FIBRINOLSIS SYSTEM Ahmad Shihada Silmi Msc, FIBMS IUG Faculty of Sciences Medical Technology Dep.

History In 1937 MaCfarlane reported that damage tissues release a substance (activator that activates the inert precursor called Plasminogen).

Fibrinolysis Process of removing fibrin from the vasculature Key Players Plasminogen Plasminogen activators (PA) Active enzyme plasmin (PLN) Fibrin Fibrin/Fibrinogen degradation products Inhibitors of Plasminogen activators and PLN

Function of Fibrinolysis in Homeostasis Fibrinolysis is the system whereby the temporary fibrin clot is systematically and gradually dissolved as the vessel heals in order to restore normal blood flow. Is the body’s defense against occlusion of blood vessels

Fibrinolytic System, cont. Sensitive to imbalances Restricts fibrin formation to area of injury Initiated when coagulation cascade begins Dissolves clot by digestion of fibrin

Overview Under the influence of thrombin. Fibrinogen cleaved into fibrin monomers Fibrin monomers are cleaved into fibrin degradation products or fibrin split products

Process summary Once clotting begins, the fibrinolytic system comes to life Plasminogen (PLG) binds to fibrin in the developing thrombus Tissue-type PLG activator (tPA) also binds to fibrin, increasing its enzymatic activity to convert plasminogen to plasmin (PLN) Complex formation of tPA, PLG and fibrin results in the break-down of fibrin PLN then further digest fibrin to soluble degradation products making fibrin fragments

Plasminogen Plasminogen is a single-chain glycoprotein with a Mwt of 90,000 d Produced in the liver Found in normal plasma Plasminogen is the inactive precursor (zymogene) of the trypsin-like serine protease plasmin.

Plasminogen Following injury, binds to fibrin during clot formation along with plasminogen activators Plasminogen: once needed converts to its active form called, Plasmin. Increased concentrations are found in association with inflammation.

When Plasminogen is activated, it unfolds to expose its potent enzymatic domains.

Activators of Fibrinolysis Contact Phase/Intrinsic Occurs by interactions of the contact factors (XIIa, HMWK, and PK) following intrinsic pathway activation(collagen exposure) Physiologic (secretions) Activators released from tissues extrinsic to the blood tPA: tissue- type PLG activator Found in endothelial cells of small vessels uPA: urokinase-type PLG activator Made in renal tubular epithelium and vascular epithelium Found in urine and plasma Exogenous activation Via medications given to lyse pathogenic clots (i.e. pulmonary emboli) Example includes Streptokinase

Activators

Another classification seen: Activation of Plasminogen can occurs due to • Intrinsic Plasminogen Activation • Extrinsic Plasminogen Activation • Exogenous Plasminogen Activation • Plasminogen Activation in Secretory Ducts. (Fig)

FIBRINOLSIS SYSTEM

Streptokinase is another agent used to activate Plasminogen to plasmin FIBRINOLSIS SYSTEM For therapeutic destruction of thrombosis, Urokinase, a trypsin-like protease, may be administered to a patient to activate plasminogen to plasmin and induce Fibrinolysis. Streptokinase is another agent used to activate Plasminogen to plasmin Tissue Plasminogen activator (tPA) is an agent used for the treatment of thrombosis. It released in vivo on endothelial cell damage and can be manufactured in vitro through recombinant DNA techniques.

FIBRINOLYTIC SYSTEM

FIBRINOLSIS SYSTEM FIG. Simple overview of Fibrinolytic pathway. Tissue plasminogen activators activate plasminogen within clot to plasmin, which slowly dissolves fibrin clot. Inhibitors are neutralized by protein C—S complex, thus enhancing fibrinolysis.

FIBRINOLSIS SYSTEM Tissue Plasminogen Activators, which convert Plasminogen to Plasmin are released from injured vessel walls. Plasmin is trapped within the clot, and clot lysis begin slowly as soon as the clot is formed, with fibrin degradation products being released in the plasma. Lysis is slow because of fibrin clot stabilization by factor XIII. Protein S and C are two substance that enhance fibrinolysis and inactivate Tissue Plasminogen Activators (tPA inhibitor).

Plasmin A non-specific Proteolytic enzyme Normally is not present in the blood in an active form. A non-specific Proteolytic enzyme Dissolves fibrin/fibrinogen clots into protein fragments that are cleared from plasma by the liver Provides a positive feedback loop for forming more Plasminogen

Plasmin Highly regulated Temporarily active Local Can digest or destroy fibrinogen, fibrin and factors V & VIII. Promotes Coagulation and activates the Kinine and Complement systems. Highly regulated Temporarily active Local

FIBRINOLSIS SYSTEM Physiologic Fibrinolysis Many similarities exist between Coagulation and Fibrinolytic systems. • AS there are checks and balances in the formation of clot • There are similar mechanism for dissolution of the clot to promote wound Healing. • Important that bleeding does not recur because of premature lysing of the clot.

Comments Plasmin's Roles FIBRINOLSIS SYSTEM Comments Plasmin's Roles Cleaves Fibrin and Fibrinogen to fibrin (ogen) degradation products X, Y and D-E Activates Fibrinolysis Factors XII----XIIa is amplified indirectly by plasmin Activates Intrinsic Coagulation Pathway Destroys factors VIII and V Interferes with intrinsic and common pathways FDP interfere with thrombin influence on fibrinogen Block Thrombin conversion of fibrinogen to fibrin

Summary FIBRINOLSIS SYSTEM Fibrinolysis is dependent on the enzyme PLASMIN Tissue Plasminogen Activators, which convert Plasminogen to Plasmin are released from injured vessel walls. Plasminogen (A zymogen) which normally present in plasma is converted to Plasmin by the action of specific enzyme called Plasminogen Activators. Activation of Plasminogen can occurs due to Intrinsic, Extrinsic, Exogenous Plasminogen Activation, andActivation in Secretory Ducts.

FIBRINOLSIS SYSTEM Plasminogen is a part of any clot because of the tendency of fibrin to absorb plasminogen from the plasma in normal circumstances. When plasminogen activators performed their function, plasmin is formed within the clot, which gradually dissolves the clot while leaving time for tissue repair. Free Plasmin is released to the plasma, however, anti-plasmin is there immediately destroy any plasmin released from the clot.

FIBRINOLSIS SYSTEM When Pathologic Coagulation processes are involved, excessive free plasmin is released to the plasma In these situation, the available anti-plasmin is depleted and plasmin begins destroying components other than fibrin, including, fibrinogen, factor V and VIII and other factors.

FIBRIN(OGEN) DEGRADATION by PLASMIN FIBRINOLSIS SYSTEM FIBRIN(OGEN) DEGRADATION by PLASMIN In the process of fibrinogen or fibrin degradation by plasmin within a clot, specific molecular fragments are produced called Fibrin (ogen) Degradation Products (FDP) or Fibrin (ogen) Split Products (FSP) These degradation products are removed by the reticuloendothelial system and other organs. The sequence of reaction in the degradation of Fibrin(ogen) by plasmin are X, Y, D (D-D dimer) and E.

Fibrinogen Structure Graphic accessed at URL http://coursewareobjects.elsevier.com/objects/elr/Rodak3e/IC/jpg/Chapter40/040006.jpg, 2008.

Fibrinolytic Mechanisms 1 - Damaged endothelia secrete TPA. 2 – TPA activates plasminogen bound to fibrin clot. 3 - Free TPA circulates bound to PAI-1 and is cleared from plasma. Plasmin systematically hydrolyzes lysine & arginine peptide bonds of the fibrin polymer. ‘P’ indicates sites where plasmin cleaves the fibrin polymer. Graphic accessed at URL http://www.nature.com/bmt/journal/v41/n8/images/1705990f1.jpg, 2008. Graphic accessed at URL http://coursewareobjects.elsevier.com/objects/elr/Rodak3e/IC/jpg/Chapter40/040006.jpg, 2008. Fibrinolytic Mechanisms

Plasmin Degradation of Fibrinogen Fragment X: small peptides from carboxyl end of α chain removed plasmin E Fragment Y + Fragment D D D plasmin E D Fragment D + Fragment E

Intrinsic/contact activation Physiologic activation Plasminogen Intrinsic/contact activation Physiologic activation Exogenous activation Plasmin Fibrin clot Fibrinogen Fibrin Degradation Fibrinogen Products Degradation Products X,Y,D=D,E X,Y,D,E,D

Fragment X and Y are referred to as early degradation products FIBRINOLSIS SYSTEM Fibrin(ogen) Degradation by Plasmin Fragment X and Y are referred to as early degradation products Fragment D and E are late degradation products Fragment X is the first and the largest fragment formed (Mwt 250,000 d) Fragment X is the results of Plasmin (P) cleavage of the terminal portion of the alpha (α) chains from a fibrin polymer Fragment X is cleaved by Plasmin (P) to form two fragments called Y (YY) and an intermediate complex (DXD)

FIBRINOLSIS SYSTEM This complex is further cleaved into intermediate complexes DED and DY/DY until finally, fragment E and D (D-D dimer) are formed. A single fragment D has Mwt 90,000 d and that the D-D dimer is 180,000 d Presence of D-D dimer is a specific indication of in vivo fibrinolysis, namely, intravascular thrombin formation leading to fibrin formation and its subsequent degradation

What is D-dimer D-dimer is a protein that represents an area of fibrin which contain cross-linked regions used as clot stabilizers. D-dimer present in circulation is used as an indicator of a blood clot being formed and broken down somewhere in the body

Pathologic Effect of FDPs The FDPs are significant because of their haemostatic effects, which include; Anti-thrombin activity Interference with polymerization of fibrin monomer Interference with platelet activity

The early and large fragments (X and Y) along with the intermediate FDPs, are important in exerting anticoagulant Effect • Fragment Y and D inhibit fibrin polymerization • Fragment E is a powerful inhibitor of thrombin • All four fragments, but particularly low molecular weight FDP, have an affinity coating platelet membrane and therefore, cause a clinically significant platelet dysfunction by inhibiting aggregation.

Fibrinolytic Inhibitors 1- Alpha-2- Anti-plasmin (α2 anti-plasmin) An (α2) glyco-protein Most important naturally occurring inhibitor The principle inhibitors of fibrinolysis by binding with plasmin that is free in the plasma (neutralizing plasmin) Inhibits the clot-promoting activities of plasma kallikrein Inhibits the serine proteases Xlla, XIa, IIa and Xa Hereditary deficiencies have been associated with, Excessive clotting (DIC) Excessive fibrinolysis

Large naturally occurring plasma GP 2- Alpha 2 Macroglobulin Large naturally occurring plasma GP Inhibits component in both the fibrinolysis and coagulation systems Inhibits plasmin after alpha 2 anti-plasmin depletion 3- Alpha 1 Antitrypsin The third most important naturally occurring inhibitor of fibrinolytic system. Inactivates plasmin slowly and does not bind plasmin until both alpha2 anti-plasmin and alpha 2 macroglobulin are saturated Inhibits coagulation by its potent inhibitory effects on factor XIa

Other Inhibitors Anti-thrombin III, inhibits fibrinolysis by inhibiting plasmin and kallikrein The C1 inactivator also inhibits plasmin.