Hematology - 11 Blood Vasculature, Megakaryocytes, and Platelets

HEMATOLOGY OVERVIEW OF HEMOSTASIS AND THROMBOSIS BLOOD VASCULATURE, MEGAKARYOCYTES & PLATELETS OUTLINE • Introduction • Platelets o Overview of Hemostasis and o Cellular Ultrastructure of a Thrombosis Mature Platelet o Platelet Kinetics Life Span & • Blood Vasculature Normal Values o Structure and Function o Functions of Platelet o The Endothelium o Chemistry of Platelets o Maintenance of Vascular o Parts of a Platelet Integrity o Additional Anomalies • Megakaryopoiesis Associated with Platelets and o The Megakaryocytic Cell Megakaryocytes Series • • • • • INTRODUCTION • • • The module tackles the blood vessels of the circulatory system, especially the microcirculation, which is composed of arterioles, the microscopic continuation of the arteries, and venules, the continuation of veins. The arterioles and venules in turn, anastomose to form the capillaries. The capillaries are the smallest of blood vessels and the part where exchange of gases and metabolites occur. So, the circulatory system is a closed system. The main role of the circulatory system, of which the blood vessels are part is to carry blood which is necessary for delivery of metabolites, gases, and others, to the different organs/ tissues for them to function. The process undergone by megakaryocytic cells in their development and maturation is unique in that, they undergo endomitosis or endoreduplication, unlike the other cell lines which proliferate through mitosis. For their optimal development, the hematopoietic growth factors and interleukins. are required. When these factors are deficient or dysfunctional, the megakaryocytic cells' development will be abnorma/ asychronistic. This will result in abnormal cells and in turn give rise to platelets which may be morphologically abnormal or fuctionally deficient. The platelets play a big role in the hemostatic mechanism, both primary and secondary. The ultrastructure of a matured platelet as well as the uses of the different parts/zones and their components are included. Circulatory system - closed system composed of systemic and pulmonary o Systemic - distribution of blood throughout the body o Pulmonary - for oxygenation of blood The maintenance of circulatory hemostasis is achieved through the process of balancing bleeding (hemorrhage) and clotting (thrombosis). Hemostasis - stoppage of bleeding Normal blood should not clot intravascularly, blood may clot extravascularly 4 Major Components of Hemostasis: o Vascular System (blood vessels) o Platelets (Thrombocytes) - cytoplasmic fragments of megakaryocytes o Blood Coagulation Factors (Procoagulants) - induce coagulation o Fibrinolysis and ultimate tissue repair (fibrinolytic component, plasmin) - reestablished blood flow Processes involved in hemostasis following injury to a small blood vessel: 1. Blood vessel spasm ▪ Due to peripheral nervous system ▪ Irritation/injury/inflammation 2. Formation of a platelet plug ▪ Loose mass ▪ End product of primary hemostasis ▪ Interaction of coagulation products 3. Contact among damaged blood vessel, blood platelet, and coagulation proteins 4. Development of a blood clot around the injury ▪ Stable fibrin clot - in vivo clot for hemostasis ▪ Fibrin polymer - in vitro clot 5. Fibrinolytic removal of excess hemostatic material to reestablish vascular integrity BLOOD VASCULATURE STRUCTURE AND FUNCTION • • Types of Blood Vessels: o Arteries and Veins ▪ Arteries - largest blood vessels carrying oxygenated blood • Aorta - largest artery ▪ Veins - carries deoxygenated blood o Arterioles and Venules ▪ Anastomose of arteries and vein ▪ Branch of arteries & veins o Capillaries ▪ Anastomose of arterioles and venules ▪ Delivery of metabolites through osmosis and diffusion ▪ Has no smooth muscles Endothelium o Covers blood vessels • Tissue Zones: o Tunica adventitia / Tunica Externa - outermost layer, where vasa vasorum that provides blood with blood supply and nutrients o Tunica media - smooth muscle o Tunica intima / Tunica interna - thinnest layer, innermost layer where endothelium is found THE ENDOTHELIUM • • The Endothelins - are produced in a variety of tissues, where they act as modulators of vasomotor tone, cell proliferation, and hormone production. 3 Members of the Endothelin Family: o Endothelin-1 - is the only family member produced in endothelial cells and is also produced in vascular smooth muscle cells (tunica media). o Endothelin-2 - is produced predominantly within the kidney and intestine, with smaller amounts produced in the myocardium, placenta, and uterus. o Endothelin-3 - has been found in high concentrations in the brain and may regulate important functions, such as proliferation and development in neurons and astrocytes. It also is found throughout the gastrointestinal tract and in the lung and kidney. THE ENDOTHELIAL DYSFUNCTION • • VASCULATURE PHYSIOLOGY • • The Role of Vasoconstriction in Hemostasis o Vasoconstriction is a reflex in which blood vessels narrow to increase blood pressure. ▪ Lessening of lumen due to vascular spasm ▪ Increase blood pressure to continue blood flow o Caused by thromboxane A2 from activated platelets and injured epithelial cells, nervous system reflexes from pain, and direct injury to vascular smooth muscle. o Epinephrine & serotonin promotes vasoconstriction The Role of Endothelium o Endothelium - epithelial cells, non contact surface o Endothelial cells shall be good enough to prevent loss of intravascular component to the outside of vessel ▪ Junctional complexes will tend to hold blood components and metabolites o Hemorrhagic events cause loose junctional complexes leading to leakage o Regulates the permeability of the inner vessel wall and provides the principal stimulus to thrombosis following injury to a blood vessel. o Involved in the clotting process by producing or storing clotting components. o Rich with plasminogen activator, which, if appropriately stimulated, is released and activates plasminogen, which ensures rapid lysis of fibrin clots. o Elaborates prostacyclin, which is synthesized by the endothelium from prostaglandin precursors and strongly inhibits platelet aggregation and adhesion. ▪ of blood components and metabolites • • The Endothelial Dysfunction - play an important role in the initiation, progression, and clinical complications of various forms of inflammatory and degenerative vascular diseases. Stimuli of Endothelial Dysfunction: o Immunoregulatory Substances (Tumor Necrosis Factor/TNF & Interleukin1/IL1 - monokines) o Viral Infection and Transformation o Bacterial Toxins o Cholesterol / Oxidatively modified lipoproteins Disruption of the endothelium directly activates all four components of hemostasis. After this event, the following events take place: o Initially, rapid vasoconstriction for up to 30 minutes reduces blood flow and promotes contact activation of platelets and coagulation factors. o In the second phase, platelets adhere immediately to the exposed subendothelial connective tissue, particularly collagen. ▪ The aggregated platelets enhance sustained vasoconstriction by releasing thromboxane A2 and vasoactive amines, including serotonin and epinephrine. o In the third phase, coagulation is initiated through both the intrinsic and extrinsic systems. o Finally, fibrinolysis occurs following the release of tissue plasminogen activators (t-PAs) from the vascular wall. Fibrinolytic removal of excess hemostatic material is necessary to reestablish vascular integrity. MAINTENANCE OF VASCULAR INTEGRITY • • Essential Factors for Vascular Integrity o Circulating functional platelets - surveillance mechanism for immediate maintenance o Adrenocorticosteroids - blood vessel tone o Ascorbic Acid - main energy source/food of fibroblasts (produces collagen for increased strength) ▪ Antioxidants - removes free radicals to prevent cell damage & eventual cancer/malignancy A lack of these factors produces fragility of the vessels, which makes them prone to disruption. • • The integrity of arterioles and venules depends on vasoconstriction, the formation of a plug of fused platelets over the injury, and the formation of a fibrin clot. Vasoconstriction is of ultimate importance in damaged arteries. Veins, which contain 70% of the blood volume, may rupture with a slight increase in hydrostatic pressure. MEGAKARYOPOIESIS • • • Hematopoietic Growth Factors and Interleukins affecting Megakaryocytes and Platelets Factors in determining age of cell o Nature of euchromatin material ▪ Mature - heterochromatin (coarse & clumped) o Number of nucleoli - decreases with maturity Mononuclear phagocyte system o Monocytes o Macrophages • Megakaryocytic cells are not seen in normal blood MEGAKARYOBLAST • • • • • • • • • Earliest recognizable precursor of platelets Size: 10-24 mcm Nucleus: Round Nucleoli: 2-6 Chromatin: Homogeneous, loosely organized euchromatin Cytoplasm: Basophilic Granules: Absent by Wright stain N/C Ratio: 3:1 Reference Interval: o Bone Marrow: 20% of megakaryocyte precursors in bone marrow o Peripheral Blood: 0% THE MEGAKARYOCYTIC CELL SERIES • • • • PROMEGAKARYOCYTE Megakaryocytopoiesis proceeds initially through a phase characterized by mitotic division of a progenitor cell, followed by a wave of nuclear endoreduplication. o Occurs through mitosis o IL-3, IL-6, IL-11 - important megakaryocytic interleukin Endoreduplication is the process in which chromosomal material (DNA) and the other events of mitosis occur without subsequent division of the cytoplasmic membrane into identical daughter cells. Thrombopoietin - hormone thought to stimulate production and maturation of megakaryocytes, which in turn produce platelets, has recently been purified and cloned. Thrombopoietin activity results from several different cytokines: erythropoietin, IL-3, and panmyeloid factor granulocyte macrophage colony-stimulating factor (GMCSF). These substances have been shown to be able to increase megakaryocyte size, maturational stage, and ploidy • • • • • • • • Size: 15-40 mcm Nucleus: Indented Nucleoli: Variable Chromatin: Condensed Cytoplasm: Basophilic Granules: Present N/C Ratio: 1:2 Reference Interval: o Bone Marrow: 25% of megakaryocyte precursors in bone marrow o Peripheral Blood: 0% MEGAKARYOCYTE • • • • PLATELETS Megakaryocytes are the largest bone marrow cells, ranging up to 160 mm in size. The nuclear- cytoplasmic (N:C) ratio can be as high as 1:12. o Large ratio in preparation for platelet formation Nucleoli are no longer visible. Distinctive feature of the megakaryocyte is that it is multilobular, not multinucleated. The fully mature lobes of the megakaryocyte shed platelets from the cytoplasm on completion of maturation. CELLULAR ULTRASTRUCTURE OF A MATURE PLATELET PLATELETS • • • • • • • Platelets have an average diameter of 2 to 4 mm, with younger platelets being larger than older ones. In contrast to megakaryocytes, platelets have no nucleus. The cytoplasm is light blue, with evenly dispersed, fine red-purple granules An inactive or unstimulated platelet circulates as a thin, smooth surfaced disc. Platelets circulate at the center of the flowing bloodstream through endothelium-lined blood vessels without interacting with other platelets or with the vessel wall. Stronger stimulation causes platelets to become sticky without losing their discoid shape; however, changes in shape to an irregular sphere with spiny pseudopods will occur with additional stimulation. This alteration in cellular shape is triggered by an increase in the level of cytoplasmic calcium. • • • • • The Glycocalyx o or a FLUFFY COAT - these surrounds the cellular membrane of the platelet externally. o unique among the cellular components of the blood. o composed of plasma proteins and carbohydrate molecules that are related to the coagulation, complement, and fibrinolytic systems. Microfilaments & Microtubules o located directly beneath the cell membrane of platelet o provide structure of the platelet to maintain its discoid shape o maintains the posin of the organelles o secondary system of microfilaments is functional in internal organization and secretion of blood coagulation products, such as fibrinogen. Granules o Alpha Granules - most abundant ▪ contains: • heparin-neutralizing platelet factor 4 (PF 4) • beta-thromboglobulin • platelet-derived growth factor • platelet fibrinogen • fibronectin • von Willebrand factor (vWF) • thrombospondin o Dense or Delta Granules ▪ contains: • serotonin • adenosine diphosphate (ADP) - potent aggregator • adenosine triphosphate (ATP) • calcium. o Lysosomes - store hydrolase enzymes Other Cytoplasmic Constituents o Contractile proteins, including actomyosin (thrombosthenin), myosin, and filamin, o Glycogen - anaerobic glycolysis o Enzymes of the glycolytic and hexose pathways PLATELET KINETICS, LIFESPAN & NORMAL VALUES • • • • • • • Average megakaryocyte produces about 1,000 to 2,000 plts. Marrow transit time / maturation period of megakaryocyte 5 days Platelets initially enter the spleen & remains for 2 days. Platelets are in either the circulating blood or the active splenic pool; Platelets exchange pools o Approximately 2/3 of the total number of platelets are in systemic circulation o The remaining 1/3 exists as pool of platelets in the spleen that free exchange with the general circulation o Life span - 9-11 days A normal person has an average of 250 × 109/L (range, 150 × 109/L to 450 × 109/L) platelets in systemic circulation. Platelet turnover or effective thrombopoiesis averages 35 × 109/L ± 4.3 × 109/L/day. The life span of a mature platelet is 9.0 days ± 1 day. o At the end of their life span, platelets are phagocytized by the liver and spleen and other tissues of the mononuclear phagocytic system. • • FUNCTIONS OF PLATELET • • • • Primary hemostasis- adhesion, secretion (release mechanism, /activation), aggregation with the end of view of forming platelet plug. Secondary hemostasis (blood clotting/coagulation) release of Pf3 or PPL (phospholipid)- which plays a big role in forming stable fibrin clot. Clot retraction- the substance responsible for extrusion of serum form the clot is the platelet contractile protein, Thrombosthenin/Actomyosin. Helps in localization of bacteria and other small objects by adhering to these objects and producing aggregates too large to pass through the capillaries. • CHEMISTRY OF PLATELETS • • More than 90 different enzymes have been associated with platelets. Platelets are composed of 60% protein, 30% lipids and 8% carbohydrates o Carbohydrates mainly glycogen and sulphate mucopolysaccharides, various minerals, water and nucleotides. o Lipids include phosphatidylethanolamine and phosphatidylserine. PARTS OF A PLATELET • Peripheral zone - the outermost zone, responsible for adhesion. and later, aggregation. o Glycocalyx - the fuzzy coating, primarily composed of glycoproteins (GP) o Plasma membrane - lies directly beneath the glycocalyx and composed of asymmetrically distributed phospholipids embedded with integral proteins where arachidonic acid is a major component. GP Ib/V/IX are receptors for vWF. GP IIb and IIIa are receptors for fibrinogen and vWF as well. o Submembranous area - site where messages from the external membrane are translated into chemical signals causing activation and a physical change in the platelets. Receptors for ADP, collagen, serotonin, epinephrine, thrombin, vWF, Factor V and Factor Xa have been identified. Sol-gel zone - lies beneath the plasma membrane. o Composed of : ▪ Microfilaments and microtubules- they are responsible for providing cytoskeleton to the platelet/ maintain the disc-shape. and the contractile system. ▪ The microfilaments contain the proteins actin and myosin which upon stimulation forms the contractile protein actomyosin or more commonly called thrombosthenin, which is responsible for clot retraction. ▪ The microtubules are composed of the protein tubulin which maintains the platelet's disc shape. Organelle zone - called the centromere., part where the secretory products come from. o Alpha granules - the most numerous of all the granules. o Dense bodies / delta bodies - play an important role in platelet aggregation especially its product, ADP. o Mitochondria - used for aerobic glycolysis, where ATP is produced (used for energy). The platelets can also do anaerobic glycolysis due to its glycogen granules. o Lysosomal granules - contain acid phosphatase and hydrolytic enzymes. Membranous system o Dense Tubular System - derived for smooth endoplasmic reticulum and sequesters calcium for platelet activation processes. It also synthesizes prostaglandin. o Surface Connected System - an invagination of the plasma membrane, acts as a canal for the release of the granule contents and cytoplasm to the exterior of the platelet. This system is also involved in platelet phagocytosis. ADDITIONAL ANOMALIES ASSOCIATED WITH PLATELETS AND MEGAKARYOCYTES • • • • • Hemmeler’s anomaly - defect when no azurophilic granules is found in cytoplasm of platelets. The absolute platelet count is within reference range. This is found in hemorrhagic diathesis (bleeding tendency) Hermansky-Pudlak syndrome - found in albinos, there is hemorrhagic diathesis, dense bodies storage pool disease. Platelets have storage pool deficiency. Atypical platelets: o giant platelets- 4-7 um in diameter o increased amount of hyalumere o granule is decreased or absent o bizarre or irregular shapes Gray platelet syndrome - defects where there is decreased number of alpha granules Platelet satellitosis/ platelet satellitism - when platelets are found encircling the borders of neutrophils, seen in patients whose blood was anticoagulated with EDTA. This phenomenon is thought to be due to a serum factor that reacts in the presence of EDTA.