Hematology 2 Lecture Notes (MTAP 2 Review) 2024-2025 PDF

HEMATOLOGY 2 LECTURE NOTES Prepared by: A.M.S. SERAFIN, RMT, C.N.L, BUCAYU, RMT, NOTES FROM: W.J.M. CAPUNO, RMT FIRST SEMESTER ACADEMIC YEAR 2024-2025 MEGAKARYOPOIESIS Production of megakaryocytes  Differentiation/maturatio...

HEMATOLOGY 2 LECTURE NOTES Prepared by: A.M.S. SERAFIN, RMT, C.N.L, BUCAYU, RMT, NOTES FROM: W.J.M. CAPUNO, RMT FIRST SEMESTER ACADEMIC YEAR 2024-2025 MEGAKARYOPOIESIS Production of megakaryocytes  Differentiation/maturation series that will eventually lead to production of platelets (via platelet shedding or thrombocytop oiesis)  THROMBOCYTOPOIESIS Dilation/delineation of the Demarcation System (DMS) forming longitudinal bundles of tubules called as proplatelet process Proplatelet process then pierce through the sinusoid-lining endothelial cells shedding platelets  Platelets arise from MEGAKARYOCYTES in the bone marrow  MEGAKARYOCYTES Largest cell in the bone marrow and are POLYPLOID (possess multiple chromosome copies) 30-50µm (Wright’s-stained bone marrow smear) with multi-lobulated nucleus Clusters with hematopoietic stem cells in vascular niches adjacent to the: venous sinusoid endothelial cells Respond to: TPO (Thrombopoietin) 1 megakaryocyte can produce: 1000-2000 to 4000 platelets  Arise from the COMMON MYELOID PROGENITOR under influence of the transcription factor GATA-1 (Globin Transcription Factor-1) regulated by its cofactor FOG-1 (Friend of Gata)  Suppressor of GATA-1: MYB  IMPORTANT NOTE: GATA-1 and MYB regulate megakaryocytopoiesis and erythropoiesis  RUNX-1: mediates switch from mitosis to endomitosis  Endomitosis: form of mitosis that lacks telophase and cytokinesis  MEGAKARYOCYTE PROGENITOR CELLS 1. BFU-Meg 2. CFU-Meg 3. LD-CFU-Meg  Growth factors/Cytokines involved in megakaryopoiesis: CSF-Meg CSF-GM FLT3-Ligand KIT ligand/Stem Cell Factor/Mast cell factor IL-3 IL-6 IL-11 TPO/Thrombopoiesis Stimulating factor/MPL ligand  IMPORTANT NOTE: CSF-Meg + TPO + IL-3  will cause CFU-GEMM to differentiate to BFU-Meg TPO + IL-11  will cause LD-CFU-Meg to differentiate to a MK-1 (Megakaryoblast) ADDITIONAL NOTES FOR MEGAKARYOCYTES:  1-4 per 100 BM Cells  1% of nucleated cell in the BM  Cells mistaken as megakaryoblasts: Plasma cell, Ferrata cell, Osteoclast, Tumor cell, RS cell  Cells mistaken as Promegakaryocyte, Megakaryocyte: Osteoblast, Multinucleated RBC, Tumor cell, Multinucleated plasma cell, Osteoclast PLATELETS:  Size: 2-4 um  Shape: Resting/Inactive: ROUND/ biconvex Active: Irregular shape with pseudopods (form during activation)  PLATELET SATELLITISM/SATELLITOSIS Phenomenon in which platelets form a ROSETTE around the periphery of neutrophils Can be due to: Excess EDTA (short draw or insufficient sample) Associated with autoantibodies against: gpIIb/ IIIa and Neutrophil Fc gamma region  Cytoplasm: Light blue to purple; very granular  Life Span: 7 to 10 days  Normal Value: 150-400 x 109/L Men: 122-350 x 109/L Women: 140-379 x 109/L  IMPORTANT NOTE: Platelet count represents only 2/3 of the available platelets in the platelet pool 1/3 of the platelets are sequestered in the spleen (serves as reservoir during injury)  Reticulated Platelets/Stress Platelets Appears in compensation to thrombocytopenia Larger than normal platelets The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES PLATELET ULTASTRUCTURE: 1. PERIPHERAL ZONE 2. SOL-GEL ZONE 3. MEMBRANE SYSTEMS 4. ORGANELLE ZONE A. PERIPHERAL ZONE (RESTING PLASMA MEMBRANE) Physical and chemical barrier between intra and extracellular environment (composed of cholesterol and phospholipids) Cholesterol: stabilizes the membrane, maintains fluidity, and helps control the transmembranous passage of materials. Phospholipids: o Plasma layer: Phosphatidylcholine, sphingomyelin o Inner cytoplasmic layer: phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine o Phosphatidylinositol – support platelet activation by supplying arachidonic acid (synthesis of prostaglandins) o Phosphatidylserine – flips to the outer surface upon activation and is the charged phospholipid surface on which the coagulation enzymes assemble: Factors Xa, Va, IXa, and VIIIa Glycocalyx o 20 to 30 nm thick (thicker than RBC and WBC) o Contains glycoproteins and proteoglycans required for platelet adhesion and aggregation o Surface for adherence of coagulation factors Submembrane area o Prevent contact between organelles and plasma membrane o Submembrane filaments  Contribute to the normal discoid shape of platelets  Interact with contractile proteins to modulate platelet adhesion and clot retraction after activation B. SOL GEL ZONE Stable gel component to regulate the arrangement of the internal organelles Microtubules: contributes to the discoid shape of an inactivated/resting platelet Microfilaments: o Contractile process (changing of shape), 50 nm o Thrombostenin (actomyosin) - major contractile protein; composed of actin and myosin; 15% of total proteins in platelets C. MEMBRANE SYSTEMS Surface Connected Canalicular System (SCCS) o Responsible for the exchange of substances in and out of the platelet o Enables the platelet to store additional quantities of the same hemostatic proteins found on the glycocalyx and raising its capacity manifold Dense Tubular System (DTS) o Found in the platelet cytoplasm; a condensed remnant of the RER o Sequesters Calcium o Synthesis of prostaglandins o “Control center” for platelet activation D. ORGANELLE ZONE Metabolic region; granular region Alpha granules (50-80 per platelet), dense granules (2-7 per platelet), mitochondria (10-60 per platelet), peroxisomes ALPHA GRANULES (300-500nm) DENSE GRANULES (250-350nm) Beta-thromboglobulin Factor VIII:vWF ATP PF 4 Thrombospondin ADP Fibrinogen Protein C Calcium Fibronectin Inhibitor Magnesium Factor V Alpha-2 Serotonin PDGF Antiplasmin Pyrophosphate TGF-B C1 Esterase HMWK Inhibitor Plasminogen PAI-1 pg. 2 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES HEMOSTASIS (PRIMARY AND SECONDARY)  Is a complex physiologic process that keeps circulating blood in a fluid state and then, when an injury occurs, produces a clot to stop the bleeding, confines the clot to the site of injury, and finally dissolves the clot as the wound heals  Is the process by which the clotting system seals a vascular injury to limit blood loss; it must be quick, localized, and well regulated.  The maintenance of circulatory hemostasis is achieved through the process of balancing bleeding (hemorrhage) and clotting (thrombosis). OVERVIEW OF HEMOSTASIS 1. Vasoconstriction of blood vessel [PRIMARY HEMOSTASIS] 2. Platelet activation 3. Activation of the coagulation mechanism 4. Fibrinolysis (Tertiary Hemostasis) [SECONDARY HEMOSTASIS] 5. Subsequent tissue repair HEMOSTASIS (TISSUE DAMAGE/ INJURY) I. Vasoconstriction and involvement of: VASCULAR ENDOTHELIUM II. Platelet activation: formation of: PLATELET PLUG/ white clot (composed of platelets and vWF) III. Coagulation mechanism (red clot): Formation of stabilized platelet-fibrin plug IV. FIBRINOLYSIS – Dissolution of Clots (lysis of fibrin) PRIMARY HEMOSTASIS Refers to the role of blood vessels and platelets in response to a vascular injury, or to the commonplace desquamation of dying or damaged endothelial cells Blood vessels contract to seal the wound or reduce the blood flow (vasoconstriction). Platelets become activated, adhere to the site of injury, secrete the contents of their granules, and aggregate with other platelets to form a platelet plug. SECONDARY HEMOSTASIS Describes the activation of a series of coagulation proteins in the plasma, mostly serine proteases, to form a fibrin clot (stabilized platelet-fibrin plug) These proteins circulate as inactive zymogens (proenzymes) that become activated during the process of coagulation and, in turn, form complexes that activate other zymogens to ultimately generate thrombin, an enzyme that converts fibrinogen to a localized fibrin clot Final event of hemostasis is fibrinolysis, the gradual digestion and removal of the fibrin clot as healing occurs. PRIMARY HEMOSTASIS  Includes the blood vessels (vascular intima) and platelets (platelet activation)  Primary Goal: Formation of platelet-plug (white clot)  BLOOD VESSELS  Capillaries  Veins  Arteries VASCULAR INTIMA  Provides the interface between circulating blood and the body tissues. The innermost lining of blood vessels is a monolayer o f metabolically active Endothelial cells IMPORTANT NOTE:  An intact vascular intima is anti-thrombotic; does not promote coagulation and activation of platelets  ENDOTHELIAL CELLS ECs form a physical barrier separating pro-coagulant proteins and platelets in blood from collagen in the internal elastic lamina that promotes platelet adhesion, and tissue factor in fibroblasts and smooth muscle cells that activates coagulation SUBSTANCES RELEASED BY ENDOTHELIAL CELLS A. ANTICOAGULANT B. PROCOAGULANT C. FIBRINOLYTIC pg. 3 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES I. ANTICOAGULANT Prostacyclin (PGI2): inhibits platelet aggregation; vasodilator Nitric oxide: induces smooth muscle relaxation; prevents platelet activation TFPI (Tissue factor pathway controls activation of the extrinsic pathway inihibitor): CD 39 (Ectonucleotidase): degrades ADP Thrombomodulin: inhibits thrombin formation; activates protein C Heparan sulfate: co-factor of anti-thrombin/anti-thrombin III 13-HODE: inhibits platelet adhesion II. PROCOAGULANT von Willebrand factor (VWF): important in platelet adhesion P-selectin (CD-62): adhesion molecule, promotes platelet and leukocyte binding ICAM’s (intercellular adhesion molecules): promotes platelet and leukocyte binding PECAM’s (platelet endothelial cell adhesion molecules): promotes platelet and leukocyte binding III. FIBRINOLYTIC TPA (Tissue plasminogen activator): activates fibrinolysis by converting plasminogen to plasmin PAI-1 (Plasminogen activator inhibitor): inhibits plasmin generation and fibrinolysis TAFI (Thrombin-activatable fibrinolysis inhibitor): activated by thrombin that binds to thrombodulin. SUMMARY OF VASULAR RESPONSE (DAMAGED VASCULAR INTIMA) 1. Vasoconstriction: Serotonin and Thromboxane A 2 2. Exposure of collagen Activates primary hemostasis: platelet adhesion Activate the intrinsic coagulation pathway/contact phase of coagulation: Activates factor XII factor XIIa 3. Release of tissue thromboplastin/tissue factor: Activates extrinsic pathway 4. Released of vWf: initiates platelet adhesion 5. Released of subendothelial cells: activates platelets 6. Initiation of fibrinolysis Release of tPA (to ensure no excessive coagulation will occur) PLATELET RESPONSE  At the time of an injury, platelets adhere, aggregate, and secrete the contents of their granules  STEPS IN THE PLATELET ACTIVATION PROCESS: 1. Platelet Adhesion 2. Platelet Aggregation 3. Platelet Shape Change 4. Platelet Release Reaction 5. Platelet-Plug Consolidation and Stabilization I. PLATELET ADHESION  Process wherein platelets adhere to a foreign surface.  Important in this process are:  gp Ib/IX/V: Primary platelet surface receptor for vWF  Gp Ia/IIa and Gp VI: Platelet surface receptor for collagen  von Willebrand Factor  Platelets adhere to collagen via:  von Willebrand factor (vWF)  Substance present in platelet granules and plasma; links platelets to collagen in high shear stress such as arteries and arterioles  VWF circulates as a globular protein, it become fibrillar as it unrolls and exposes sites that partially bind to the GP Ib/ IX/V receptor  ADAMTS-13/vWF-cleaving protease: Digests “unused’ vWF  gp Ib/IX/V: Primary platelet surface receptor for vWF  DISORDERS OF PLATELET ADHESION  Deficiency in VWF: von Willebrand’s disease  Deficiency in gp Ib/IX/V receptor: Bernard-Soulier syndrome  13-HODE (13-hydroxyoctadecadienoic acid): derived from endothelial cells; inhibits platelet adhesion  12-HETE (12-hydroxyeicosatetraenoic acid): derived from cytosol of platelets; promotes platelet adhesion II. PLATELET AGGREGATION  Aggregation is the property by which platelets bind to one another  Is a key part of primary hemostasis, which in arteries may end with the formation of a: “white clot,” (platelet-plug)  MECHANISMS OF PLATELET AGGREGATION: 1. RELEASE OF ADP pg. 4 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES  Released from dense granules in response to collagen, epinephrine, thrombin, and TxA2  Mediates ADP-induced aggregation: gp IIb/IIIa + Ca2+ + Fibrinogen (calcium bridges)  Deficiency in Gp IIb/IIIa receptor: GLANZMANN’S THROMBASTHENIA 2. THROMBIN 1) Stimulate platelets to release ADP (ADP-induced aggregation) 2) Formation of COAT platelet (collagen and thrombin activated)  integral to the cell-based coagulation model 3. THROMBOXANE A2 (TxA2) 3) Promotes platelet aggregation directly and acts synergistically with ADP 4) Also promotes vasoconstriction and activation of platelets 5) Arachidonic acid will be oxidized to endoperoxides (PGG 2 and PGH2)  Enzyme: CYCLOOXYGENASE 6) Endoperoxides  Thromboxane A2  Enzyme: THROMBOXANE SYNTHETASE III. PLATELET SHAPE CHANGE  Contraction of microfilaments  Platelet pseudopod formation  Requires Calcium and Thrombostenin/Actomyosin IV. PLATELET RELEASE REACTION  Platelets secrete the contents of their granules during adhesion and aggregation, with most secretion occurring late in the platelet activation process  Contents of a-granules and lysosomes flow through the SCCS, while dense granules migrate to the plasma membrane, where their contents are secreted  Stimulus of platelet release (also stimulate in-vitro platelet aggregation): Collagen, thrombin, epinephrine, thromboxane A2, arachidonic acid, P-selectin (CD 62)  Substances released: Factor V, VWF, Factor VIII, Fibrinogen, Ca2+, and ADP V. PLATELET-PLUG CONSOLIDATION AND STABILIZATION  Coagulation is initiated on tissue factor–bearing cells (such as fibroblasts) with the formation of the extrinsic tenase complex TF:VIIa:calcium, which activates factors IX and X and produces enough thrombin to activate platelets and factors V, VIII, and XI in a feedback loop  Coagulation is then propagated on the surface of the platelet with the formation of the intrinsic tenase complex (IXa:VIIIa:phospholipid:calcium) and t he prothrombinase complex (Xa:Va:phospholipid:Calcium), ultimately generating a burst of thrombin at the site of injury  Thrombin will then activate fibrinogen to fibrin (stabilized platelet-fibrin plug) SECONDARY HEMOTASIS Procoagulants; also known as coagulation factors  Glycoproteins in which majority are produced in the liver  Other sites of production:  Monocytes  Endothelial cells (especially the vWF)  Megakaryocytes (Factor V, Fibrinogen, vWF, and HMWK) NOMENCLATURE OF COTTING FACTORS In 1958 the International Committee for the Standardization of the Nomenclature of the Blood Clotting Factors officially name d the plasma procoagulants (clotting factor) using Roman numerals in the order of their initial description or discovery When a procoagulant becomes activated, a lowercase a appears behind the numeral; for instance, activated factor VII is VIIa  Factor VII: inactive  Factor VIIa: activated Factors I, II, III, IV are seldom called with their corresponding roman numerals  Factor I: Fibrinogen  Factor II: Prothrombin  Factor III: Tissue thromboplastin/Tissue factor  Factor IV: Calcium Factor VI: was later known as activated Factor V (Va); removed in the naming system Prekallikrein and HMWK: not given roman numeral designation because they belong to kallikrein and kinin systems  HMWK – high-molecular-weight kininogen  When activated they become bradykinins (part of kinin system)  Prekallikrein and HMWK does not have a roman number because they are part of respective systems.  Prekallikrein: kallikrein system  HMWK: kinin system pg. 5 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES von Willebrand factor:  Important in platelet adhesion (binds to collagen and platelets through GP Ib/IX/V receptor)  Other function: carrier of Factor VIII (for transport)  Factor VIII cannot travel to plasma w/o carrier Platelet phospholipids (Phosphatidylserine) or Platelet factor 3 (PF3)  No roman numeral designation  Platelet plays a role in coagulation complexes. Phosphatidylserine (PS) will flip to the other surface of cell membrane allowing clotting factors to assemble eventually forming complexes.  Older references: it is called Platelet factor 3 (PF3)  Platelet factor 3 assay: assess phospholipids present in platelets Remember: This is the part of platelet function that is included in secondary hemostasis. Note: There are 16 clotting factors.  Factor I to XIII (except 6, because this is later known as Factor Va)  vWF, prekallikrein, HMWK, platelet factor 3 (PF3) Factor V (Labile factor): fast to deteriorate especially if prolonged storage Factor VIII: in older references, Factor VIII contains c Factor IX: Trivia – first person to be diagnosed of Christmas disease is Stephen Christmas OTHER NAMES Factor II  Prethrombin Factor III  Tissue thromboplastin Factor V  Proaccelerin  Labile factor accelerator globulin (aCg) Factor VII  Proconvertin  Serum prothrombin conversion accelerator (SPCA), Autoprothrombin I Factor VIII (Also known as  Anti-hemophilic globulin (AHG) Factor VIII:C)  Anti-hemophilic factor A  Platelet cofactor 1 Factor IX  Plasma Thromboplastin component (PTC)  Anti-hemophilic factor B  Platelet cofactor 2 Factor X  Stuart factor  Prower factor  Auto-prothrombin II Factor XI  Anti-hemophilic factor C Factor XII  Glass factor  Contact factor High Molecular weight  Williams factor kininogen  Flaujeac factor  Contact activation cofactor Factor XIII  Laki-lorand factor  Fibrinase  Fibrinoligase Note:  Factor VIII, IX, XI: Anti-hemophilic factors pg. 6 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES  Deficiency of these factors will lead to hemophilia (ABC)  Factor VIII Deficiency: Hemophilia A (Classic Hemophilia, Royal’s disease)  Factor IX Deficiency: Hemophilia B (Christmas disease)  Factor XI Deficiency: Hemophilia C (Rosenthal disease)  Factor IX-XI  Factor IX: Plasma Thromboplastin component (PTC)  Factor XI: Plasma Thromboplastin Antecedent (PTA) TYPES OF CLOTTING FACTORS ACCORDING TO FUNCTION ENZYMES  ZYMOGENS: inactive enzymes  SERINE PROTEASES:  active enzymes; proteolytic enzymes of the trypsin family  are synthesized as zymogens but activation occurs by the action of another protease during the coagulation process Important Note: Factor XIII (transglutaminase): cross-linking of fibrin polymer  Factor XIII is neither zymogen nor serine proteases. It is a transglutaminase. CO-FACTORS  Bind, stabilize, and enhance the activity of their respective enzymes; participate in complex formation SUBSTRATE  Factors activated by serine proteases and their cofactors FIBRINOGEN  Ultimate substrate of coagulation pathway; when activated by thrombin forms the primary structural protein of the FIBRIN CLOT and stabilized by Factor XIII (stabilized fibrin clot/stabilized platelet-fibrin plug) Remember: The goal of coagulation mechanism is to produce a stabilized platelet-fibrin clot. CALCIUM  required for the assembly of coagulation complexes on platelet or cell membrane phospholipids  Serine proteases bind to negatively charged phospholipid surfaces, predominantly phosphatidylserine, through positively charged calcium ions TYPES OF CLOTTING FACTORS ACCORDING TO PHYSICAL PROPERTIES A. VITAMIN K-DEPENDENT GROUP/ PROTHROMBIN GROUP Vitamin K-dependent Group/Prothrombin group named the prothrombin group because of their structural resemblance to prothrombin (All seven proteins have 10 to 12 glutamic acid units near their amino termini) Absent in adsorbed plasma: Barium sulfate and Aluminum hydroxide Procoagulants: Factors IX, X, VII, II Regulatory proteins: Protein S, C, Z Vitamin K: is a quinone found in green leafy vegetables and is produced by the intestinal organisms Bacteroides fragilis and Escherichia coli Vitamin K catalyzes an essential posttranslational modification of the prothrombin group proteins: g-carboxylation of amino-terminal glutamic acid (allows clotting factors to bind to calcium and platelet phospholipids during complex formation) PIVKA (des-g-carboxyl proteins or proteins in vitamin K antagonism)  Formed during Vitamin K deficiency and presence of warfarin (Vitamin K antagonist)  Warfarin: oral anticoagulant; if taken, vitamin K deficiency occurs.  If deficient in vit. K, there will be no g-carboxylation  Procoagulants lack the second carboxyl group thus they cannot bind to calcium and platelet phospholipids thus preventing them from participating in the coagulation reaction  Examples of warfarin: coumarin/Coumadin B. CONTACT GROUP/ CONTACT PHASE GROUP Also called as intrinsic accessory pathway proteins or contact factors pg. 7 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES Procoagulants: Factor XII, HMWK, Prekallikrein, Factor XI Named because they are activated by contact with negatively charged surfaces (Collagen, glass, APTT reagent → kaoline, cellite, elagic acid) o Exposure to collagen will activate intrinsic pathway of coagulation o Once exposed to negatively charged surface, Factor XII will be activated (Factor XIIa) Factor XIIa transforms pre-Kallikrein, a glycoprotein that circulates bound to HMWK, into its active form kallikrein, which cleaves HMWK to form bradykinin The contact factor complex HMWK:pre-K:FXIIa activates factor XI Factor XIa is an activator of factor IX C. FIBRINOGEN GROUP Procoagulants: Factors I, V, VIII, XIII Highest molecular weight because of factor VIII Factor VIII: 260,000 daltons Most labile → deteriorate because of Factors V and VIII: heat labile clotting factors In blood banking, prolonged storage of Factors V and VIII will make them easily degraded. It should be transfused immediately because the levels deteriorate instantly (especially Factor VIII) Factors V and VIII are inactivated by protein C (protein C pathway) Thrombomodulin will activate Factor C. In time, Factor C will inactivate Factors V and VIII. If there is no Factors V and VIII, thrombin will not be activated. Consumed in coagulation (Fibrinogen) If blood is clotted, there is no fibrinogen but fibrin. Fibrinogen is converted to fibrin Factor VIII binds to vWF in the plasma (VIII:vWF): activated by thrombin during coagulation vWF: Factor VIII carrier and important in platelet adhesion Factor VIII can’t travel to site of injury without vWF. Factor VIII will be activated (Factor VIIIa) by thrombin.  Thrombin will dissociate VIII:vWF complex to form Factor VIIIa. Summary of the Types of Clotting Factors according to Physical Properties (Procoagulants and Regulatory Proteins) Procoagulants Regulatory proteins Vitamin L-Dependent Factors IX, X, VII, II Protein S, C, Z Group/Prothrombin Group Contact Group/Contact Phase Group Factors XII, HMWK, Prekallikrein, Factor XI Fibrinogen Group Factors I, V, VIII, XIII COAGULATION MECHANISM (SECONDARY HEMOSTASIS) PLASMA BASED (IN-VITRO COAGULATION) Intrinsic, Extrinsic and Common Pathway Used extensively to interpret in vitro laboratory testing and to identify factor deficiencies: however, they do not adequately describe the complex interdependent reactions that occur in vivo. EXTRINSIC PATHWAY Formation of TF:VIIa has since proven to be the primary in vivo initiation mechanism for coagulation. Because tissue factor is not present in blood, the tissue factor pathway has been called the extrinsic pathway Factors involved: Tissue factor, Factor VII, Calcium, Factor X, Factor V, Prothrombin, Fibrinogen Extrinsic tenase complex: TF: VIIa + PL + Calcium Test to assess deficiency: PT (Prothrombin time) INTRINSIC PATHWAY Consequently, the reaction system that begins with factor XII and culminates in fibrin polymerization has been called the intrinsic pathway. Factors involved: Factor XII, Pre-K, HMWK, Factor XI, Factor IX, Factor VIII, Factor X, Factor V, Prothrombin, Fibrinogen Intrinsic tenase complex: IXa: VIIIa + PL + Calcium Test to Assess Deficiency: APTT (Activated Partial Thromboplastin Time) COMMON PATHWAY Since the two pathways have the common factor X, factor V, prothrombin and fibrinogen; this portion of the coagulation pathway is often called the common pathway Prothrombinase complex: Xa: Va + PL + Calcium Test to assess deficiency: PT and PTT pg. 8 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES CELL BASED (IN VIVO, PHYSIOLOGIC COAGULATION) In addition to procoagulant and anticoagulant plasma proteins, normal physiologic coagulation requires the presence of two cell types for formation of coagulation complexes:  Cells that express tissue factor (usually extravascular)  Platelets (intravascular) In-vivo coagulation can be described as occurring in two phases:  Initiation: occurs on tissue factor–expressing cells and produces 3% to 5% of the total thrombin generated  Propagation: occurring on platelets, which produces 95% or more of the total thrombin INITIATION In vivo, the principle mechanism for generating thrombin is begun by formation of the extrinsic tenase complex, rather than the intrinsic pathway  Extrinsic tenase complex then intrinsic pathway  Initiation is like the formation of Extrinsic tenase compex  Refers to extrinsic tenase complex formation and generation of small amounts of factor Xa, factor IXa, and thrombin  About 1% to 2% of factor VIIa is present normally in blood in the activated form, but it is inert until bound to tissue factor PROPAGATION In this phase the reactions occur on the surface of the activated platelet, which now has all the components needed for coagulation STEPS INVOLVED:  Platelets are activated at the site of injury by both the low-level thrombin generated in the initiation phase and by adhering to exposed collagen  These partially activated COAT-platelets (Collagen and Thrombin platelets) have a higher level of procoagulant activity than platelets exposed to collagen alone and provide a surface for the formation and amplification of the intrinsic and prothrombinase complexes  The cofactors Va and VIIIa activated by thrombin in the initiation phase bind to platelet membranes and become receptors for Xa and IXa  IXa generated in the initiation phase binds to VIIIa on the platelet membrane to form the intrinsic tenase complex IXa:VIIIa  Intrinsic tenase complex (IXa:VIIIa)  activates factor X at a 50- to 100-fold higher rate than the extrinsic tenase complex  Factor Xa binds to Va to form the prothrombinase complex  Prothrombinase tenase (Xa:Va)  activates prothrombin and generates a burst of thrombin (Factor IIa)  Thrombin cleaves fibrinogen into a fibrin clot, activates factor XIII to stabilize the clot, binds to thrombomodulin to activate the protein C control pathway, and activates TAFI (Thrombin activatable Fibrinolysis inhibitor) to inhibit fibrinolysis. pg. 9 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES THROMBIN: The primary function of thrombin is to cleave fibrinopeptides A and B from the a and b chains of the fibrinogen molecule, triggering spontaneous fibrin polymerization Other functions:  Activates Factors V, VIII, XI (by a positive feedback mechanism)  Factors Va: Cofactor of Factor Xa (prothrombinase complex)  Factor VIIIa: Cofactor of Factor IXa (Intrinsic Tenase)  Factor XI: Cofactor of Factor IXa  Activates Factor XIII (forms covalent bonds between the D domains of the fibrin polymer to cross-link and stabilize the fibrin clot)  Together with calcium  Initiates platelet aggregation (via platelet release)  Thrombin bound to thrombomodulin activates the protein C pathway to suppress coagulation (coagulation control)  Protein C pathway will inhibit Factors V, VIII so that thrombin will not produce.  activates TAFI to suppress fibrinolysis (fibrinolysis control) Primary action of thrombin is to activate fibrinogen and, usually the activation of fibrinogen starts by cleaving the fibrinopeptides A and B. Once the fibrinopeptides A and B is removed, the fibrinogen is activated to form monomers, polymers, and stabilized by XIIIa and calcium. FIBRINOGEN (FACTOR I) primary substrate of thrombin, which converts soluble fibrinogen to insoluble fibrin to produce a clot is also essential for platelet aggregation because it links activated platelets through their GP IIb/IIIa platelet fibrinogen receptor Normal value: 200-400 mg/dL  25 mL - 19-gauge needle is required A 23-gauge needle is acceptable for pediatric patients or patients whose veins are small, but the negative collection pressure must be reduced pg. 15 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES ANTICOAGULANTS USED FOR HEMOSTASIS TESTING 3.2% BUFFERED SODIUM CITRATE (0.105-0.109 M) Anticoagulant used for hemostasis testing Binds calcium ions to prevent coagulation, and the buffer stabilizes specimen pH as long as the tube stopper remains in place Blood to anticoagulant ratio: 9:1 (0.3 mL of anticoagulant is mixed with 2.7 mL of whole blood) OTHER ANTICOAGULANTS USED FOR HEMOSTASIS TESTING A. EDTA (Ethylenediaminetetraacetic Acid) are not used for coagulation testing because calcium ion chelation by EDTA is irreversible, interfering with coagulation assays Inhibits: thrombin-fibrinogen reaction anticoagulant used in collecting specimens for complete blood counts, including platelet counts may be required for specimens used for molecular diagnostic testing, such as testing for factor V Leiden mutation or the prothrombin G20210A mutation B. Heparin Heparinized specimens have never been validated for use in plasma coagulation testing but may be necessary in cases of platelet satellitosis (satellitism) as a substitute for specimens collected in EDTA or sodium citrate Also used for platelet retention test/glass bead retention test (test for platelet adhesion) C. CITRATE THEOPHYLLINE ADENOSINE DIPYRIDAMOLE (CTAD, blue stopper) are used to halt in vitro platelet or coagulation activation for specialty assays such as those for the platelet activation markers platelet factor 4 (PF4) and platelet surface membrane P-selectin (measured by flow cytometry) or the coagulation activation markers prothrombin fragment 1+2 and thrombin-antithrombin complex D. Oxalates: Shortened clotting time because it forms insoluble complexes/precipitates HEMOSTASIS SPECIMEN STORAGE TEMPERATURE Sodium citrate-anticoagulated whole blood specimens are placed in a rack and allowed to stand in a vertical position with the stopper intact and uppermost pH remains constant as long as the specimen is sealed: increased pH deteriorates clotting factors (prolonged coagulation tests) Specimens are maintained at 18° C to 24° C (ambient temperature), never at refrigerator temperatures IMPORTANT NOTE: Storage at 1° C to 6° C activates factor VII and XI, destroys platelet activity through uncontrolled activation, and causes the cryoprecipitation of large VWF multimers Never be stored at temperatures greater than 24° C because heat causes deterioration of factors V and VIII Specimens collected for PT testing may be held at 18° C to 24° C and tested within 24 hours of the time of collection Specimens collected for APTT testing also may be held at 18° C to 24° C, but they must be tested within 4 hours of the time of collection, provided that the specimen does not contain unfractionated heparin anticoagulant If a patient is getting unfractionated heparin therapy (UFH), specimens for APTT and PT testing must be centrifuged within 1 hour of the time of collection, and the plasma, which should be PPP, must be tested within 4 hours of the time of collection. PREPARATION OF HEMOSTASIS SPECIMENS FOR ASSAY WHOLE-BLOOD SPECIMENS USED FOR PLATELET AGGREGOMETRY Blood for whole-blood platelet aggregometry or lumiaggregometry must be collected with 3.2% sodium citrate and held at 18° C to 24° C until testing Chilling destroys platelet activity; aggregometry should be started immediately and must be completed within 4 hours of specimen collection. PLATELET RICH PLASMA SPECIMENS USED FOR PLATELET AGGREGOMETRY Platelet count: 200,000 to 300,000/uL Sodium citrate–anticoagulated blood is first checked visually for clots and then centrifuged at 50 x g for 30 minutes with the stopper in place to maintain the pH. The supernatant PRP is transferred by a plastic pipette to a clean plastic tube, and the tube is sealed and stored at 18° C to 24° C (ambient temperature) until the test is begun PLATELET POOR PLASMA (PPP) USED FOR COAGULATION STUDIES Clot-based plasma coagulation tests require PPP-plasma Platelet count: less than 10,000/uL Sodium citrate-anticoagulated whole blood is centrifuged at 1500 xg for 15 minutes in a swinging bucket centrifuge to produce supernatant PPP Alternatively, the angle-head StatSpin Express 2 generates 4400 xg and can produce PPP within 3 minutes. pg. 16 The information in this material is solely intended for educational purposes only and does not guarantee accuracy, completeness, or timeliness and without any warranties implicated. These handouts are not intended for sale. HEMATOLOGY 2 LECTURE NOTES LABORATORY EVALUATION OF PRIMARY HEMOSTASIS COMMON TESTS FOR PRIMARY HEMOSTASIS A.) PLATELET COUNT  Reasons why platelets hard to count: Platelets adhere to foreign surfaces Platelets easily disintegrate. They are hard to differentiate from debris. Platelets are unevenly distributed in the blood because they tend to clump.  Normal Value: 150,000 – 400,000/uL  Platelet count of