Thrombopoiesis - notes.pdf

MLS 323 1 Thrombopoiesis, Platelets structure and functions. 2 Objectives  Describe the morphological features of the mature stages of development in the megakaryocyte series.  Describe the process of formation of platelets from a megakaryocyte.  L...

MLS 323 1 Thrombopoiesis, Platelets structure and functions. 2 Objectives  Describe the morphological features of the mature stages of development in the megakaryocyte series.  Describe the process of formation of platelets from a megakaryocyte.  List the ultrastructural components and cytoplasmic constituents of a mature platelet and describe the overall function of each.  Explain the function of platelets in response to vascular damage.  Define generally the terms platelet adhesion and platelet aggregation. Platelets  Platelets are small fragments of cytoplasm derived from 3 megakaryocytes. On average they are 2– 4 μm in diameter but may be larger in some disease states.  Platelet production:  Platelets are produced in the BM by fragmentation of the cytoplasm of megakaryocytes, one of the largest cells in the body.  The precursor of the megakaryocyte- the megakaryoblast-arises by a process of differentiation from the hemopoietic stem cell.  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. 4  5  The megakaryocyte matures by endomitotic synchronous replication(endoreduplication) enlarging the cytoplasmic volume as the number of nuclear lobes increase in multiples of two.  Recognizable megakaryocytes have ploidy values of 4n, 8n, 16n, and 32n.  Approximately each megakaryocyte giving rise to 1000-5000 platelets*.  6  The time interval from differentiation of the human stem cell to the production of platelets averages approximately 10 days.  Thrombopoietin is the major regulator of platelet production and is constitutively produced by the liver and kidneys.  The normal platelet count is approximately is between (150000-450000 cell/uL) and the normal platelet lifespan is 7-10 days.  Up to one-third of the marrow output of platelets may be trapped at anyone time in the normal spleen but this rises to 90% in cases of massive splenomegaly  7  An inactive or un-stimulated platelet circulates as a thin, smooth- surfaced disc.  This discoid shape is maintained by the microtubular cytoskeleton beneath the cytoplasmic membrane.  Platelets circulate at the center of the flowing bloodstream through endothelium-lined blood vessels without interacting with other platelets or with vessel wall.  Cellular Ultrastructure of a Mature Platelet The Glycocalyx  It is the external fluffy coat which surrounds the cellular membrane.  It is composed of plasma proteins and carbohydrate molecules that are related to the coagulation, complement, and fibrinolytic systems. Glycoprotein receptors  The glycoprotein receptors of the glycocalyx mediate the membrane contact reactions of platelet adherence, change of cellular shape, internal contraction, and aggregation 8  9 Cytoplasmic membrane  Adjacent to the glycocalyx is the cytoplasmic membrane  Extending through the plasma membrane and into the interior of the platelet is an open canalicular or surface connecting system into which plasma clotting factors are selectively adsorbed. Canaliculi system  the canaliculi penetrate the cytoplasm in a random manner, they are generally in close proximity to granules and other organelles.  Therefore, products released by the granular or cytoplasm can be transported to the exterior environment through the canaliculi.  10 Microfilaments and Microtubules  Directly beneath the cell membrane is a series of sub- membrane filaments and microtubules that form the cellular cytoskeletone.  In addition to providing the structure for maintaining the circulating discoid shape of the cell, the cytoskeleton also maintains the position of the organelles.  A secondary system of microfilaments is functional in internal organization and secretion of blood coagulation products, such as fibrinogen.  These subcellular and cytoplasmic filaments make up the contractile system of the platelet.  11 Granules  Three different types of strong granules related to hemostasis are present in the mature platelet.  These granules are alpha granules, dense or delta granules, and lysosomes.  The alpha granules are the most abundant. Alpha granules contain heparin –neutralizing platelet factor 4 ( PF 4), beta thromboglobulin, platelet-derived growth factor, platelet fibrinogen, fibronectin, von Willebrand factor (vWF), and thrombospondin.  12  Dense bodies, named because of their appearance when viewed by electron microscopy, contain serotonin, ADP, ATP, and calcium.  Lysosomes, the third type of granule, store hydrolase enzymes.  Extrusion of the contents of these storage granules requires internal, cellular contraction.  Secretions from the granules are released into the open canalicular system.  13 Other cytoplasmic Constituents  Contractile proteins, including actomycin, myosin, and filamin.  Glycogen and enzymes of the glycolytic and hexose pathways.  The platelet is a very high-energy cell with a metabolic rate 10 times that of an erythrocytes 14  15 Platelet's role in hemostasis 1. Adhesion and aggregation forming the primary haemostatic plug 2. Release of platelet activating and procoagulant molecules 3. Provision of a procoagulant surface for the reactions of the coagulation system  The adherence and aggregation of platelets at the sites of vascular damage allow for the release of molecules involved in hemostasis and wound healing and provide a membrane surface for the assembly of coagulation enzymes that lead to fibrin formation.  Platelet Adhesion  Platelet adhesion to subendothelial connective tissues, especially collagen, within 1 to 2 minutes after a break in the endothelium.  ADP increases the adhesiveness of platelets.  The transformation of the platelet from a disc to a sphere with pseudopods produces surface membrane reorganization.  Internal contraction of the platelet results in release of granular contents of the alpha and dense granules and the lysosomal contents. 16  17  Platelets adhere at sites of mechanical vascular injury and then undergo activation and express functional glycoprotein IIb/IIIa receptors (also referred to as integrin alpha beta) for circulating adhesive ligand proteins (primarily fibrinogen).  These functional glycoprotein IIb/IIIa receptors mediate the recruitment of local platelets by forming fibrinogen bridges between platelets—a process called platelet cohesion.  18 Platelet Aggregation  Platelet aggregation represents a multistep adhesion process involving distinct receptors and adhesive ligands, with the contribution of individual receptor-ligand interactions to the aggregation process dependent on the prevailing blood flow conditions.  A variety of agents are capable of producing platelet aggregation, an energy-dependent process.  These agents include particulate material such as collagen, proteolytic enzymes such as thrombin, and biological amines such as epinephrine and serotonin.  19  It is believed that bridges formed by fibrinogen in the presence of calcium produce a sticky surface on platelets. This results in aggregation.  If these aggregates are reinforced by fibrin, they are referred to as a thrombus.  Aggregation of platelets by at least one pathway can be blocked by substances such as prostaglandin E (PGE), adenosine, and nonsteroidal anti-inflammatory agents (e.g., aspirin).  Aspirin induces a long-lasting functional defect in platelets. It is clinically detectable as a prolongation of the bleeding time. 20 21 22 23  24 Laboratory Assessment of Platelets Quantitative Determination of platelets  Can be determined in a blood sample using an electronic particle counter or by manual methods.  Examination of a stained blood film provides a rapid estimate of platelet numbers. Normally, there are 8 to 20 platelets / 100 oil immersion field in a properly prepared smear where the erythrocytes barely touch or just overlap. At least 10 different fields should be carefully examined for platelet estimation. The average number can be multiplied by a factor of 20,000 to arrive at an approximation of quantitative platelet concentration. 25 Qualitative Assessment of Platelets  If platelet count is normal but a patient has a suggestive bleeding history, an assessment of platelet function should be conducted.  Methods of evaluation include bleeding time, aggregating agents, and lumi-aggregation.  26 Bleeding Time  The bleeding time test is an in vivo measurement of platelet adhesion and aggregation on locally injured vascular subendothelium.  This test provides an estimate of the integrity of the platelet plug and thereby measures the interaction between the capillaries and platelets.  As the platelet count drops below 100 X 109/L , the bleeding time increases progressively to more than 30 minutes.  A prolonged bleeding time in a patient with a platelet count above 100 X 109/L indicates either impaired platelet function or a defect of subendothelial factor. Results between 8 and 11 minutes are usually not clinically significant.  Clot Retraction  The contractile abilities of platelets also result in the contraction of formed clots.  Clot retraction reflects the number and quality of platelets, fibrinogen concentration, fibrinolytic activity, and packed red cell volume.  Since the fibrin clot enmeshes the cellular elements of the blood, primarily erythrocytes , the degree of clot retraction is limited to the extent that fibrin contracts by the volume of erythrocytes (hematocrit).  Therefore, the smaller the hematocrit, the greater the degree of clot retraction.  The degree of clot retraction is directly proportional to the number of platelets and inversely proportional to the hematocrit and blood fibrinogen.  When clot dissolution ( fibrinolysis ) is very active, the fibrin clot may be dissolved almost as quickly as it is formed, and clot 27 retraction will be impaired.  28 Platelet Aggregation  Agents such as ADP, collagen, epinephrine, snake venom, thrombin can be used to aggregate platelets.  The principle of the test is that platelet –rich plasma (PRP)is treated with a known aggregating agent.  If aggregated, cloudiness or turbidity can be measured using a spectrophotometer. Depending on the type of aggregating agent used, a curve that can be used to assess platelet function is obtained.  29 Platelet Adhesion  Platelet adhesion in vivo occurs as platelets attach either to a damaged vessel wall or to each other.  Methods of in vitro analysis rely on the adherence of platelet to glass surfaces.  The amount of adherence of platelets in a blood sample to a glass surface can be measured by counting the number of platelets before and after exposure to glass beads. 30 Antiplatelet Antibody Assay  Antibody against platelets may appear in the plasma of patients in certain clinical conditions, although it may be difficult to demonstrate these antibodies in cases of immune thrombocytopenia.  Available techniques can include complement fixation methods, lysis of chromium 51-labeled platelets, assays of platelet-bound immunoglobulins, and competitive inhibition assays. 31 References  Clinical Hematology :- Theory and Procedures, Fifth Edition, by Mary Louise Turgeon  Essential Haematology: By: Hoffbrand, A. V. Malden, Mass.: Blackwell Pub., 2011.  Dacie and Lewis practical haematology 10th ed., Indian ed. Author Lewis, S. M. (Shirley Mitchell).