Hemostasis Mike Clark, M.D.

Hemostasis A series of reactions for stoppage of bleeding During hemostasis, three phases occur in rapid sequence Vascular spasms – immediate vasoconstriction in response to injury Platelet plug formation Coagulation (blood clotting)

Vascular Spasm The first step is immediate constriction of damaged vessels caused by (1) disturbance of myogenic tone and (2) vasoconstrictive paracrine secretions released by the endothelium – like endothelin and thromboxane. Vasoconstriction temporarily decreases blood flow and pressure within the vessel. When you put pressure on a bleeding wound, you also decrease flow within the damaged vessel.

Platelet Plug Formation Platelets do not stick to each other or to blood vessels Upon damage to blood vessel endothelium platelets: With the help of von Willebrand factor (VWF) adhere to collagen Are stimulated by thromboxane A2 Stick to exposed collagen fibers and form a platelet plug Release serotonin and ADP, which attract still more platelets The platelet plug is limited to the immediate area of injury by prostacyclin

Coagulation A set of reactions in which blood is transformed from a liquid to a gel Coagulation follows intrinsic (new name contact activation pathway) and extrinsic pathways (new name tissue factor pathway) and a common pathway The final three steps of this series of reactions are: Prothrombin activator is formed Prothrombin is converted into thrombin Thrombin catalyzes the joining of fibrinogen into a fibrin mesh

Extrinsic Intrinsic Pathway Pathway Common Pathway The Actual Resultant Clot

Classes of Clotting Factors Thrombin sensitive – Factors I, V, VIII and XIII Vitamin K Dependent – II, VII, IX, X

Coagulation versus Anticoagulation It is important that we do not form inappropriate blood clots. Inappropriate blood clots can lead to sudden death. If a clot (thrombus) forms in the circulation, it could break free and travel (embolus) and block the circulation in vital organs. However, if we get cut we do not want to bleed to death. So we need hemostasis to occur quickly. NOTE: A thrombus is a sitting clot and an embolus is a moving clot.

Coagulation versus Anticoagulation So that we do not form inappropriate blood clots, we have anticoagulants in the blood. This along with other mechanisms to deter hemostasis, is in place prevent inappropriate blood clotting. When we do get cut, we wish for clotting to occur quickly, thus clotting occurs using a physiologic enzymatically converted cascade that involves positive feedback. Clotting Occurs Quickly!

Proenzyme A Inactive Clotting Factor A Something Activates it Activated Enzyme A Active Clotting Factor A Inactive Enzyme B Inactive Clotting Factor B Active Enzyme B Active Clotting Factor B Inactive Enzyme C Inactive Clotting Factor C Active Enzyme C Active Clotting Factor C NOTE: As an inactive enzyme is activated, it becomes an enzyme for the subsequent reaction. This is the enzyme cascade action.

Proenzyme A Inactive Clotting Factor A Something Activates it Activated Enzyme A Active Clotting Factor A Inactive Enzyme B Inactive Clotting Factor B Active Enzyme B Active Clotting Factor B Inactive Enzyme C Inactive Clotting Factor C Active Enzyme C Active Clotting Factor C NOTE: In some cases, a subsequent enzyme can retrospectively act as an enzyme for a previous reaction. This is positive feedback; the accelerates the clotting cascade allowing for quick clotting.

Coagulation Figure 17.13a

Coenzymes and Cofactors Calcium (Ca++) is absolutely necessary in all clotting reactions, except two reactions. It is acting as a cofactor in the enzymatic reactions. Certain substances, such as tissue factor and tissue phospholipid will act as coenzymes. NOTE: If a coenzyme or cofactor is required in an enzymatic reaction, the reaction will not occur if the substances are not present.

Detailed Events of Coagulation Figure 17.13b

Coagulation Phase 1: Two Pathways to Prothrombin Activator May be initiated by either the intrinsic or extrinsic pathway Triggered by tissue-damaging events Involves a series of procoagulants Each pathway cascades toward factor X Once factor X has been activated, it complexes with calcium ions, PF3, and factor V to form prothrombin activator

Coagulation Phase 2: Pathway to Thrombin Prothrombin activator catalyzes the transformation of prothrombin to the active enzyme thrombin

Coagulation Phase 3: Common Pathways to the Fibrin Mesh Thrombin catalyzes the polymerization of fibrinogen into fibrin Insoluble fibrin strands form the structural basis of a clot Fibrin causes plasma to become a gel-like trap Fibrin in the presence of calcium ions activates factor XIII that: Cross-links fibrin Strengthens and stabilizes the clot

Extrinsic Pathway (1) In order to activate the extrinsic pathway a hole must be poked all the way through the blood vessel wall. When that occurs a chemical called “tissue factor” leaks in from the tissue fluids outside of the blood vessel. That chemical along with some circulating calcium in the blood stream activate an enzyme in the bloodstream that converts inactive clotting factor 7 to an active factor 7.

Extrinsic Pathway (2) Activated Factor 7 complexes with circulating calcium and acts as an enzyme to convert inactive Factor 10 to active factor 10. Activated Factor 10 complexes with a already active Factor 5 along with a platelet phospholipid and circulating calcium to from “Prothrombin Activator.” Prothrombin Activator starts the “common pathway.”

Intrinsic Pathway (1) The intrinsic pathway will initiate if the lining endothelial blood vessel cells are disturbed or blood touches a wettable surface, like metal or glass. Foreign bodies and pathogens in the circulation can activate the intrinsic pathway. It is the intrinsic clotting pathway that causes blood to clot in a glass test tube.

Intrinsic Pathway (2) The blood vessel wall injury or foreign bodies activate an inactive Clotting Factor 12 to an active one. Active clotting factor 12, then acts as an enzyme to activate inactive clotting factor 11. These two activations (12,11) do not require calcium. Factor 11 plus circulating calcium activate an inactive factor 9 to an active 9. Remember, I said that the clotting factor numbers are not in order, because the numbers were given in time of clotting factor discovery and not the process.

Intrinsic Pathway (3) Active Clotting Factor 9, complexes with an already active Clotting Factor 8 plus some calcium to act as an enzyme to activate an inactive clotting factor 10 to an active one. As in the Extrinsic pathway, once Factor 10 is activated, it will assist in forming “Prothrombin Activator”, thus starting the Common Pathway.

Common Pathway Activated Factor 10 plus an already active clotting factor 5, plus platelet phospholipid and calcium will form “Prothrombin Activator.” Prothrombin activator acts to convert Clotting Factor 2 (Prothrombin) to Thrombin. Thrombin plus calcium act to convert clotting factor 1 (Fibrinogen) to Fibrin. Fibrin is the actual stringy clot substance.

Tightening and Strengthening the Clot Once the clot forms, it needs to tighten to give a good plug over the blood vessel damage. Two things help this: 1. platelet contraction and 2. the action of Clotting Factor 13, termed fibrin stabilizing factor. Fibrin stabilizing factor gives better covalent bonding within the clot.

Factor XIII action

Clot Retraction and Repair Clot retraction – stabilization of the clot by squeezing serum from the fibrin strands Repair Platelet-derived growth factor (PDGF) stimulates rebuilding of blood vessel wall Fibroblasts form a connective tissue patch Stimulated by vascular endothelial growth factor (VEGF), endothelial cells multiply and restore the endothelial lining

Factors Limiting Clot Growth or Formation Two homeostatic mechanisms prevent clots from becoming large Swift removal of clotting factors Inhibition of activated clotting factors Protein C – glycoprotein produced in the liver and is the major inhibitor of coagulation. It degrades factors V and VIII. It needs Protein S to work. Protein S – Produced in liver and acts as a cofactor for Protein C. Thrombomodulin – binds to thrombin and activates and activates Protein C Antithrombin III – produced in liver and is major inhibitor of Thrombin -

Heparin Produced by mast cells, basophils and endothelial cells. Complexes with Antithrombin III to inhibit Thrombin – but also inhibits XII, XI, X, IX

Plasminogen

Inhibition of Clotting Factors Thrombin not absorbed to fibrin is inactivated by antithrombin III Heparin, another anticoagulant, also inhibits thrombin activity

Inhibition of Clotting Factors Fibrin acts as an anticoagulant by binding thrombin and preventing its: Positive feedback effects of coagulation Ability to speed up the production of prothrombin activator via factor V Acceleration of the intrinsic pathway by activating platelets

Factors Preventing Undesirable Clotting Unnecessary clotting is prevented by endothelial lining the blood vessels Platelet adhesion is prevented by: The smooth endothelial lining of blood vessels Heparin and PGI2 secreted by endothelial cells Vitamin E quinone, a potent anticoagulant

Commercial Anticoagulants Heparin – acts as an inhibitor of the intrinsic pathway – measured by the PTT Coumadin- acts an a vitamin K antagonist. (Factors 2, 7, 9,10 inhibited) Extrinsic Pathway- PT NOTE: Factor 7 has the shortest serum half-life NEW ONE SEEN OF TV – XARELTO It blocks factor X (10).

Hemostasis Disorders: Thromboembolytic Conditions Thrombus – a clot that develops and persists in an unbroken blood vessel Thrombi can block circulation, resulting in tissue death Coronary thrombosis – thrombus in blood vessel of the heart

Hemostasis Disorders: Thromboembolytic Conditions Embolus – a thrombus freely floating in the blood stream Pulmonary emboli can impair the ability of the body to obtain oxygen Cerebral emboli can cause strokes

Hemostasis Disorders: Thromboembolytic Conditions Embolus – a thrombus freely floating in the blood stream Pulmonary emboli can impair the ability of the body to obtain oxygen Cerebral emboli can cause strokes

Hemostasis Disorders Disseminated Intravascular Coagulation (DIC): widespread clotting in intact blood vessels Residual blood cannot clot Blockage of blood flow and severe bleeding follows Most common as: A complication of pregnancy A result of septicemia or incompatible blood transfusions

Hemostasis Disorders: Bleeding Disorders Thrombocytopenia – condition where the number of circulating platelets is deficient Patients show petechiae due to spontaneous, widespread hemorrhage Caused by suppression or destruction of bone marrow (e.g., malignancy, radiation) Platelet counts less than 50,000/mm3 is diagnostic for this condition Treated with whole blood transfusions

Hemostasis Disorders: Bleeding Disorders Inability to synthesize procoagulants by the liver results in severe bleeding disorders Causes can range from vitamin K deficiency to hepatitis and cirrhosis Inability to absorb fat can lead to vitamin K deficiencies as it is a fat-soluble substance and is absorbed along with fat Liver disease can also prevent the liver from producing bile, which is required for fat and vitamin K absorption

Hemostasis Disorders: Bleeding Disorders Hemophilias – hereditary bleeding disorders caused by lack of clotting factors Hemophilia A – most common type (83% of all cases) due to a deficiency of factor VIII Hemophilia B – due to a deficiency of factor IX Hemophilia C – mild type, due to a deficiency of factor XI

Hemostasis Disorders: Bleeding Disorders Symptoms include prolonged bleeding and painful and disabled joints Treatment is with blood transfusions and the injection of missing factors PT (Prothrombin Time) measures extrinsic pathway https://en.wikipedia.org/wiki/Prothrombin PTT (Partial Thromboplastin Time) measures intrinsic pathway https://en.wikipedia.org/wiki/Partial_thromboplastin_time INR (International Normalized Ratio)

Diagnostic Blood Tests Laboratory examination of blood can assess an individual’s state of health Microscopic examination: Variations in size and shape of RBCs – predictions of anemias Type and number of WBCs – diagnostic of various diseases Chemical analysis can provide a comprehensive picture of one’s general health status in relation to normal values