Hematology 2 LECTURE PDF

PLATELETS The megakaryocytes differentiate into the megakaryocyte lineage under the influence of...

PLATELETS The megakaryocytes differentiate into the megakaryocyte lineage under the influence of a hormone and a series of cytokines. MEGAKARYOPOIESIS ○ This hormone is the TPO (Thrombopoietin) ❖ Process of producing megakaryocytes inside ○ These cytokines are IL-3,6,11 the bone marrow. Note: With the presence of these cytokines & hormone, ❖ As the cell matures, the cell becomes bigger megakaryocytes will no longer differentiate into and bigger. erythrocytes. There are three megakaryocyte MEGAKARYOCYTE lineage-committed progenitor stages, defined by their culture colony characteristics. These ❖ Megakaryocytes are among the largest cells in are the: the body and are polypoids—(Multiple set of 1. BFU-Meg (Burst forming unit chromosomes) megakaryocyte) ❖ 30 to 50 μm in diameter with a multilobulated 2. CFU-Meg (Colony forming unit nucleus and abundant granular cytoplasm on megakaryocyte) a Wright-stained bone marrow aspirate. 3. LD-CFU-Meg (Light density colony ❖ Myelocytic and erythrocytic precursor cells, forming unit megakaryocyte) which locate further from the endothelial cells, All three progenitor stages resemble small may cross the megakaryocyte cytoplasm to lymphocytes. reach the sinusoid lumen through a faux phagocytosis. How do we differentiate these 3 stages? This process is called EMPERIPOLESIS The BFU-Meg and CFU-Meg are diploid and (Pseudo phagocytosis) participate in normal mitosis, maintaining a pool of megakaryocyte progenitors. Their ENDOMITOSIS proliferative (mitotic) properties are reflected in their ability to form colonies of hundreds of cells ❖ Maturation is marked by a mysterious form of (BFUs) or scores (CFUs) of progeny in culture. mitosis that lacks telophase & cytokinesis (no ○ Scores means up to 20 cells* cell division) LD-CFU-Meg has little proliferative capacity ❖ In endomitosis, DNA replication proceeds to and produces few cells, but begins the the production of 8N, 16N, or 32N ploidy with progress through endomitosis to reach duplicated sets of chromosomes, but no cell increased nuclear ploidy. division. Some megakaryocyte nuclei replicate The LD-CFU-Meg may be a transitional, or 5 times, reaching 128N; this level of ploidy is “promegakaryoblast” stage in which unusual, however, and may signal hematologic polyploidy is first established, but its morphology disease. is indistinguishable from that of small ❖ The key to endomitosis is the loss of spindle fiber lymphocytes. orientation at the point of telophase. ○ May be considered the start of ❖ NC ratio of endomitosis: (4:1 1:12) megakaryopoiesis. At the LD-CFU-Meg point of development, MEGAKARYOCYTE PROGENITORS megakaryocyte progenitors enter terminal differentiation, as they lose their ability to undergo normal mitosis but continue with endomitosis. 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 or hematoxylin and eosin–stained bone marrow biopsy sections. NATHALIA COLEEN FAVILA | HEMATOLOGY 2 LEC 1  In short, no proliferation will occur. The cytoplasm is azurophilic (lavender), Megakaryopoiesis process last up to 5 granular, and platelet-like, because of the to 7 days spread of the DMS and α-granules. ○ Mepacrine is the stain/dye used to identify the lobularity of the megakaryocytes with the use of a megakaryocyte flow cytometer. After they release the platelets, megakaryocytes will be phagocytized by the macrophages. THROMBOCYTOPOIESIS ❖ The DMS dilates, longitudinal bundles of tubules form, cytoplasmic extensions called proplatelet process develop (last step before the platelets shed), and transverse constriction appear throughout the processes. ❖ The proplatelet processes pierce through or between sinusoid lining endothelial cells, MK-I STAGE extend into the venous blood, and release Also called megakaryoblast. platelets. It is the least differentiated megakaryocyte Note: For a megakaryocyte to produce platelets (in precursor. inactive form), it should have >4 nucleus. Cannot be reliably distinguished from myeloblast or pronormoblast using light HORMONES AND CYTOKINES OF microscopy. MEGAKARYOCYTOPOIESIS At this stage, the megakaryocyte begins to develop most of its cytoplasmic ultrastructure, including procoagulant-laden α-granules, δ- THROMBOPOIETIN granules (dense bodies), and the demarcation system (DMS). ❖ It is the one responsible for signaling our body Demarcation system - Biologically identical to to produce platelets or megakaryocytes. the plasma membrane and ultimately ❖ Found in the kidney, liver, stromal cells, and delineates the individual platelets during smooth muscle cells, though the liver has the thrombocytopoiesis. most copies. ❖ The concentration of TPO is inversely MK-II STAGE proportional to platelet and megakaryocyte Nuclear lobularity first becomes apparent as mass. indentation at the 4N replication stage, TPO= platelet rendering the cell identifiable. ❖ TPO induces stem cells to differentiate into Cytoplasm is abundant, and the nucleus shows megakaryocyte progenitors in synergy with minimal lobularity. cytokines and that it further induces The megakaryocyte reaches its full ploidy level differentiation of megakaryocyte progenitors (32N) by the end of the MK-II stage. into megakaryocytes, induces the proliferation and maturation of megakaryocytes, and MK-III STAGE induces platelet release. At the MK-III stage, the megakaryocyte is easily recognized at 10× magnification on the basis CELL-DERIVED STIMULATORS of its 30 to 50 μm diameter. The nucleus is intensely indented, or lobulated, ❖ Includes IL-3, IL-6, and IL-11 and the degree of lobulation is imprecisely ❖ IL-3 seems to act in synergy with TPO to induce proportional to ploidy early differentiation of stem cells. The chromatin is variably condensed with light ❖ IL-6 and IL-11 act in the presence of TPO to and dark patches. enhance the later phenomena of endomitosis, megakaryocyte maturation, and platelet release. NATHALIA COLEEN FAVILA | HEMATOLOGY 2 LEC 2  ❖ Other cytokines and hormones include stem expanded reactive surface to which plasma cell factor (kit ligand or mast cell growth clotting factors are selectively adsorbed. factor), granulocyte-macrophage colony- ❖ Microfilaments, microtubules - Maintain the stimulating factor (GM-CSF); granulocyte discoid shape of the cell as well as maintain colony-stimulating factor (G-CSF); and the position of the organelles. A secondary erythropoietin (EPO). system of microfilaments is functional in internal organization and secretion of blood INHIBITORS coagulation products, such as fibrinogen. The microfilaments interact with the dense tubular ❖ Platelet factor 4 (PF4), β-thromboglobulin, system in sequestering calcium, which initially neutrophil activating peptide 2, IL-8, and other causes centralization of internal organelles. factors inhibit in vitro megakaryocyte growth, These subcellular and cytoplasmic filaments which indicates that they may have a role in make up the contractile system (sol gel zone) the control of megakaryocytopoiesis in vivo. of the platelet. ❖ Internally, reduction in the transcription factors Sol gel zone is responsible for: FOG, GATA-1, and NF-E2 diminish Cytoskeletal rearrangement megakaryocytopoiesis at the progenitor, Platelet shape change endomitosis, and terminal maturation phases. Platelet contraction during clot activation PLATELETS ❖ Granules – Related to hemostasis Alpha granules - Most abundant granules in platelets and contain ❖ Life span: 7 to 10 days heparin-neutralizing factor 4, ❖ Are anucleated blood cells that circulate in beta-thromboglobulin, platelet- amounts of 150 to 400 x 10⁹/L, with mean derived growth factor, platelet counts slightly higher in women than in men. fibrinogen, fibronectin, vWF, and These numbers only represent ⅔ of thrombospondin. platelets available in our body. Dense granules - Contain serotonin, Remaining ⅓ is sequestered by the adenosine diphosphate (ADP), spleen. adenosine triphosphate (ATP), and Because women have menstruation calcium. Also recruits other platelets. etc. and has a major role in stopping the ❖ Trigger primary hemostasis on exposure to lost of blood during an injury. endothelial, subendothelial, and plasma Lysosomes - Store hydrolase enzymes procoagulants in blood vessel injury. ❖ Other cytoplasmic content: ❖ They have an average diameter of 2.5 μm, Actomyosin (thrombosthenin): Essential corresponding to a mean platelet volume for force generation & platelet (MPV) of 8 to 10 fL in an isotonic suspension, as contraction determined using laboratory profiling Myosin: Converts chemical energy to instruments. mechanical energy (generates ❖ Arise from unique bone marrow cells called movement and force) megakaryocytes. Filamin: Cytoskeleton remodeling ❖ RETICULATED PLATELETS: “Stress platelets” are Glycogen and enzymes of the larger than ordinary platelets, 6 μm in diameter, glycolytic and hexose pathways MPV is 12 to 14 fL. PLATELET ULTRASTRUCTURE ❖ Glycocalyx - Unique among the cellular components of the blood. It is composed of plasma proteins and carbohydrate molecules that are related to the coagulation, complement, and fibrinolytic systems. ❖ Cytoplasmic membrane - Contains the open canalicular system. ❖ Canalicular system - Forms the invaginate, sponge-like portion of the cell that provides an NATHALIA COLEEN FAVILA | HEMATOLOGY 2 LEC 3 HEMOSTASIS This vasoconstriction and platelet plug formation will comprise the initial, rapid, short-lived response to vessel HEMOSTASIS damage. With the help of fibrin for the blood to be reinforced. ❖ An important function that our platelets are part of. MAIN FACTORS INVOLVED IN PRIMARY HEMOSTASIS ❖ Hemostasis is a complex physiologic process that keeps circulating blood in a fluid state and Blood vessels then, when an injury occurs, produces a clot to Platelets stop the bleeding, confines the clot to the site of injury, and finally dissolves the clot as the wound heals. VASCULAR INTIMA IN HEMOSTASIS When this process is out of balance, it may result to hemorrhage ❖ A blood vessel is structured into three layers: an (uncontrolled bleeding), and inner layer (vascular intima), a middle layer thrombosis might be life threatening. (vascular media), and an outer layer (vascular When there is an absent of single adventitia). procoagulant, or coagulating factor, ❖ The vascular intima provides the interface the individual may suffer from life long between circulating blood and the body hemorrhage, chronic inflammation, tissues. and transfusion dependence. ❖ Vascular intima is the most important layer in Conversely, absence of a control hemostasis. protein allows coagulation to proceed, It is composed of a monolayer of and results into thrombosis, stroke, metabolically active endothelial cells. pulmonary embolism, and cardiovascular events ❖ Hemostasis involves the interaction of vasoconstriction, platelet adhesion and aggregation, and coagulation enzyme activation to stop bleeding. The coagulation system, similar to other humoral amplification mechanisms is complex because it translates a diminutive physical or chemical stimulus into a profound lifesaving event. ❖ The key cellular elements of hemostasis are the VASCULAR INTIMA COMPONENTS cells of the vascular intima (inner lining), extravascular tissue factor (TF) bearing cells, ❖ Endothelial cells – form a smooth, unbroken and platelets. surface that eases the fluid passage of blood. ❖ The plasma components include the These endothelial cells play an coagulation and fibrinolytic proteins and their essential role in immune response, inhibitors. vascular permeability, proliferation, hemostasis. PRIMARY HEMOSTASIS ❖ Basement membrane – an elastin-rich internal elastic lamina, and its surrounding layer of ❖ Refers to the role of blood vessels and platelets connective tissues support the ECs. in the initial response to a vascular injury or to ❖ Fibroblasts – occupy the connective tissue the commonplace desquamation of dying or layer and produce collagen. damaged endothelial cells. Blood vessels ❖ Smooth muscle cells – contract when an injury contract to seal the wound or reduce the occurs and primary hemostasis is initiated. blood flow (through vasoconstriction). Helps in vasoconstriction Vasoconstriction reduces the chances of excessive blood loss. ❖ In this primary hemostasis, platelets will become activated to adhere on the sites of injury, then secret their contents to aggregate with other platelets to form platelet plugs. NATHALIA COLEEN FAVILA | HEMATOLOGY 2 LEC 1 ANTICOAGULANT PROPERTIES OF INTACT VASCULAR ❖ VWF has been described as a “carpet” on INTIMA which activated platelets assemble. This is a large multimeric glycoprotein, ❖ Endothelial cells – present a smooth, and it acts as a bridge that binds contiguous surface platelets to expose subendothelial It also prevents harmful turbulence that collagen in arteries and veins. otherwise may activate platelets and ASAMts 13 is also secreted in coagulation enzymes. endothelial cells, which is also imporin ❖ Prostacyclin – platelet inhibitor VWF as it cleaves it into shorter chains Prevents unnecessary platelet to support normal platelet adhesion. activation ❖ On activation, ECs secrete and coat ❖ Nitric oxide – vascular relaxing factor themselves with P-selectin, an adhesion Involved in vasodilation molecule that promotes platelet and leukocyte It also inhibits platelet activation and binding. They also secrete immunoglobulin-like promote angiogenesis for a healthy adhesion molecules like icams (intercellular arteriole. adhesion molecules) & pecams ❖ Tissue factor pathway inhibitor – controls the ❖ subendothelial smooth muscle cells and activation of extrinsic pathway fibroblasts support the constitutive membrane Limits the activation of tissue factor VII protein tissue factor a & X a complex Endothelial cell disruption exposes ❖ Endothelial protein C along with tissue factor in subendothelial cells and thrombomodulin – activates C protein which activates the coagulation system downgrades coagulation by digesting through the contact with Factor VII activated factor V and VIII, inhibiting thrombin leading to fibrin formation. formation. ❖ Heparan sulfate - enhances the activity of FIBRINOLYTIC PROPERTIES OF INTACT VASCULAR INTIMA antithrombin. ❖ ECs support fibrinolysis, the removal of fibrin to PROCOAGULANT PROPERTIES OF INTACT VASCULAR restore vessel patency, with the secretion of a INTIMA protein. This protein is tissue plasminogen ❖ Although the intact endothelium has activator that is released by anticoagulant properties, when damaged, the endothelial cells to support fibrinolysis vascular intima (ECs and the subendothelial or removal of fibrin clots matrix) promotes coagulation through several ❖ This protein activates fibrinolysis by converting procoagulant properties. plasminogen to plasmin, which gradually ❖ Smooth muscle cells contract, the vascular digests fibrin and restores blood flow. ECs also lumen narrows or closes, and blood flow regulate fibrinolysis by providing inhibitors to through the injured site is minimized (A.K.A. prevent excessive plasmin generation. Vasoconstriction) Plasminogen activator inhibitor 1 (PAI Although veins and capillaries do not 1) prevents plasmin generation and have smooth muscle cells, bleeding fibrinolysis into surrounding tissues creates ❖ Another inhibitor of plasmin generation, extravascular pressure on the blood thrombin activatable fibrinolysis inhibitor (TAFI), vessels, effectively minimizing escape is activated by thrombin bound to EC of blood. membrane thrombomodulin ❖ Second, the subendothelial connective tissues ❖ Elevation of PAI-1 and TAFI can slow fibrinolysis of arteries and veins are rich in collagen, a and increase the tendency for thrombosis. flexible, elastic structural protein. Exposed with injury to the vessel, collagen binds and PLATELET FUNCTION DURING PRIMARY HEMOSTASIS activates platelets. ❖ Third, ECs secrete von Willebrand factor (VWF) 1. Adhesion from storage sites called Weibel-Palade bodies 2. Aggregation when activated by vasoactive agents such as 3. Secretion thrombin. NATHALIA COLEEN FAVILA | HEMATOLOGY 2 LEC 2  PLATELET ADHESION SUMMARY OF PRIMARY HEMOSTASIS ❖ Adhesion is the property by which platelets bind non platelet surfaces such as subendothelial collagen. ❖ VWF links platelets to collagen in areas of high shear stress such as arteries and arterioles, whereas platelets may bind directly via specific receptors to collagen in damaged veins and capillaries. This VWF binds platelets through their glycoprotein. Specifically 1B, 5, 9 membrane receptor ❖ The whole process of platelet adhesion is reversible ❖ It also seals the endothelial gaps caused by the injury ❖ During this process, it also secretes growth factors PLATELET AGGREGATION ❖ is the property by which platelets bind to one another ❖ When platelets are activated, a change in the GP IIb/IIIa receptor allows binding of fibrinogen, as well as VWF and fibronectin. ❖ Fibrinogen binds to GP IIb/IIIa on adjacent platelets and joins them together in the presence of ionized calcium (Ca2+). ❖ Aggregation process is irreversible ❖ During this process, platelet plugs are formed PLATELET SECRETION ❖ Platelets secrete the contents of their granules during adhesion and aggregation, with most secretion occurring late in the platelet activation process. ❖ Platelets secrete procoagulants, such as factor V, VWF, factor VIII, and fibrinogen, as well as control proteins, Ca2+, ADP, and other hemostatic molecules. ❖ This process is irreversible and occurs during the late aggregation phase. ❖ This secretes procoagulant that make it necessary for coagulation NATHALIA COLEEN FAVILA | HEMATOLOGY 2 LEC 3