Chapter 13: PLATELET PRODUCTION, STRUCTURE AND FUNCTION MEGAKARYOCYTES - Platelets are anucleate blood cells - 1-4 um with

life span 9-12 days - Mean counts slightly higher in women than in men - Platelets arise from unique bone marrow cells called megakaryocytes - Among the largest cells in the body and are polyploid - Other hematopoietic cells may cross the megakaryocyte cytoplasm to reach the sinusoid lumen, a faux phagocytosis known as emperopolesis Platelets - 60% protein - 30% lipids - 10% CHO Chromomere - Granular portion - Central Hyalomere - Peripheral; Non-granular MEGAKARYOCYTE PROGENITORS - Hematopoietic stem cell (CFU-GEMM) differentiates to the megakaryocytes lineage under the influence of the hormone thrombopoietin (TPO) and a series of cytokines - Three megakaryocyte lineage committed progenitor stages a. BFU-Meg are diploid and participate in normal mitosis, maintaining a pool of b. CFU-Meg megakaryocyte progenitors c. LD-CFU-Meg has little proliferative capacity and produces few cells but progresses to increased nuclear ploidy - may be a transitional, or promegakaryoblast stage in which polyploidy is established but the morphology is indistinguishable from small lymphocytes - Useful megakaryocyte specific and platelet- specific immunologic markers are Platelet Factor 4 (PF 4), Von Willebrand Factor (VWF), and Platelet Glycoproteins 1b (GP 1b, CD42b), and IIb/IIIa (GP IIb, IIIa, CD 41) TERMINAL MEGAKARYOCYTE DIFFERENTIATION - MK I (Megakaryoblast) - MK II (Promegakaryocyte) - MK III (Megakaryocyte) - MK IV characterized by multilobed; highly condensed molecule a. MK I - Plasma membrane blebs, blunt projection that resemble platelets or nuclear lobulation that reflects polyploidy - Greater diameter than the other two blasts

- The nucleus, although essentially round, reaches its full ploidy - Cytoplasm possesses alpha-granules and demarcation system (DMS) - DMS a series of membrane-lined channels, invades from the plasma membrane and grows over the course of terminal differentiation to subdivide the entire cytoplasm b. MK II - Easily recognized as megakaryocytic on the basis of its 15-40 um diameter - Nucleus is indented or lobulated c. MK III - The largest cell in the bone marrow, easily detected with the 10x objective - Nucleus is intensely lobulated and chromatin is condensed - Cytoplasm is eosinophilic, granular, and platelet-like - The stage from which platelets shedding or thrombopoiesis proceeds ENDOMITOSIS/ENDOREDUPLICATION - Megakaryocyte maturation is marked by mysterious form of mitosis that lacks telophase and cytokinesis - DNA synthesis proceeds to the production of 8N, 16N, and 32N ploidy with completely duplicated sets of chromosomes but no cell division - A single megakaryocyte can shed 2000-4000 platelets - Mitosis ends at the progenitor stage, endomitosis is complete at MK I THROMBOPOIESIS - The DMS dilates longitudinal bundles of tubules form, cytoplasmic extensions called proplatelet processes - Leaves behind naked megakaryocyte nuclei to be consumed by marrow macrophages, although these are rarely seen in bone marrow aspirate films HORMONES AND CYTOKINES OF MEGAKARYOPOIESIS - TPO is a 70-kD molecule with 23% homology to EPO mRNA found in the kidney, liver, and smooth muscle cells Primarily produced in the liver and is the ligand that binds to a megakaryocyte and plately membrane receptor protein Inversely proportional to platelet and megakaryocyte mass, implying that binding and disposal of TPO by platelets is the primary control mechanism - Cell division stimulators of megakaryocytopoeisis include: Interleukin (IL) 3, IL-6, and IL-11 used to stimulate platelet production in chemotherapy induced thrombocytopenia IL- 3 seems to act in synergy with TPO to induce early differentiation of stem cells IL-6 and IL-11 act in the presence of TPO to enhance the later phenomena of endomitosis, megakaryocyte maturation and platelet release - Kit ligand or mast cell growth ligand Other cytokines and hormones that participate synergistically with TPO and the IL are stem cell factors PLATELETS - Biconvex, even and they flow smoothly through the blood vessels

Move laterally with the leukocytes into the white pulp of the spleen where both become sequestered The normal peripheral blood platelet count is 150-400 x 109/L Reticulated platelets Stress platelets Markedly larger than ordinary mature circulating platelets Diameter in blood films exceeding 6 um and their MPV reaching 12-14 fL Cylindrical and beaded, resembling megakaryocyte proplatelet processes Carryfree ribosomes and fragments of rER, analogous to reticulocytes, triggering speculation that they rise from early and rapid proplatelet extension and release

RESTING PLATELET PLASMA MEMBRANE - Phospholipids which form the basic structure and cholesterol, which distributes asymmetrically throughout the phospholipids - Phosphoinositol support platelet activation by supplying arachidonic acid Arachidonic acid unsaturated fatty acid that becomes converted to the eicosanoids, prostaglandin, and thromboxane during platelet activation - Cholesterol stabilizes the membrane, maintains fluidity, and helps control the transmembranous passage of materials - Glycocalyx Platelet membrane surface; absorbs albumin, fibrinogen; endocytosis; thicker (platelet) SURFACE-CONNECTED CANALICULAR SYSTEM - Twists spongelike throughout the platelet, enabling the platelet to store additional quantities of the hemostatic proteins of the glycocalyx - The route for endocytosis and for secretion of granular contents of activation DENSE TUBULAR SYSTEM - A dense remnant of ER - Sequesters calcium and bears a series of enzymes that support platelet activation - Enzymes: Phospholipase A, Cyclooxygenase A, and Thromboxane synthase - Control center for platelet activation PLATELET ACTIVATION a. ADHESION - Platelets reversibly bind elements of the vascular matrix - Touch the surface - Platelets adhere to fibrillar VWF through their GP 1B/IX/V receptor b. AGGREGATION - Platelets irreversibly bind each other - Defect (GP IIb/IIIa) touch the surface Binds the RGD sequence of any plasma protein; the protein most readily available is fibrinogen which is the major player - Part of primary hemostasis and is irreversible end point is the white clot of Platelet-VWF plug - White clots arteries - Red clots Essential to wound repair but is characteristic of inappropriate coagulation in venules and veins, resulting in deep vein thrombosis and pulmonary emboli

c. SECRETION - Activated platelets release granular contents - Delta granule contents are vasoconstrictions and platelet agonist that amplify primary hemostasis

PLATELET ACTIVATION PATHWAYS G Proteins - Control cellular activation for all cells (not just platelets) at the membrane - Are alpha-beta-gamma (aBy) heterotrimers that bind GDP when inactive a. Eicosanoid Synthesis - Alternatively named the prostaglandin, cyclooxygenase or thromboxane pathway - One of the two essential platelet activation pathways triggered by G protein - Arachidonic acid Phospholipid whose number 2 carbon binds numerous unsaturated fatty acids - Cyclooxygenase converts arachidonic acid to prostaglandin G2 and Prostaglandin H2 - The cyclooxygenase pathway in endothelial cells incorporates the enzyme prostacyclin synthase in place of the thromboxane synthase in platelets - Endpoint: Prostaglandin I2 or Prostacyclin b. Inositol Triphosphate and Diacylglycerol -G protein activation triggers phospholipase C - phospholipase C cleaves membrane phosphatidylinositol 4,5 biphosphate to form IP3 and DAG - IP3 and DAG second messengers for intracellular activation - IP3 promotes release of ionic calcium from the DTS, triggering actin microfilament contraction; also activates phospholipase A - DAG triggers a multistep process: activation of phosphokinase C -> triggers phosphorylation of pleckstrin - Pleckstrin regulates actin microfilament contraction

Chapter 11: IRON METABOLISM IRON -

Essential for all living organisms Most functional iron in humans is in the form of Hb and Mb In Hb, an atom of ferrous iron is incorporated into protoporphyrin IX, a ring of C, H, and N of iron storage form Most nonheme iron is stored in ferritin or hemosiderin in hepatocytes or macrophages Carrier of electrons and is used to bind with cofactors essential to basic metabolic oxidation and reduction reactions Catalyst for oxygenation, hydroxylation and other crucial metabolic processes Plays a key role in the formation of harmful oxygen radicals that can damage cellular structures

DIETARY IRON - Iron is absorbed in 2 forms: Heme and nonheme iron - Heme iron - Absorbed more effectively than nonheme iron - Present in forms of Hb, Mb, and heme enzymes in meat sources - 5-35% - Non heme iron legumes and leafy vegetables - Accounts for approx 90% of the dietary iron but only 2% to 20% of it is absorbed IRON ABSORPTION AND EXCRETION - Duodenum and upper jejunum are sites of maximal absorption of iron - Transport of O2 iron must be in Ferrous state or converted from ferric-nonheme iron to the soluble ferrous form by a duodenum-specific cytochrome b-like protein: DCYTB - Ferrous iron is transported across the duodenal epithelium by the apical divalent metal transporter (DMT1) Basolateral membrane, where it is believed the ferrous iron is exported to the portal circulation (liver) and mediated by ferroportin, a basolateral transport protein - Ferroportin may work in conjunction with a copper-containing iron oxidase hephaestin - Ferritin storage form of iron while the cells are exfoliated - Hepcidin an antimicrobial peptide produced in the liver seems to act as a negative regulator of intestinal iron absorption and release from macrophage - Transferrin is taken into the cell by endocytosis - Iron is released from transferring by acidification of the endosome to a pH of about 5.5 - Iron is transported across the endosomal membrane by DMT1 and used in the synthesis of iron containing proteins - The amount of iron absorbed is inversely related to the amount of iron stores and the rate of eythropoiesis - Normal iron absorption 1-2 mg/d

CYCLE AND TRANSPORT - Iron cycles moving from absorption in the GI tract via the circulation to the bone marrow, where it is incorporated with protoporphyrin IX in the mitochondria of the erythroid precursors to make heme - Hb synthesis reticulocyte stage

Ferrokinetics involve the proteins: transferrin, transferrin receptor and ferritin - they are regulated by an iron-responsive protein (IRP) Most transferrin is produced by hepatocytes, transport iron from the plasma to the normoblasts in the marrow. It has a half life of 8 days and migrates in the B-fraction in serum electrophoresis The transferrin molecule can exist as apoferritin, a single chain glycoprotein with no iron attached The transferrin receptors affinity for transferring depends on the iron content and the physiologic pH (7.4) When transferrin is fully saturated, iron absorbed by the intestine is deposited in the liver. Iron enters a chelatable soluble pool in the cell, where it is used for synthesis of essential cellular or for deposition as ferritin, a nontoxix storage form of iron. IRPs are messenger RNA binding proteins that coordinate the intracellular expression of transferrin receptor, ferritin and other proteins important for iron metabolism IRPs bind to IRES when iron supply is decreased and dissociate from IREs when iron supply is increased

STORAGE - Iron may be stored in accessible reverse form as ferritin or as a partially degraded or precipitated ferritin called hemosiderin (Prussian blue) - Apoferritin, the protein component of the ferritin molecule without iron, is a spherical protein shell about 12 nm in diameter and 1 nm in width and is composed of 24 subunits which are mixture of light (L) and heavy (H) subunits - The liver and spleen, which function as major iron storage deposits, have a large amount of L subunits - Iron storage sites have a higher proportion of H subunits - The gene for 2 types of L chains is on chromosome 19; the gene for heavy chains is on chromosome 11 - Hemosiderin A degradation product of ferritin produced by the partial digestion of protein and release of iron micelles, which form insoluble ferritin aggregates - Ferritin and Hemosiderin stores are found in liver, bone marrow and spleen, with most in the liver LABORATORY ASSESSMENT OF IRON STORES - Serum iron concentration Refers to the Fe+3 bound to serum transferrin and does not include any iron contained in serum as free Hb Chromogenspectrophotometic methods 1/3 of the iron binding sites of transferrin are occupied by Fe+3, 2/3 of the iron binding sites do not carry iron; this is known as the serum unsaturated iron binding capacity Total iron binding capacity total number of available sites Percent saturation is calculated by dividing the serum iron by the total iron-binding capacity and multiplying the result by 100 Ferritin present in the blood at low concentrations Serum transferrin sensitive immunologic methods; appears to be a truncated form of the cellular receptor and circulates bound to transferrin

Erythrocyte protoporphyrin - is an intermediate product of the Hb production and may be seem in excess in conditions in which heme production is incomplete - Free erythrocyte protoporphyrin combines with zinc when iron is unanvailable and can be measured directly by measuring the fluorescence of zinc protoporphyrin in hematofluorometer Tissue iron concentration can be assessed by a tissue biopsy of the bone marrow or liver Hemosiderin, the degradation product of ferritin molecules that has been incorporated into lysozomes of the macrophages In the normal bone marrow smear, iron granules are found in 10% or more of the nucleated RBCs and contain one to three blue inclusions that represent iron These cells are called sideroblasts; granules are siderosomes; reticulocytes in the bone marrow that contain iron - siderocytes