Their proliferative (mitotic) properties are reflected in their ability to form colonies of hundreds (BFUs) or scores (CFUs) of progeny in culture (Figure 13-2). In contrast to the BFU-Meg and CFU-Meg, the LD-CFU-Meg has little proliferative capacity and produces few cells, but begins the progress through endomitosis to reach increased nuclear ploidy.

The LD-CFU-Meg may be a transitional, or “promegakaryoblast,” stage in which polyploidy is first established, but its morphology is indistinguishable from that of small lymphocytes. At the LD-CFU-Meg point of development, megakaryocyte progenitors enter terminal differentiation, as they lose their ability to undergo normal mitosis but continue with endomitosis.

In specialty laboratories, immunologic probes and flow cytometry are employed to identify megakaryocyte progenitors. Useful flow cytometric progenitor markers are the general stem cell marker CD34, HLA-DR, and platelet glycoprotein IIIa (GP IIb/IIIa, CD41).⁸ Platelet peroxidase, localized in the endoplasmic reticulum of progenitors and megakaryoblasts, also may be identified by cytochemical stain in transmission electron microscopy. Identical peroxidase activity is localized to the dense tubular system of mature platelets.⁹

Terminal Megakaryocyte Differentiation

Megakaryocyte progenitors leave the proliferative phase and enter terminal differentiation, a series of stages in which microscopists begin to recognize their unique Wright-stained morphology in bone marrow aspirate films (Figure 13-3) or hematoxylin and eosin–stained bone marrow biopsy sections (Table 13-1).

Morphologists call the least differentiated megakaryocyte precursor the MK-I stage or megakaryoblast. Although they no longer look like small lymphocytes, megakaryoblasts cannot be reliably distinguished from myeloblasts or pronormoblasts using light microscopy (Figure 13-4). The morphologist may see plasma membrane blebs, blunt projections from the margin that resemble platelets. At this stage the megakaryocyte begins to develop most of its cytoplasmic ultrastructure, including procoagulant-laden α-granules, δ-granules (dense bodies), and the demarcation system (DMS).¹⁰

The later section on mature platelet ultrastructure discusses α-granules and δ-granules with their contents. The DMS is a series of membrane-lined channels that invade from the plasma membrane and grow over the course of terminal differentiation to subdivide the entire cytoplasm. The DMS is biologically identical to the plasma membrane and ultimately delineates the individual platelets during thrombocytopoiesis.

If there is a clinical need, immunologic probes or cytochemical markers are employed for definitive identification by flow cytometry (Table 13-2). In addition to the progenitor markers listed previously, the megakaryoblast may be identified using immunologic probes for the more differentiated membrane structures GP Ib, which is part of the GP Ib/IX/V von Willebrand factor (VWF) adhesion receptor (CD42); mpl, which is the TPO receptor site; or cytoplasmic VWF, detected by histochemical immunostaining rather than flow cytometry. CD36, which is GP IV, becomes visible in the developing MK-II stage, and fibrinogen may be detected by immunostaining in the fully developed megakaryocyte.

Nuclear lobularity first becomes apparent as indentation at the 4N replication stage, rendering the cell identifiable as an MK-II stage megakaryocyte by light microscopy. The morphologist seldom makes the effort to distinguish the MK-II and MK-III stages during a routine examination of a bone marrow aspirate film.

The megakaryocyte reaches its full ploidy level by the end of the MK-II stage. At the MK-III stage, the megakaryocyte is easily recognized at 10× magnification on the basis of its 30- to 50-µm diameter (Figures 13-5 and 13-6). The nucleus is intensely indented or lobulated, and the degree of lobulation is imprecisely proportional to ploidy. (When necessary, ploidy levels are measured using mepacrine, a nucleic acid dye, in megakaryocyte flow cytometry.¹¹) The chromatin is variably condensed with light and dark patches. The cytoplasm is azurophilic (lavender), granular, and platelet-like, because of the spread of the DMS and α-granules. At full maturation, platelet shedding, or thrombocytopoiesis, proceeds.

Thrombocytopoiesis (Platelet Shedding)

Figure 13-7 appears to show platelet shedding. One cannot find reliable evidence for platelet budding or shedding simply by examining megakaryocytes in situ, even in well-structured bone marrow biopsy preparations. In megakaryocyte cultures examined by transmission electron microscopy, the DMS dilates, longitudinal bundles of tubules form, cytoplasmic extensions called proplatelet processes develop, and transverse constrictions appear throughout the processes.¹² The proplatelet processes pierce through or between sinusoid-lining endothelial cells, extend into the venous blood, and release platelets (Figure 13-8). Sometimes whole megakaryocytes escape the marrow in this fashion to lodge in other organs, such as the lungs. Morphologists presume that thrombopoiesis leaves behind naked megakaryocyte nuclei to be consumed by marrow macrophages, although these are rarely seen in bone marrow aspirate films. Their absence leads a few morphologists to speculate that the lung is the primary site of thrombopoiesis.¹³

Hormones and Cytokines of Megakaryocytopoiesis

TPO is a 70-kD molecule with 23% homology with erythropoietin.¹⁴ Messenger ribonucleic acid (RNA) for TPO has been found in the kidney, liver, stromal cells, and smooth muscle cells, though liver has the most copies. TPO circulates in plasma and is the ligand that binds a megakaryocyte and platelet membrane receptor protein, mpl, named for v-mpl, a viral oncogene associated with murine myeloproliferative leukemia. The concentration of TPO is inversely proportional to platelet and megakaryocyte mass, which implies that membrane binding and consequent disposal of TPO by platelets is the primary control mechanism.¹⁵ Investigators have used in vitro and in vivo experiments to show that TPO induces stem cells to differentiate into megakaryocyte progenitors in synergy with cytokines and that it further induces differentiation of megakaryocyte progenitors into megakaryocytes, induces the proliferation and maturation of megakaryocytes, and induces platelet release (Table 13-3).^16,17 Recombinant TPO in several forms elevates the platelet count in healthy donors and in patients treated for a variety of neoplasms, including acute leukemia, and the commercial form NPlate (romiplostim, Amgen) is effective in raising the platelet count in immune thrombocytopenic purpura.¹⁸

Cell-derived stimulators of megakaryocytopoiesis include interleukin-3 (IL-3), IL-6, and IL-11. IL-3 seems to act in synergy with TPO to induce early differentiation of stem cells, whereas IL-6 and IL-11 act in the presence of TPO to enhance the later phenomena of endomitosis, megakaryocyte maturation, and platelet release. IL-11 has been synthesized and marketed by Genetics Institute as Neumega (oprelvekin) and used to stimulate platelet production in patients with chemotherapy-induced thrombocytopenia.¹⁹ Other cytokines and hormones that participate synergistically with TPO and the interleukins are stem cell factor, also called kit ligand or mast cell growth factor; granulocyte-macrophage colony-stimulating factor (GM-CSF); granulocyte colony-stimulating factor (G-CSF); and erythropoietin (EPO). The list continues to grow.

Platelet factor 4 (PF4), β-thromboglobulin, neutrophil-activating peptide 2, IL-8, and other factors inhibit in vitro megakaryocyte growth, which indicates that they may have a role in the control of megakaryocytopoiesis in vivo. Internally, reduction in the transcription factors FOG, GATA-1, and NF-E2 diminish megakaryocytopoiesis at the progenitor, endomitosis, and terminal maturation phases.²⁰

Resting Platelet Plasma Membrane

The platelet plasma membrane resembles any biologic membrane: a bilayer composed of proteins and lipids, as diagrammed in Figure 13-9. The predominant lipids are phospholipids, which form the basic structure, and cholesterol, which distributes asymmetrically throughout the phospholipids. The phospholipids form a bilayer with their polar heads oriented toward aqueous environments—toward the plasma externally and the cytoplasm internally. Their fatty acid chains, esterified to carbons 1 and 2 of the phospholipid triglyceride backbone, orient toward each other, perpendicular to the plane of the membrane, to form a hydrophobic barrier sandwiched within the hydrophilic layers.

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Introduction to Leukocyte Neoplasms Bone Marrow Failure Coagulation Instrumentation Acute Leukemias Myelodysplastic Syndromes Nonmalignant Leukocyte Disorders

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