HEMA LEC 🩸 4 PDF - Hemolytic Anemia Lecture Notes

MLS 417 – LEC | HEMATOLOGY-2-LECTURE F1−3: Hemolytic Anemia Professor: Lee-An Anayon, RMT, ASCPI / Dr. Chesa Belandres / Dr. Ismael Maminggen / Rodak’s 6th Edition Date: April 28, 2024 INTRODUCTION TO INCREASED DESTRUCTION OF RBCs Hemolysis/Hemolytic Disorder 1. Includes: 1. 2. 3. 4. Increased rate...

MLS 417 – LEC | HEMATOLOGY-2-LECTURE F1−3: Hemolytic Anemia Professor: Lee-An Anayon, RMT, ASCPI / Dr. Chesa Belandres / Dr. Ismael Maminggen / Rodak’s 6th Edition Date: April 28, 2024 INTRODUCTION TO INCREASED DESTRUCTION OF RBCs Hemolysis/Hemolytic Disorder 1. Includes: 1. 2. 3. 4. Increased rate of destruction (lysis) of RBCs, 2. Shortening its lifespan 3. Reduced tissue oxygenation 4. Increased Erythropoiesis (EPO) 5. RBC Production Hemolytic Anemia RBC Destruction > RBC Production experience intermittent episodes of hemolysis DISAPPEARS OR SUBSIDES BETWEEN EPISODES ○ Px condition may return to normal Classification Anemias of Secondary Hemolytic Component Hemolysis is NOT THE PRIMARY UNDERLYING CAUSE Anemias develop as a result of the inability of the bone marrow to INCREASE RBC production Rapid onset and isolated (sudden), episodic or paroxysmal Paroxysmal Cold Hemoglobinuria hemolysis after exposure to cold G6PD Compensation returns upon removal of drug Thalassemia major severe anemia + BM cannot generate cells fast enough Inadequate globin chains → decrease functional HGB → O2carrying capacity chronically low Passed to offspring by mutant genes from parents Ex. thalassemia Acquired Intrinsic Chronic shortened → BM compensates Challenged (oxidized) → dramatic acute hemolytic event Inherited Comparing Primary Hemolytic Anemias Acute Deficiency Excess normal globin chains precipitate intracellularly → cell lysis Iron Deficiency Anemia B9 and B12 Deficiency Anemia of Chronic Inflammation Anemia of Renal Disease (Systemic) Primary Hemolytic Anemias Hemolysis is THE PRIMARY UNDERLYING CAUSE Classified as 1. Acute vs Chronic 2. Intrinsic vs Extrinsic 3. Intravascular vs Extravascular 4. Fragmentation vs Macrophage-mediated Paroxysmal Nocturnal Hemoglobinuria not evident if Bone Marrow is still able to compensate but over time may develop hemolytic crisis that cause anemia RBC lifespan is chronically abnormalities of RBC membranes, enzymatic pathways or HGB molecule ○ Inherited - mostly ○ Subclassifications: Membrane defects, enzyme defects, hemoglobinopathies previously hematologically normal but acquired an agent or condition that causes lysis of RBCs; Ex. Malaria Extrinsic due to external agents ○ Substances in plasma or conditions affecting the anatomy of the circulation system Acquired - mostly Subclassifications: Immunohemolytic, traumatic, microangiopathic, infectious agents, chemical agents, physical agents Ex. Malaria, antibodies against RBC antigen, prosthetic heart valves Copy for: HAW, SPENCER O. | 1 Intravascular Occurs inside the vessels by fragmentation ○ Fragmentation can occur in the spleen and bone marrow as well Extravascular Occurs outside the vessels Lysis within the macrophage (macrophage-mediated) Hemolysis Normal Macrophage-Mediated Hemolysis and Bilirubin Metabolism Bilirubin: prominent product of RBC hemolysis RBC lifespan: 120 days Reticuloendothelial system (Mononuclear phagocyte System): recognize changes and aged RBCs leading to a Macrophage-mediated hemolytic process Sequestration Areas: macrophages in the liver and spleen(mostly), BM, LNs, monocytes Copy for: HAW, SPENCER O. | 2 Normal Catabolism of Hemoglobin. Macrophages lyse ingested red blood cells (RBCs) and separate hemoglobin (Hb) into globin chains and heme components. Amino acids from the globin chains are reused. Heme is degraded to iron and protoporphyrin. ○ Iron is returned to the blood to be reused. ○ Protoporphyrin is degraded to unconjugated bilirubin. Visualizing the Color Changes of Hemoglobin Degradation The degradation of heme can be seen in bruises in fair-skinned individuals or in the eye's sclera after a vascular bleed. The same process that macrophages facilitate can occur in tissues. At first, the extravasated but deoxygenated blood gives the injury the purple-red appearance of hemoglobin. As the hemoglobin is degraded, the color changes to a greenish hue due to the biliverdin, but ultimately it becomes yellow due to the bilirubin. Catabolism of Heme to Bilirubin. In cells containing heme oxygenase, iron is removed from heme, and the protoporphyrin ring is opened up to form an intermediate, biliverdin. Biliverdin is converted to unconjugated bilirubin by biliverdin reductase. The unconjugated bilirubin is secreted into the blood and binds to albumin for transport to the liver. When unconjugated bilirubin enters the hepatocyte, UGT1A1 (uridine diphosphate glucuronosyltransferase family 1 member A1, formerly glucuronyl transferase) adds two molecules of glucuronic acid to form bisglucuronosyl bilirubin, also called conjugated bilirubin Typical reference intervals Total serum bilirubin level 0.5–1.0 mg/dL Direct (conjugated) serum bilirubin level 0–0.2 mg/dL Indirect (unconjugated) serum bilirubin level 0–0.8 mg/dL Copy for: HAW, SPENCER O. | 3 Normal Macrophage-Mediated Hemolysis 1. 2. 3. 4. 5. 6. 7. 8. Plasma HGB salvaged during normal Fragmentation Hemolysis Fragmentation Hemolysis Result of trauma to the RBC membrane that causes a breach to spill cell contents like HGB 10-20% of normal RBC destruction via fragmentation Secondary to turbulence and anatomic restriction in the vessels Normal Macrophage-Mediated Hemolysis. In a macrophage, hemoglobin is degraded to heme, the iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin. Macrophages release unconjugated bilirubin into the blood, where it binds to albumin for transport to the liver. Unconjugated bilirubin enters the hepatocyte. The hepatocyte converts unconjugated bilirubin to conjugated bilirubin. Conjugated bilirubin leaves the liver in the bile and enters the small intestine. Bacteria in the large intestine convert conjugated bilirubin to urobilinogen, most of which is excreted in the stool. Some of the water-soluble urobilinogen is reabsorbed in the portal circulation, and most is recycled through the liver for excretion. A small component of the reabsorbed urobilinogen is filtered and excreted in the urine. Copy for: HAW, SPENCER O. | 4 (PICTURE ABOVE) NORMAL FRAGMENTATION HEMOLYSIS 1. Normally a small number of red blood cells lyse within the circulation, forming schistocytes and releasing hemoglobin (Hb) into the blood, mostly as a/b dimers. 2. The plasma protein haptoglobin (Hpt) binds a hemoglobin dimer in a complex. 3. The hemoglobin-haptoglobin complex binds to CD163 on the surface of macrophages in various organs. 4. The complex is internalized into the macrophage, where the hemoglobin dimer is released. The hemoglobin dimer is degraded to heme, the iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin. 5. The haptoglobin is degraded. 6. The unconjugated bilirubin released into the blood is bound to albumin and processed through the liver. 7. When free hemoglobin is released into the blood with fragmentation, the iron is rapidly oxidized, forming methemoglobin, and the heme (metheme) molecule dissociates from the globin. 8. The plasma protein hemopexin (Hpx) binds free metheme into a complex. 9. The hemopexin-metheme complex binds to CD91 on the surface of hepatocytes. 10. The complex is internalized into the hepatocyte. 11. The iron is released from the metheme, and the protoporphyrin ring is converted to unconjugated bilirubin, ready for conjugation and further processing. Excessive Hemolysis Excessive Macrophage-Mediated Extravascular) Hemolysis Anemia develops if: Senescent+affected cells is more than what is normally removed and BM cannot compensate “Extravascular” ○ RBC ingested by Macrophage and contents rare degraded here ○ RBC contents not detected in plasma Normal Aged RBC Display surface markers recognized by macrophages for removal Pathologic Processes Expression of same markers → removal 1. 2. 3. 4. 5. 6. (PICTURE ABOVE) EXCESS MACROPHAGE-MEDIATED HEMOLYSIS More than the usual number of red blood cells are ingested each day by macrophages. An increased amount of unconjugated bilirubin is produced, released into the blood, and binds to albumin. When increased unconjugated bilirubin is presented to the liver, an increased amount of conjugated bilirubin is made and excreted into the intestine. When an increased amount of conjugated bilirubin is present in the intestine, an increased amount of urobilinogen is formed and excreted in the stool. Increased urobilinogen in the intestine results in increased urobilinogen reabsorbed into the blood. Increased urobilinogen in the blood results in increased urobilinogen filtered and excreted in the urine Excessive Fragmentation Intravascular) Hemolysis Major feature in pathologic processes Ex. dramatic hemolysis Copy for: HAW, SPENCER O. | 5 ○ Physical lysis of RBCs by prosthetic heart valves ○ Exit of malaria protozoa bursting out of cells Appearance of RBC contents in plasma (HGB) LEGEND EXCESS FRAGMENTATION HEMOLYSIS: The Role of Macrophages 1. 2. 3. 4. 5. When an increased number of red blood cells lyse by fragmentation, more than the usual amount of hemoglobin (Hb) is released into the blood, mostly as a/b dimers. Haptoglobin (Hpt) binds the increased hemoglobin dimers, forming more than usual numbers of complexes. The hemoglobin-haptoglobin complexes are taken up by macrophages bearing the CD163 receptor in various organs. An increased amount of hemoglobin dimers is released from the complexes. The hemoglobin is degraded to heme, the iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin. The increased amount of unconjugated bilirubin is then transported to the liver and processed as with excess macrophage-mediated hemolysis Degradation of haptoglobin is accelerated compared with normal. Copy for: HAW, SPENCER O. | 6 The Role of the Liver 1. 2. 3. 4. If the amount of hemoglobin released from lysing red blood cells exceeds the capacity of haptoglobin, the unbound free hemoglobin is rapidly oxidized, forming methemoglobin, and the metheme molecule dissociates from the globin. Hemopexin binds to metheme, and the complex is captured by the CD91 receptor on hepatocytes. The complex is internalized by the hepatocyte, the iron is released from the metheme, and the protoporphyrin ring is converted to unconjugated bilirubin and ultimately to conjugated bilirubin to be processed, as in Figure 20.5, steps 3 to 6. Although a small of amount of hemopexin is recycled to the blood, most is degraded. Metheme can also temporarily bind to albumin, forming metheme-albumin (not shown), but metheme is rapidly transferred to hemopexin. The Role of the Kidney 1. 2. 3. 4. 5. 6. When excess red blood cells lyse by fragmentation and other systems are saturated, free (met)hemoglobin enters the urinary filtrate. Cubilin (Cb) on the luminal side of the proximal tubular cells binds proteins for reabsorption, includ- ing hemoglobin. Cubilin carries hemoglobin into the proximal tubular cells. The hemoglobin is degraded to heme, the iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin as in a macrophage and conjugated as in a hepatocyte. The fate of the bilirubin is uncertain, as it may be secreted to the filtrate or reabsorbed into the blood. When the amount of hemoglobin exceeds the capacity of the proximal tubular cells to absorb it from the filtrate, hemoglobinuria occurs. Copy for: HAW, SPENCER O. | 7 Fate of Iron Removed from Salvaged Hemoglobin in the Kidney ○ There is predominance of unconjugated bilirubin Icterus yellow plasma and tissue Kernicterus yellow coloring of the brain tissues Lipid solubility of unconjugated bilirubin → deposit in the brain Chronic hemolysis 1. 2. Iron (Fe) salvaged from absorbed hemoglobin can be transported into the circulation by ferroportin on the basolaminal side of the tubular cell. In the blood it will be bound to transferrin (Tf) for transport. Iron in excess of what can be transported into the circulation is stored as ferritin, and some is converted to hemosiderin. If the tubular cell is sloughed into the filtrate and appears in the urine sediment, the hemosiderin can be detected using the Prussian blue stain CLINICAL FEATURES If hemolysis is sufficient to cause anemia: ○ General anemias s/s: fatigue, dyspnea, dizziness, pallor, tachycardia Jaundice Splenomegaly develop with chronic macrophage-mediated hemolytic processes Gallstones (Cholelithiasis) constantly increased amount of bilirubin in the bile leads to stone formation Bone deformities in children persistent compensatory BM hyperplasia → bone deformities in growing bones Acquired, acute hemolytic Associated s/s: malaise, aches, vomiting, fever (confused with infection) Acute fragmentation hemolysis Profound prostration and shock ○ Brown urine associated with methemoglobinuria Heme toxicity Flank pain, oliguria, anuria → Acute renal failure yellow color of the skin and sclera Other causes of jaundice: Hepatitis, gallstones If Hemolytic cause = “HEMOLYTIC JAUNDICE” or “PREHEPATIC JAUNDICE” Copy for: HAW, SPENCER O. | 8 LABORATORY FINDINGS Hemolytic Anemia LOW VALUES MAY MEAN IMPAIRED SYNTHESIS OF HAPTOGLOBIN CAUSED BY LIVER DISEASE, ALWAYS DOUBLE-CHECK INC erythrocyte destruction Compensatory INCREASE in the rate of erythropoiesis Tests of Accelerated RBC Destruction Bilirubin Plasma HGB, Urine HGB and Urine Hemosiderin due to INC rate of HGB destruction → INC unconjugated bilirubin ○ Icteric serum or plasma ○ INCREASED indirect bilirubin → INCREASED total bilirubin Gross examination of plasma and urine may suggest fragmentation hemolysis Coffee-brown color plasma and root beer or beer-colored urine ○ MetHGB ○ Methemalbumin ○ Hemopexin-heme Normal physiologic fragmentation hemolysis produce plasma HGB 10% Few spherocytes on smear Evident during pregnancy, splenomegaly, againg (declining erythropoiesis) Spherocytes Jaundice (Sometimes during viral infxn) Mutations that Alter Membrane Structure Hereditary Spherocytosis HS Pathophysiology Gene mutations in which the defective proteins disrupt the vertical interactions between transmembrane proteins and the underlying cytoskeleton 5 known gene mutations in Cytoskeletal proteins: ANK1 codes of Ankyrin (US 40-65% cases) SPTA1 a-spectrin (25-50 IMAGE SIZE (um) MORPHOLOGY NUCLEUS: Single, centrally located or multiple round and oval nuclei; lobed CYTOPLASM: Diffuse blue-absence of specific granules, irregular in shape, and has cytoplasmic tags; basophilic cytoplasmi DESCRIPTION “PRIMITIVE CELL” TPO → responsible for the commitment of the megakaryocyte EARLIEST RECOGNIZABLE STAGE NUCLEUS: Increased nuclear lobes CYTOPLASM: Cell membrane retains its characteristic cytoplasmic tags and is rich in polyribosomes. Capable of protein synthesis DEMARCATING MEMBRANE SYSTEM (DMS) → developed more forms by invagination of the plasma membrane Round and expanded in volume NUCLEUS: Multiple nuclei and peripheral margins CYTOPLASM: Contains numerous small, rather uniformly distributed granules with a reddish-blue hue Polyribosomes and mitochondria located in the central area of the cell NUCLEUS: Pyknotic CYTOPLASM: Aggregations of granular material, groups of 10-12 azurophilic granules Separated by Demaracating Membrane System or vesicle May be seen near the periphery VERY LARGE CELL INCOMPLETE ENDOPLASMIC RETICULAR SYSTEM Copy for: HAW, SPENCER Y. | 3 PLATELET KINETICS Megakaryocyte produces 40-60 platelets per day until cytoplasm is depleted 50% more decrease of platelet may be caused by: 1. Defective platelet formation 2. Ineffective platelet delivery to the circulation 3. Intramedullary destruction 4. Interference caused by Bone marrow tumors Thrombopoietin ○ Maintains megakaryocyte mass ○ Regulates the presence and rate of cytoplasmic maturation and platelet release LIFE SPAN: 2-10 days LARGER PLATELETS: Due to severe bleeding SMALLER PLATELETS: Due to inflammation and iron deficiency anemia PLATELET STRUCTURE RETICULATED PLATELETS Also known as “STRESS PLATELETS” Compensational to thrombocyopenia SIZE: 8um MPV (Mean Platelet Volume): 12 to 14 fL EDTA: Like ordinary platelets, they round up CITRATE: Cylindrical and beaded, resembling fragments of megakaryocyte proplatelet processes Carry free ribosomes and fragments of the Rough Endoplasmic Reticulum Small, discoid shape packages of cytoplasm surrounded by a membrane similar structure to the normal cell membrane CANALICULI – platelet channels that give it a sponge-like appearance Platelet is divided into 4 major areas: 1. Plasma Membrane (Peripheral Zone) 2. Submembrane Area (Membranous Zone) 3. Platelet Cytoskeleton (Sol-gel zone) 4. Organelle Zone 1 PLASMA MEMBRANE OUTERMOST LAYER 7.5 nm in thickness; has a trilaminar unit structure GLYCOCALYX (Surface coat) ○ Smooth and contain pore-like indentations = distinct connection between the inside of the platelet and its surroundings ○ 10-15 um thickness ○ Number of glycoproteins are incorporated = important in platelet adhesion and aggregation ○ Provides a surface → coagulation factors may adhere Serves as the physical and chemical barrier between intracellular and extracellular constituents of the platelet Sodium/Potassium ATPAse ionic pump → maintains a transmembrane ionic gradient Phospholipid constituents and other fatty acid pools are also located Copy for: HAW, SPENCER Y. | 4 Von Willebrand Factor → also located ○ Peripheral Zone of Platelet → von Willebrand Factor + GP IIb + GP IIIa 2 SUBMEMBRANE AREA 3 PLATELET CYTOSKELETON Contains organized system of filaments Prevents contact between organelles and the cell membrane Submembrane filaments; contribute to the regulation of the normal platelet ⇒ DISCOID Act as a base for pseudopod formation Interacts with other contractile proteins → Modulate platelet adhesion and clot retraction Also known as SOL-GEL ZONE Maintains hape of resting platelet ⇒ DISCOID (Psuedopod) Shape change → Platelet activation 3 Filaments ○ Microtubules – maintains discoid shape ○ Microfilaments – mediates contractile events ○ Intermediate filaments 4 ORGANELLE ZONE ALPHA GRANULES GTP (Guanosine Triphosphate) Responsible for signal transduction Calcium Regulates platelet activation/aggregation for both primary and secondary hemostasis Magnesium Has a role in vasodilation Inhibits platelet aggregation and adherence Serotonin Responsible for vasoconstriction In injury, helps constrict first Spherical and larger (300 - 500 nm in diameter) Contains GP IIb and IIIa PF4 (Platelet Factor 4) Responsible for metabolic activity of platelets Mitochondria is present, lacks nucleus, golgi apparatus and rough Endoplasmic reticulum 3 TYPES OF GRANULES ○ Alpha Granules ○ Dense Granules ○ Lysosomal Granules Beta TG (Beta Thromboglobulin) Neutralizes heparin (the normal anticoagulant) to produce clot Promotes smooth muscle growth for vessel repair Promotes platelet interaction PDGF (Platelet-derived growth factor) Thrombospondin Platelet Granules LYSOSOMES DENSE GRANULES (DENSE BODIES) 250-350 nm in diameter ADP (Adenosine Diphosphate) Most important component Promotes platelet aggregation ATP (Adenosine Triphosphate) Responsible for energy GDP (Guanosine Diphosphate) Responsible for signal transduction Contains enzymes known as hydrolases ○ Elastase, collagenase, cathepsin, heparinase, and enzymes that degrade polysaccharides Acts to digest materials brought into the platelet by endocytsosis Increased clot lysis; results to Microangiopathic Hemolytic Anemia (MAHA) → Schistocyte formation → Hemolysis Copy for: HAW, SPENCER Y. | 5 PLATELET FUNCTION 1. Adhesion 2. Activation and Shape CHange 3. Secretion and Release 4. Aggregation ROLE OF PLATELETS IN THE CIRCULATION 1. Surveillance of blood vessel continuity 2. Formation of Primary hemostatic plug 3. Surface for coagulation factors to make secondary hemostatic plug 4. Aid in healing injured tissues Recall: Primary Hemostasis → Blood vessel, platelet Vascular and platelet response to vessel injury Form platelet plug Secondary Hemostasis → Produces stable clot due to coagulation factors Response of the coagulation process ultimately leads to stable fibrin-platelet plug HEMOSTASIS “The stoppage of blood flow” Involves the interaction of blood vessels, platelets, the coagulation mechanism, fibrinolysis, and tissue repair 3 COMPONENTS 1. Extravascular – tissues surrounding blood vessels 2. Vascular – the vessels through which blood flows 3. Intravascular – platelets and many biochemicals (procoagulants) in the plasma PLATELET MEMBRANE SYSTEM OPEN CANALICULAR SYSTEM OR SURFACE-CONNECTED CANALICULAR SYSTEM (SCCS) Uptake extracellular substances (Ca2+) and release intracellular substances 1 Vasoconstriction (P) Controlled by vessel smooth muscle; enhanced by chemicals secreted by platelets 2 Platelet adhesion (P) Adhesion to exposed subendothelial connective tissue 3 Platelet aggregation (P) Interaction and adhesion of platelets to one another to form initial plug at injury site 4 Fibrin-platelet plug formation (S) 5 Fibrin stabilization (S) DENSE TUBULAR SYSTEM Sequester Ca2+ Control center of platelet activation Site of Thromboxane synthesis (TXA2) Coagulation factors interact on platelet surface to produce fibrin; fibrin-platelet plug then forms at site of vessel injury Fibrin clot must be stabilized by coagulation factor XIII Copy for: HAW, SPENCER Y. | 6 ROLE OF THE BLOOD VESSELS ARTERIES AND VEINS 1. 2. 3. 4. RBCs and platelets usually leave Pericytes - cells beneath endothelium that may differentiate into vessel-related cells when needed ○ do not the Vasoconstriction and vasodilation are controlled by smooth muscle in these vessels 3 STRUCTURAL LAYERS Tunica adventitia - outer part of the vessel wall; with connective tissue fibroblasts and collagen fibers Tunica media - smooth muscle cells and connective tissue, collagen, occasional fibroblasts Tunica intima - inner endothelial lining comes into contact with blood cells separating them from the sub endothelium (basement membrane, collagen. elastic fibers) Blood is maintained in a fluid state as it flows through intact vessels VASCONSTRICTION → neurogenic response PLATELET ACTIVATION → platelet plug formation = platelet-fibrin clot formation COAGULATION → collagen exposure + tissue factor exposure = activation of extrinsic pathway then intrinsic pathway = common pathway = formation of fibrin clot FIBRINOLYSIS → release of TPA (Tissue Plasminogen Activator) → Plasminogen → Plasmin = Clot dissociation CAPILLARIES Smallest and most numerous blood vessel Where metabolic exchange between blood and tissues takes place Lumen is just large enough for a single RBCS or WBCS Junctions - found along the walls and allow the passage of WBCs, oxygen and nutrients Copy for: HAW, SPENCER Y. | 7 ROLE OF COAGULATION Antithrombotic, Fibrinolytic, and Coagulant Substances Released from or Found on the Surface of Intact Endothelial Cells SUBSTANCE ACTION HEMOSTATIC ROLE Numeral Preferred Name I Fibrinogen Synonyms II Prothrombin Prethrombin Prostacyclin (Prostaglandin I2 (PGI2)) Inhibits platelet adhesion and aggregation; stimulates vasodilation Anticoagulant Reduces blood flow rate III Tissue factor Tissue thromboplastin Adenosine (metabolic product of ATP and ADP) Stimulates vasodilation Reduces blood flow rate IV Calcium Ca2+ V Proaccelerin ADPase Destroys ADP, limits platelet activation Anticoagulant Labile factor Accelerator globulin (AcG) Thrombomodulin Endothelial surface receptor for thrombin Binds and inactivates thrombin and enhances anticoagulant and fibrinolytic action of protein C found in the plasma Anticoagulant Fibrinolytic VII Proconvertin Stable factor Serum prothrombin conversion accelerator (SPCA) Autoprothrombin I Antihemophilic factor (AHF) Heparan sulfate Coats the endothelial cell surface and weakly enhances activity of antithrombin-Ill, a plasma anticoagulant Anticoagulant Antihemophilic globulin (AHG) Antihemophilic factor A Platelet cofactor 1 IX Plasma thromboplastin component (PTC) Tissue plasminogen activator (TPA) Converts plasminogen to plasmin, which plays an important role in fibrinolysis Released only on appropriate stimulus, such as vessel injury, to prevent excessive clot formation at the site of injury and begin slow clot dissolution as the injured vessel heals Fibrinolytic Christmas factor Antihemophilic factor B Platelet cofactor 2 X Stuart-Prower factor Stuart factor Prower factor Autoprothrombin III Thrombokinase XI Plasma thromboplastin antecedent (PTA) Antihemophilic factor C XII Hageman factor Glass factor Contact factor XIII Fibrin stabilizing factor Laki-Lorand Factor (LLF) Fibrinase Plasma transglutaminase Fibrinoligase - Prekallikrein Fletcher factor - High-molecular-weight kininogen (HMWK) Fitzgerald factor Contact activation cofactor Williams factor Flaujeac factor Plasminogen activator inhibitor-1 Regulatory protein for fibrinolytic system von Willebrand factor (vWF) Protein produced in endothelium and stored in subendothelium (in the form of Weibel-Palade bodies) before secretion into the plasma and attachment to factor VIII:C VIII-C Antifibrinolytic Coagulation and platelet adhesion Copy for: HAW, SPENCER Y. | 8 EXTRINSIC PATHWAY Activated following vascular endothelial cell injury → exposure of TF (Transmembrane receptor Tissue Factor) TF and Plasma Factor VII bind to form the TF:VII complex = TF:VIIa complex TF:VIIa complex, along with Ca2+ activates Factor X to Xa in the common pathway The extrinsic and common pathways are evaluated in the PROTHROMBIN TIME (PT) test INTRINSIC PATHWAY COMMON PATHWAY Begins with activation of Factor X to Xa Factor Xa with a cofactor, Va, lipid, and Ca2+ converts prothrombin (II) to thrombin (IIa) Factor IIa converts fibrinogen (I), to fibrin Factor XII then stabilizes the fibrin clot Activation in vitro: when factor XII is exposed to a negatively charged surface such as glass or kaolin = contact phase of coagulation (XII, XI, high-molecular-weight kininogen (HMWK), and prekallikrein) ⇒ XIIa and XIa Facotr XIa, with Ca2+, in turn, converts Factor IX to IXa Factor IXa + platelet phospholipid, Ca2+ and a cofactor: Factor VIIIa, converts Factor X to Xa The intrinsic and common pathways are evaluated in the PTT Copy for: HAW, SPENCER Y. | 9 SUMMARY OF IMPORTANT SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN HEMOSTASIS ROLE IN HEMOSTASIS SUBSTANCE SOURCE HMWK Promote coagulation Fibrinogen Contact activation of intrinsic coagulation pathway a-granules Cofactor in fibrin clot formation vWF Assists platelet adhesion to subendothelium to provide coagulation surface Dense bodies a-granules Thrombospondin Promote vascular repair Serotonin Dense bodies Promotes vasoconstriction at injury site Thromboxane A2 precursors Membrane phospholipids Same; promotes platelet release reaction Platelet-derived growth factor a-granules Promotes smooth muscle growth for vessel repair Plasminogen PLATELET’S ROLE IN HEMOSTASIS 1. 2. 3. 4. 5. Adhesion to injured vessels Aggregation at the injury site Promotion of coagulation on their phospholipid surface Release of biochemicals important to hemostasis from their alpha granules and dense bodies Induction of clot retraction Same; also inhibits heparin Same ß-thromboglobulin Other systems affected Promotes platelet aggregation Calcium Platelet factor 4 Promote vasoconstriction Converted to fibrin for clot formation Factor V ADP Promote aggregation COMMENTS ON PRINCIPAL FUNCTION Chemotactic for fibroblasts to help in vessel repair; inhibits heparin a-granules Precursor to plasmin, which induces clot lysis a2-antiplasmin Plasmin inhibitor; inhibits clot lysis Protease nexin II Inhibits factor XIa and thus factor IX activation Platelet inhibitor of factor XI (PIXI) C1 esterase inhibitor Complement system inhibitor Copy for: HAW, SPENCER Y. | 10 FIBRINOLYSIS the system whereby the temporary fibrin clot is systematically and gradually dissolved as the vessel heals in order to restore normal blood flow Plasmin is capable of degrading fibrin as well as factors I, V, and VIII: PLATELET AGGREGATION PLATELET ADHESION Normally, platelets do not adhere to intact endothelial surface When damaged, they adhere to the exposed subendothelial structures Collagen, fibronectin, thrombospondin, laminin, vitronectin, and tissue factor → May trigger adhesion Different platelet response in different vessel wall components Platelet adhesion to collagen requires von Willebrand Factor, which links platelets to Glycoprotein Ib-IX COMPLEX and Subendothelial connective tissue ○ Which can be followed by the activation of Glycoprotein IIB-IIIA BERNARD SOULIER SYNDROME Cx: PLATELET SECRETION VON WILLEBRAND DISEASE Rare, autosomal recessive disorder GP Ib receptor is absent, sometimes; GP IX is absent Prolonged bleeding time Thrombocytopenia Gian platelets Autosomal dominant/recessive Qualitative or quantitative abnormalities in vWF glycoprotein No abnormality to platelet themselves = cryoprecipitate as treatment Triggered by ADP, Thrombin, Thromboxane A2 (TXA2) Preceded by a change in platelet shape ⇒ pseudopod formation ADP acts on a specific receptor site and causes secondary, irreversible platelet aggregation and recruit more platelets ○ ADP is dependent on the presence of a specific platelet glycoprotein receptor known as GP IIb-IIIa GLANZMANN THROMBASTHENIA - GP IIb-IIIA receptor is absent or mutated, platelets do not aggregate with ADP, thrombin, collagen, epinephrine, or arachidonic acid as normal platelets do THROMBIN Least of 3 mechanisms Stimulates ADP release Activates platelets phospholipases initiating formation of TxA2 May directly aggregate platelets THROMBOXANE A2 Metabolite of arachidonic acid Activated by thrombin, endotoxin, epinephrine = release of arachidonic acid, oxidized by CYCLOOXYGENASE = PGG2 and PGH2 = TxA2 = Prostaglandin Prostaglandins induce platelet aggregation Also a vasoconstrictor In the vascular endothelium, arachidonic acid is metabolized to prostacyclin or PGI2 ⇒ inhibits aggregation of platelets and vasodilation Free arachidonic acid via LYPOOXYGENASE = 12-HETE, (12-Hydroxyeicosatetraenoic acid) which is important for platelet adhesion With relatively weak stimuli, only the contents of the a-granules are released, but with increased stimuli from higher concentrations of these agents, the contents of the dense bodies are also released Alpha granules = platelet aggregation and activate the coagulation system ○ GRAY PLATELET SYNDROME → deficiency of a-granules causing lifelong bleeding tendency Copy for: HAW, SPENCER Y. | 11 HEMATOLOGY-2-LECTURE F5: Mechanism of Coagulation and Fibrinolysis Professor: Lee-An Anayon, RMT Date: May 18, 2024 INTRODUCTION BLOOD EXTRAVASATION is mediated by: 1. The lining of endothelial cells in the blood vessels 2. Platelets 3. Plasma coagulation proteins 4. Protease inhibitors 5. The fibrinolytic system COAGULATION PATHWAY COAGULATION FACTORS/ZYMOGENS EXTRINSIC Prothrombin Time evaluates extrinsic and common pathway Calcium Fibrinogen Also known as Factor I Phospholipid Platelets Factor II, VII, IX, X, XI, XII, XIII* and Prekallikrein Factor XIII is cysteine-rich NON-ENZYMATIC COFACTORS Zymogens are substrates that have no biologic activity until converted by enzymes to active enzymes. Factors V and VIII, TF, Ca2+, High Molecular Weight Kininogen Cofactors assist in activation of zymogens by either ○ Altering zymogen conformation to permit more efficient cleavage by the serine protease, or ○ Binding the zymogen and appropriate serine protease on a platelet phospholipid surface to enhance and accelerate the zymogen activation process, or both TISSUE FACTOR ○ Also known as TF, Tissue thromboplastin, or factor III ○ Phospholipid containing ○ Found in the plasma membrane of many cell types EXCEPT ENDOTHELIAL CELLS ○ Activates EXTRINSIC COAGULATION PATHWAY ○ Do not normally circulate in the blood → Copy for: HAW, S.Y. | 1 COAGULATION AND KININS KININS III Tissue factor Tissue thromboplastin IV Calcium Ca2+ V Proaccelerin Labile factor Accelerator globulin (AcG) VII Proconvertin Stable factor Serum prothrombin conversion accelerator (SPCA) Autoprothrombin I Antihemophilic factor (AHF) Antihemophilic globulin (AHG) Antihemophilic factor A Platelet cofactor 1 IX Plasma thromboplastin component (PTC) Christmas factor Antihemophilic factor B Platelet cofactor 2 X Stuart-Prower factor Stuart factor Prower factor Autoprothrombin III Thrombokinase XI Plasma thromboplastin antecedent (PTA) Antihemophilic factor C XII Hageman factor Glass factor Contact factor XIII Fibrin stabilizing factor Laki-Lorand Factor (LLF) Fibrinase Plasma transglutaminase Fibrinoligase - Prekallikrein Fletcher factor - High-molecular-weight kininogen (HMWK) Fitzgerald factor Contact activation cofactor Williams factor Flaujeac factor VIII-C Prekallikrein (a.k.a Fletcher factor); Kallikrein (the serine protease or activated form of prekallikrein); high molecular weight kininogen (HMWK [Fitzgerald Factor]) Plays a role in CHEMOTAXIS and the sensation of pain Mediate inflammatory responses, increase vascular permeability, cause vasodilation and hypotension, and induce contraction of smooth muscle (Prekallikrein + HMWK) Prekallikrein → Kallikrein Leads to acceleration factor XII activation Kallikrein and activated factor XII (XIIa) form a complex known as a plasminogen activator → which converts plasminogen to its active form plasmin NOMENCLATURE Each Roman numeral was assigned in the order of factor discovery, not its place in the reaction sequence ‘A’ denotes activated, “f” is for fragmented KININS NAMED BY THEIR NAMES, NO NUMBER Numeral Preferred Name I Fibrinogen II Prothrombin Synonyms Prethrombin Copy for: HAW, S.Y. | 2 1. 2. 3. THE COAGULATION FACTOR GROUPS The contact group; The prothrombin or vitamin K-dependent group; The fibrinogen group CONTACT GROUP PROTHROMBIN FIBRINOGEN GROUP Prekallikrein and HMWK, factors XII and XI Activated with contact to negative glass surfaces (in vitro) or collagen, or the subendothelium in vivo Contact factors are also involved in kinin formation and activation of fibrinolysis and the complement system FACTOR XII FACTOR XI PARTIAL THROMBOPLASTIN TIME PTT Evaluating the intrinsic and common coagulation pathways RECALL: Intrinsic system requires a complex of phospholipid, Ca2+, activated factor IX (IXa), and cofactor VIII (VIIIa) in order to activate Factor X in the common pathway. Requires the use of platelet-poor plasma IN DEPTH DISCUSSION OF THE DIFFERENT FACTORS AKA Vitamin K-Dependent Group Factors II, VII, IX and X Dietary Vitamin K deficiency; diseases causing malabsorption of vitamin K; administration of antibiotics that sterilise the intestinal tract; oral anticoagulant therapy Factor VIII (VIII/vWF) has two principal parts: ○ The anticoagulant portion (VIII:C) and the von Willebrand portion (wVF), which is important to normal platelet function Factors I, V, VIII, and XIII Highest molecular weights, most labile Only group that acts as substrates for the fibrinolytic enzyme plasmin Factors I and V are found in platelet a-granules Factor XIII is found in the general platelet cytoplasm Adsorbed in negative glass surface with Prekallikrein and HMWK Role in coagulation: Factor XIIa is an initiator of the intrinsic coagulation pathway Factors XIIA and XIIf can initiate the extrinsic coagulation pathway Factors XIIa and XIIf initiate fibrinolysis Factor XIIf initiates the kinin and complement systems. Factor XIIa, with HMWK, activates Factor XI to the serine protease XIa Activated directly by contact activation Factor XIa also activates plasminogen 2 ALTERNATE PATHWAYS 1. Activation of thrombin 2. Activation of XIa FACTOR IX TISSUE FACTOR The activation of factor IX to the serine protease IXa by factor XIa requires Ca2+ Kallikrein is also capable of directly activating factor IX Present in the plasma membrane of many cell types and has a high affinity for plasma factor VII Exposure of TF → to plasma factor VII → tissue factor: factor VII (TF:VII) complex on the cell surface Factor VII is Vitamin K dependent TF: VIIa-Ca2+ complex on a cell surface converts factor X to Xa in the common pathway Copy for: HAW, S.Y. | 3 FIBRINOLYTIC SYSTEM Fibrinolysis relies on converting plasminogen into plasmin, an enzyme that breaks down fibrin. Plasminogen is made in the liver, stored in eosinophils, and increases during inflammation. Fibrin clots absorb plasminogen, incorporating it into the clot. Plasminogen is then converted to plasmin by activators, leading to clot breakdown. Plasmin also breaks down coagulation factors, but anti-plasmins in plasma control its activity. In liver disease or certain cancers, plasmin levels can build up, causing excessive coagulation factor destruction. In liver disease or certain cancers, plasmin levels can build up, causing excessive coagulation factor destruction. 2 most rapidly acting antiplasmins: ○ a2-antiplasmin and a2 macroglobulin INTRINSIC PLASMINOGEN ACTIVATION Factors Xlla, Xllf, XIa, kallikrein, HMWK, and a specific plasma protein (proactivator) EXTRINSIC PLASMINOGEN ACTIVATION Tissue plasminogen activators (TPA) (found in endothelial cells) EXOGENOUS PLASMINOGEN ACTIVATION Urokinase Streptokinase tissue plasminogen activator (TPA) 4 INHIBITORS OF COAGULATION ANTITHROMBIN III inhibits several clotting factors, including thrombin, Xlla, XIa, Xa, and IXa. Also inhibits plasmin and kallikrein. It plays a crucial role in regulating the coagulation, fibrinolytic, kallikrein-kinin, and C1 INACTIVATOR C1 inactivator is a major inhibitor of the contact system, inhibiting factors XIIa, XIIf, and XIa, accounting for 95% of factor XIIa inhibitory capacity. major inhibitor of the contact system Copy for: HAW, S.Y. | 4 NATURALLY OCCURING INHIBITORS OF FIBRINOLYSIS complement systems. ALPHA-2-MACROGLOBULIN is a large plasma glycoprotein that inhibits various proteolytic enzymes, including thrombin, but its inhibition of thrombin is slower than that of AT-III. It does not completely inhibit its target enzymes binds with the thrombin, slower than AT III also inhibit kallikrein and fibrinolysis PROTEIN C AND S Protein C and its cofactor protein S are glycoproteins dependent on vitamin K. The activated protein C complexed with protein S inhibits coagulation by destroying factors Va and VIIIa. PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) ALPHA-2 ANTIPLASMIN ALPHA-1 MACROGLOBULIN A1 ANTITRYPSIN also known as a1-antiprotease potent inhibitor of factor Xla. weak inhibitor of trypsin (a= substance that activates factor XII) and fibrinolytic system deficiency may be found in liver disease and pulmonary disease; not associated with thrombotic disorders Inhibitor of TPA (Tissue plasminogen activator) and urokinase Found in plasma, platelets (alpha granules) and endothelium Released in response to thrombin formation Form a 1:1 complex with both Tissue plasminogen activator and urokinase, thus inhibiting fibrinolysis Can inhibit plasmin, activated protein C and thrombin Congenital deficiency of PAI-1 → cause a hemorrhagic disorder to unopposed fibrinolysis Inhibited by XIa Principal inhibitor of fibrinolysis Binds 1:1 irreversible complex with plasmin that is free in plasma Crosslinked to fibrin by XIIa = assist in the inhibitory mechanism Inhibits components of fibrinolytic and coagulation systems Rapidly inhibits plasma after Alpha-2 Antiplasmin depletion ALPHA-1 ANTITRYPSIN (ALPHA-1 PROTEASE) Inactivates plasmin slowly Does not bind plasmin until both alpha-2 antiplasmin and macroglobulins are saturated ANTITHROMBIN III Inhibits plasma and kallikrein C1 INACTIVATOR Inhibits plasmin PHYSIOLOGIC COAGULATION CONTROL MECHANISM Antithrombotic, Fibrinolytic, and Coagulant Substances Released from or Found on the Surface of Intact Endothelial Cells SUBSTANCE Prostacyclin ACTION Inhibits platelet adhesion and HEMOSTATIC ROLE Anticoagulant Copy for: HAW, S.Y. | 5 (Prostaglandin I2 (PGI2)) aggregation; stimulates vasodilation Reduces blood flow rate Adenosine (metabolic product of ATP and ADP) Stimulates vasodilation Reduces blood flow rate ADPase Destroys ADP, limits platelet activation Anticoagulant Thrombomodulin Endothelial surface receptor for thrombin Binds and inactivates thrombin and enhances anticoagulant and fibrinolytic action of protein C found in the plasma Anticoagulant Fibrinolytic Heparan sulfate Coats the endothelial cell surface and weakly enhances activity of antithrombin-Ill, a plasma anticoagulant Anticoagulant Tissue plasminogen activator (TPA) Converts plasminogen to plasmin, which plays an important role in fibrinolysis Released only on appropriate stimulus, such as vessel injury, to prevent excessive clot formation at the site of injury and begin slow clot dissolution as the injured vessel heals Fibrinolytic Plasminogen activator inhibitor-1 Regulatory protein for fibrinolytic system Antifibrinolytic von Willebrand factor (vWF) Protein produced in endothelium and stored in subendothelium (in the form of Weibel-Palade bodies) before secretion into the plasma and attachment to factor VIII:C Coagulation and platelet adhesion PLATELET DISORDERS QUANTITATIVE DEFECTS THROMBOCYTOPENIA AND THROMBOCYTOSIS THROMBOCYTOPENIA DECREASE IN CIRCULATING PLATELETS platelet count: