Adv Pathophysiology Unit 4 Page 1 of 10. Learning Objectives for this file:

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1 Adv Pathophysiology Unit 4 Page 1 of 10 Learning Objectives for this file: Topics include structure & function of: 1. Coagulation process platelets, clotting factors 2. Coagulation cascade formation of fibrin clot, initiation (extrinsic, intrinsic pathway) 3. Importance of calcium 4. Thrombo-resistance of normal vascular compartment 5. Lysis of clots

2 Adv Pathophysiology Unit 4 Page 2 of 10 COAGULATION (CLOTTING): There is always a balance between clotting and fibrinolysis (dissolution of clot). Circulating natural substances in blood are procoagulants or anticoagulants. Hemostasis a Combination of Platelet aggregation & Clotting Factor activity: Hemostasis means to stop bleeding must also fix the damaged blood vessel Summary of hemostasis: o vasospasm (intense vasoconstriction) of blood vessel due to smooth muscle contraction in response to the injury o platelet cells adhere to exposed collagen protein of vessel endothelium o platelets release cytokines that call other platelets and induce platelet aggregation o there is formation of a platelet plug o meanwhile, tissue factor is produced that activates the coagulation cascade (activation of coagulation factor proteins via the extrinsic pathway) to form the enzyme thrombin o thrombin breaks inactive fibrinogen into active fibrin o fibrin protein reinforces the platelet plug and platelets contract and squeeze out fluid o now we have a CLOT o the intrinsic pathway of the coagulation cascade is also activated to maintain the clotting process and platelets continue to release ADP, 5HT (serotonin), and thromboxane-a2 (TXA2) to stimulate further platelet aggregation and vasoconstriction What balances the above activity? o at the same time as above, cytokines are released by the platelets to oppose vasoconstriction release of the vasodilator prostacyclin (epoprostenol,pgi2) o and at the same time, there is fibrinolysis (breakdown of clot) tpa & urokinase enzymes break down plasminogen to form the active enzyme plasmin that chews up fibrin Thus there is always a BALANCE of clotting/vasoconstriction and fibrinolysis/vasodilatation Normal thrombo-resistance of the blood vessels: undamaged blood vessels are NOT thrombogenic (do not initiate clotting) o endothelial cells that make up the blood vessel wall are smooth and covered with glycocalyx that resists platelet adhesion o if there is no injury to the blood vessel, the collagen is not exposed to trigger hemostasis events there are natural ANTI-COAGULANTS that inhibit coagulation o alpha-antiprotease, alpha-macroglobulin, & antithrombin-iii, whose cofactor is heparin o Thus, heparin combines with antithrombin-iii to reduce clotting, and is a naturally occurring substance in the body there is background activity of plasmin enzyme chewing up fibrin o plasmin enzyme is created by tpa (tissue plasminogen activator) enzyme and Urokinase enzyme

3 Adv Pathophysiology Unit 4 Page 3 of 10 Platelet Activity: blood vessel damage vasospasm platelet adherence to exposed collagen platelet aggregation & degranulation of cytokines more platelets called to site platelet fusing (platelet plug) Injury Collagen exposed Platelets aggregate and respond to cytokines Fibrin meshwork Electron Micrograph of platelet degranulation Clot Platelets cause clot retraction and seal hole in vessel Platelet Degranulation and Aggregation

4 Adv Pathophysiology Unit 4 Page 4 of 10 Don t confuse: Platelet activity: o this is activity of the platelet cells to support vasoconstriction and platelet aggregation o drugs working AGAINST platelet activity are called anti-thrombotics or antiplatelet drugs Clotting factor activity: o this is creation of activated clotting factors to produce fibrin as the end product of the coagulation cascade o remember that the clotting factors are made in an INACTIVE form and need to be activated to function o when they are activated a little a is placed after the factor (such as Xa for activated clotting factor ten) o drugs working AGAINST clotting factor activity are called Anti-coagulants Coagulation (Clotting): tissue factors activate coagulation cascade (extrinsic path) INACTIVE proteins that can become inactive in the inactive form that are zymogens are activated in turn, in a cascade, by enzymes in sequential steps fibrin produced to reinforce platelet plug intrinsic path activated Interaction of platelets & Coagulation Cascade: thrombin causes further platelet degranulation platelet factors (ADP, 5-HT, TXA2) vasoconstrict vessel & attract more platelet aggregation. Platelet activity is thrombogenic. Other platelet activity is anti-thrombotic release of vasodilator PG (prostacyclin, PGI2) Plasmin and PAI: FIBRINOLYSIS Balance to the system at the same time we clot, we are destroy clots through fibrinolysis Plasminogen activators released by vascular endothelial cells due to presence of fibrin (tpa & urokinase) Plasmin enzym formed & breaks down fibrin into small pieces (swept away by the bloodstream) Yet another part of the balance things inhibiting fibrinolysis thrombogenic o Plasminogen activator inhibitors (e.g. PAI) more likely to clot Clinical: lots of PAI more thrombogenic thrombosis (CVA, MI) Excessive PAI contributes risk for CV disease o and plasmin inhibitors also present and prevent fibrinolysis

5 Adv Pathophysiology Unit 4 Page 5 of 10 COAGULATION CASCADE THE CLOTTING PATHWAY -- OVERVIEW: A. COAGULATION CASCADE & CLOT FORMATION (OVERVIEW): proteins are "pro" enzymes that are inactive, called zymogens Most are made in the liver circulating around, waiting to be activated Coagulation cascade (overview): In response to rupture of vessel or damage to blood, a cascade of chemical reactions occurs in blood involving coagulation factors. This results in the formation of a complex of activated substances Normally the fibrinogen stays in the vascular space, so interstitial fluid doesn't clot. If the capillaries are leaky, fibrinogen can enter any tissue area that needs clotting. Process: o Calcium + prothrombin activator converts prothrombin thrombin enzyme o Thrombin converts fibrinogen fibrin mesh o Fibrin enmeshes cells & plasma blood clot AND, thrombin can make more of itself: (one of the few healthy positive feedback cycles) Thrombin prothrombin more thrombin (it makes more of itself). Fibrin clotting (overview): Strengthening this fibrin clot is fibrin-stabilizing factor, released by the platelets. The clot continues to grow, then retracts (platelet microtubule action), squeezing out serum (plasma with all the clotting factors removed). This causes apposition of the broken wall of the vessel, closing off the wound. Stopping the process: To stop clotting, or stop a clot from growing, thrombin must be removed from the area. Natural anticoagulants: o Thrombin + antithrombin III (with heparin cofactor) = inactive thrombin. o Heparin is released by mast cells (tissues) or basophils (blood). o Alpha-2-macroglobulin + other clotting factors = inactive clotting factors. BALANCE to the system See picture next page

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7 Adv Pathophysiology Unit 4 Page 7 of 10 B. INITIATION OF COAGULATION = FORMATION OF PROTHROMBIN ACTIVATOR: there are two pathways to initiate clotting they converge at their last step (FINAL COMMON STEP). These pathways are usually occurring simultaneously. 1. Extrinsic Pathway (very FAST pathway): traumatized (damaged) tissue releases tissue factor (tissue thromboplastin), mostly phospholipids from tissue membranes and a lipoprotein proteolytic enzyme. This complexes with factor VII to make VIIa (the "little a" after the factors means "activated"). This in turn activates factor X. Factor Xa + factor V + platelet phospholipids = prothrombin activator complex. This is the final common step (making prothrombin activator complex -- see above -- it will act on the prothrombin to make thrombin). 2. Intrinsic Pathway (slower pathway): trauma to blood or exposure of blood to collagen activates this pathway. Factor XII is activated by presence of collagen or a smooth surface (such as a glass test tube). Factor XIIa then activates factor XI (requires prekallikrein and kinogen), and then the factor XIa goes on to activate factor IX. Factor IgA + factor VIII + platelet phospholipids is a complex that activates factor X. Once you have activated factor X, it is the same last step as in the extrinsic pathway (you made prothrombin activator complex). 3. Importance of Calcium ions: are necessary for most of the steps in both pathways. Clinical: o Your lavender top test tubes contain chelating agents or precipitating agents (citrate ion, oxalate ion, or EDTA) that remove Ca from solution and prevent initiation of the clotting pathway o used for complete blood count which requires that the blood sample remains liquid and not clotted

8 Adv Pathophysiology Unit 4 Page 8 of 10 C. THROMBO-RESISTANCE OF THE NORMAL VASCULAR COMPARTMENT: Regulatory factors: endothelial cells are smooth, and are coated with glycocalyx that repels platelets & clotting factors. There are several proteins that bind with and inactivate the circulating thrombin. Vessel wall damage removes these protections. Normal anti-coagulants: heparin, alpha-2-macroglobulin, and antithrombin III work to remove thrombin to prevent formation of large clots Vasodilatation & platelet inhibition in the normal blood vessel: One of the PGs (PGI2, prostacyclin, epoprostenol) released by platelets is a potent vasodilator Also inhibits platelet aggregation SO, platelet activity is a balance of activity based on the chemicals released by activated platelets o actually inhibits itself o actually stimulates MORE platelet aggregation Platelet factors: at the same time, platelets release Procoagulants that stimulate platelet aggregation & vessel vasoconstriction (TXA2, serotonin, ADP), AND Anti-coagulants that inhibit aggregation and promote vasodilatation (PGI2. Clinical: disruption of homeostasis (disruption of this normal balance) Atherogenesis: Atherosclerotic heart disease (ASHD) o some theories of atherogenesis assume endothelial damage from oxidized-ldl-c (cholesterol) molecules that accumulate in abnormal "foam cells" o resulting in platelet aggregation and cause growth of atherosclerotic plaques over many years. Aspirin therapy: o Aspirin in low doses binds irreversibly to platelets and inhibits their aggregation by reducing their production of thromboxane A2 (TXA2) (COX enzyme inhibition) o explains aspirin's success in primary & secondary prevention of MI. o If aspirin is given in higher doses, unfortunately it will inhibit (the good) vasodilator prostaglandin (epoprostenol) synthesis, & net effect on capillaries may be to cause vasoconstriction and possibly enhance the clotting process. o Therefore, the aspirin doses used for atherogenesis protection is usually a very low dosage inhibits thromoboxane A2 synthesis to inhibit platelet aggregation, but doesn't inhibit the naturally occurring prostaglandin vasodilator. Prostacyclin (PGI2): vasodilating prostaglandin o medication for therapy of primary pulmonary hypertension as an IV drug to dilate the pulmonary circulatory tree.

9 Adv Pathophysiology Unit 4 Page 9 of 10 D. LYSIS OF BLOOD CLOTS (FIBRINOLYSIS): blood vessel lumen is kept open by activation of the fibrinolytic system, which occurs whenever there is fibrin deposition BALANCE between fibrin deposition & lysis allow clots time to stabilize for vessel hemostasis & repair The enzyme used to break down fibrin is called plasmin, and it is formed in its OWN cascade (using plasminogen activators like tpa & urokinase)!! Breakdown of fibrin yields fibrin degradation products. As well, there are substances that inhibit the activity of both plasminogen activators or plasmin (like PAI); this will allow clots time to stabilize for vessel hemostasis & repair 1. Plasmin formation (fibrinolysis): Plasminogen (plasma protein) binds to fibrin (at lysine sites) Plasminogen activators released by vascular endothelium (due to fibrin) These include tissue plasminogen activator (tpa) & urokinase. (Streptokinase is a bacterial plasminogen activator that is also used clinically.) o Drug therapy that are thrombolytics ( clot busters ) Plasminogen + plasminogen activators plasmin Plasmin breaks down fibrin into small pieces (swept away in circulation) 2. Plasmin inhibition (inhibition of fibrinolysis): to prevent excessive fibrinolysis & to allow clots enough time to stabilize, there are also inhibitors of fibrinolysis, called plasminogen activator inhibitors (PAI) as well as plasmin inhibitors. Clinical Correlate: overproduction of PAI may be etiologic in atherosclerotic heart disease.

10 Adv Pathophysiology Unit 4 Page 10 of 10 INTERACTION AMONG THE THREE PROTEIN SYSTEMS: when one system is activated, the other two become activated thus complement, clotting & kinins are all LINKED in their action Control of all three systems: o accomplished by enzymes (present in tissues to degrade the clotting proteins) o prostaglandins o C1 esterase enzyme (which blocks activation of all three protein systems) INTEGRATED IMMUNE & INFLAMMATORY RESPONSE interaction of the different systems -- there is always an interaction of the immune, fibrinolytic (clotting) and inflammatory response Clinical correlate: o any tissue damage, whether it be infectious, injury, pathological (e.g. atherosclerosis, ischemic) will cause a general response that affects ALL these systems and result in complex clinical findings. o treatment of pathologic conditions that seem unrelated must be thought of globally how it affects the entire body in order to more perfectly restore the body to homeostasis.