Hemostasis and Blood Coagulation PDF

UNIT VI Chapter 37: Hemostasis and Blood Coagulation Ebaa M Alzayadneh, PhD Associate Professor of Physiology  Vascular Constriction Local myogenic spasm-most effective Local...

UNIT VI Chapter 37: Hemostasis and Blood Coagulation Ebaa M Alzayadneh, PhD Associate Professor of Physiology  Vascular Constriction Local myogenic spasm-most effective Local autacoid factors Nervous reflexes-from pain Events in receptors Hemostasis Formation of a Platelet Plug Small cut in a vessel; have a plug instead of the complete clotting mechanism Platelet Plug Formation Von Willebrand factor (vWF) serves as an adhesion bridge between subendothelial collagen and the glycoprotein Ib (GpIb) platelet receptor. University of Jordan 3  Blood Clotting 3. Blood clotting Serum is blood plasma minus clotting proteins Clotting – series of chemical reactions culminating in formation of fibrin threads Clotting (coagulation) factors – Ca2+, several inactive enzymes, various molecules associated with platelets or released by damaged tissues University of Jordan 4 Blood Clotting Exposure of blood to vasc wall- release of TF (III or thromboplastin) from endo cells , phospholipids, thrombin activation- ----------Fibrin Events in Hemostasis Platelets Fragmented megakaryocytes; after released via capillaries 150,000-300,000 Do not have nuclei and cannot reproduce Contain actin and myosin (thrombosthenin) Mito-Produce ATP Release prostaglandins Release endothelial cell growth factor Surface glycoproteins for adherence to damaged Vessels Half-live of 8-12 days  Events in Hemostasis Mechanism of the Platelet Plug a. Platelets swell; irregular shape with pseudopods b. Become sticky and adhere to collagen c. Thromboxane A2 and ADP enhance adherence d. Damaged wall activates increasing numbers of platelets e. Important in closing small tears or ruptures in very small vessels (petechiae)  Blood Coagulation Basic Theory a. Depends on the state of balance of 50 or more possible blood procoagulants and anticoagulants b. Procoaglants overrides, formation of prothrombin activator c. Conversion of prothrombin to thrombin d. Conversion of fibrinogen to fibrin procoagulants anticoagulants Clotting Factors  3 Stages of Clotting 1. Extrinsic or intrinsic pathways lead to formation of prothrombinase 2. Prothrombinase converts prothrombin into thrombin 3. Thrombin converts fibrinogen (soluble) into fibrin (insoluble) forming the threads of the clot Thus, the rate-limiting factor in causing blood coagulation is usually the formation of prothrombin activator  Hemostasis: Prevention of Blood Loss Vascular constriction Formation of a platelet plug Formation of a blood clot Healing of vascular damage ± re- canalization  Vascular Constriction Myogenic spasm Local autocoid factors from damaged tissues and platelets Nervous reflexes Smaller vessels: thromboxane A2 released by platelets  Platelets (Thrombocytes) 1- 4 µm discs Released by fragmentation of megakaryocytes 150-300,000 per µL Half-life in blood of 8-12 days Platelet Functions Contractile capabilities actin, myosin, thrombosthenin Residual ER and Golgi synthesize enzymes, prostaglandins, fibrin-stabilizing factor, PDGF, store Ca++ Mitochondria / enzymes produce ATP, ADP Platelet Plug Formation Von Willebrand factor (vWF) serves as an adhesion bridge between subendothelial collagen and the glycoprotein Ib (GpIb) platelet receptor. University of Jordan 15  Platelet Membranes Surface glycoprotein Repels intact endothelium Adheres to injured endothelium and exposed collagen Membrane phospholipids Activate blood clotting  Formation of the Platelet Plug Contact with damaged endothelium Assume irregular forms Contract and release granules (ADP, thromboxane A2) Adhere to collagen and vWF Other platelets accumulate, adhere, and contract, form plug, initiate clotting Very low platelets → petechaiae, bleeding gums  Platelet Plug Figs. 7.15a and 7.14b, Stevens & Lowe Human Histology, 4th edition  Begins in 15- 20 seconds in severe vascular trauma Occlusive clot within 3-6 minutes Clot unless very large vascular defect Formation and Progression 20-60 minutes: Clot retraction 1- 2 weeks : Invasion by fibroblasts Organization into fibrous tissue Key Steps in Blood Clotting  Effector Proteins for Clotting Prothrombin α 2 globulin, MW 68,700; 15 mg/dl in plasma Vitamin K-dependent synthesis in liver Cleaved by PT activator to thrombin, MW 33,700 Fibrinogen MW 340,000; 100-700 mg/dl in plasma Synthesized in the liver (acute phase reactant) Usually intravascular; can extravasate with increased vascular permeability  Fibrin Production Thrombin (weak protease) cleaves four small peptides from fibrinogen → fibrin monomer → spontaneous polymerization Long fibers form clot reticulum Fibrin stabilizing factor In plasma and released from platelets Activated by thrombin Covalent bonds, and cross-linking of fibrin monomers and adjacent fibrin fibers  Clot Extension Thrombin is bound to platelets and trapped in the clot Can act on prothrombin to generate more thrombin (positive feedback) Thrombin also produces more prothrombin activator by acting on other clotting factors Additional fibrin monomers and polymers are generated at the periphery of the clot  Clot Retraction Begins within 20-60 minutes Fibrin binds to damaged vessel wall Platelets bind to multiple fibrin fibers contract via actin, myosin, thrombosthenin, and FSF (FactorVIII), Ca++ from organells Clot tightens, expressing serum, and closing the vascular defect  Generating Prothrombin Activator Two pathways Extrinsic pathway – Trauma to vessel wall and adjacent tissues Intrinsic pathway – Trauma to the blood or exposure of the blood to collagen Both pathways involve “clotting factors”— mostly inactive proteases that are activated in cascades Extrinsic Pathway of Blood Clotting  Blood Coagulation Extrinsic Pathway a. Release of tissue factor b. Activation of Factor X- role of Factor VII and tissue factor c. Effect of Xa to form prothrombin activator -role of Factor V in the presence of calcium and phospholipids to split prothrombin to thrombin  Blood Coagulation Intrinsic Pathway a. Blood trauma causes activation Factor XII and release of platelet phospholipids b. Activation of Factor XI c. Activation of Factor IX by activated XI d. Activation of Factor X-role of Factor VIII e. Action of activated Factor X to form prothrombin activator-role of Factor V  Intrinsic Pathway of Blood Clotting VIII or platelets if def, 4 is def  Synergy between the Intrinsic and Extrinsic Pathways Tissue injury… Tissue factor activates the Extrinsic Pathway Exposure of Factor XII and platelets to collagen activates the Intrinsic Pathway Extrinsic pathway can be explosive, with clotting in < 15 seconds The Intrinsic pathway is slower → 1 – 6 minutes  Prevention of Clotting Smoothness of the endothelial surface Mucopolysaccharide coating (glycocalyx) repels platelets and clotting factors Thrombomodulin bound to endothelium binds (competes for) thrombin Thrombin-thrombomodulin activates Protein C→ inactivates factors V and VIII Damage to glycocalyx activates factor XII, platelets (intrinsic pathway). If collagen is exposed→ even more robust  Negative Feedback Fibrin fibers bind 85-90% of thrombin and localize it to the clot Antithrombin III combines with the remainder and inactivates it over 12-20 minutes  Heparin Physiologically, availability is limited Used therapeutically Highly negatively charged Binds anti-thrombin III and increases its effectiveness 100- to 1000-fold Heparin-antithrombin III removes free thrombin from the blood almost instantly Also removes XIIa, XIa, Xa, and IXa Mast cells, basophils particularly abundant in pericapillary regions of liver and lung  Clot Lysis Plasminogen is trapped in the clot Over several days, injured tissues release tissue plasminogen activator (tPA) Plasminogen is activated to plasmin, a protease resembling trypsin Plasmin digests fibrin fibers and several other clotting factors Often results in re-opening repaired small blood vessels  Causes of Excessive Bleeding Hepatocellular disease Vitamin K deficiency Hemophilia Low platelet count (thrombocytopenia)  Vitamin K Deficiency Essential to carboxylate glutamic acid in five important clotting factors: prothrombin and factors VII, IX, X, and protein C In this process vitamin K is oxidized and inactivated Vitamin K epoxide reductase complex 1 (VKOR c1) reduces vitamin K and reactivates it  Vitamin K Produced in the intestine by bacteria Fat-soluble: malabsorption of fats can lead to deficiency Lack of bile production or delivery can cause fat malabsorption and vitamin K deficiency In patients with liver or biliary disease, vitamin K can be injected 4-8 hours before surgery  Hemophilia Hemophilia A – Deficiency of factor VIII 85% of hemophilia cases 1 / 10,000 males Hemophilia B – Deficiency of factor IX 15% of cases About 1 / 60,000 males Both impair Intrinsic Pathway activation Both genes are on the X chromosome (males only get one copy) Clinically: Bleeding after minor trauma  Factor VIII Deficiency Factor VIII has two components… Large: MW > 106 Small: MW ~ 230,000 Deficiency of the small component causes hemophilia A → treat bleeding with factor VIII replacement Deficiency of the large component causes von Willebrand disease (resembles decreased platelet function)  Thrombocytopenia Low numbers of platelets Bleeding from small venules or capillaries Petechaiae, thrombocytopenic purpura Often idiopathic < 50,000 platelets / µL – usually modest bleeding < 10,000 platelets / µL – life-threatening Treated with platelet infusions → effective for 1 – 4 days each time  Thrombi and Emboli An abnormal clot is a thrombus When it floats it’s an embolus Caused by… Endothelial roughening (e.g. atherosclerosis) Slow flow (e.g. prolonged air travel) Treatment… tPA Embolectomy  Pulmonary Embolus Usually from deep leg veins Part of thrombus disengages ~10% of the time Occludes pulmonary arteries—potentially fatal tPA can be life-saving  Disseminated Intravascular Coagulation (DIC) Occurs in the setting of massive tissue damage or sepsis Wide-spread coagulation in small vessels Manifested as bleeding from multiple sites because of depletion of clotting factors  Clinically Useful Anticoagulants Heparin Binds, potentiates antithrombin III Works rapidly, generally used acutely Coumarins Inhibit VKOR c1 Deplete active vitamin K → deplete active prothrombin, factors VII, IX, X Slower acting (days); used chronically Over-anticoagulation – Treat with FFP and vitamin K  In vitro Anti-coagulation Siliconized containers prevent activation of factor VII and platelets Heparin – used in blood collection, heart-lung and kidney machines Calcium chelators (citrate, EDTA) used in blood collection, blood storage  Blood Coagulation Tests Bleeding Time (from small cut) normally 1 - 6 minutes Largely reflects platelet function Clotting time Invert tube every 30 seconds Normally 6 – 10 minutes Not reproducible, generally not used  Prothrombin Time Add excess calcium and tissue factor to oxylated blood, measure time to clot Assesses Extrinsic and Common Pathways Usually about 12 seconds Tissue factor batches have to be standardized (activity expressed as “International Sensitivity Index (ISI)” ) Prothrombin Concentration and Function International Normalized Ratio (INR) Normal INR: 0.9 – 1.3 Therapeutic range: 2.0 - 3.0  Tests of Other Clotting Factors Mix the patient’s plasma with a large excess of all needed components except the factor being tested Compare time to coagulation with that for pooled plasma of healthy volunteers  aPTT The activated partial thromboplastin time (aPTT) is a test performed to investigate bleeding disorders and to monitor patients taking an anticlotting drug such as heparin which inhibits factors X and thrombin, while activating anti-thrombin. The aPTT test uses blood which is decalcified to prevent clotting before the test begins. The plasma is separated by centrifugation. (Ionized) Calcium and activating substances are added to the plasma to start the intrinsic pathway of the coagulation cascade. The substances are: kaolin (hydrated aluminum silicate) and cephalin. Kaolin serves to activate the contact-dependent Factor XII, and cephalin substitutes for platelet phospholipids. The partial thromboplastin time is the time it takes for a clot to form, measured in seconds. Normally, the sample will clot in 35 seconds. PTT measures the integrity of the intrinsic system (Factors XII, XI, VIII, IX) and common clotting pathways. Increased levels in a person with a bleeding disorder indicate a clotting factor may be missing or defective. At this point, further investigation is needed and warrants the use of sensitive assays for specific coagulation factors. Liver disease decreases production of factors, increasing the PTT.  aPT PT