Hematology - 14 Fibrinolysis

HEMATOLOGY FIBRINOLYSIS • OUTLINE • Fibrinolysis o Introduction o Fibrinolysis o Plasmin o Fibrinolysis Process o Components of the Plasma Fibrinolytic System o Proteins of the Fibrinolysis Pathway o Laboratory Evaluation of Fibrinolysis • FIBRINOLYSIS monomer polymerization and interfere with platelet aggregation which end up in bleeding. In the blood circulation, the predisposition to thrombosis depends on the balance between procoagulant and anticoagulant factors. Several important biological activities normally protect the body against thrombosis. These activities include, the normal blood flow, the removal of activated clotting factors and particulate material, natural anticoagulant system known to be operative in vivo (AT-III, Heparin Cofactor II {HC-II}, Protein C and its cofactor Protein S, and cellular regulators. Clearly, fibrinolysis is important in the over-all hemostasis. When impaired, either overactivity or underactivity, the patient will be affected. INTRODUCTION FIBRINOLYSIS • • • • • • Fibrin clots are temporary structures that seal off the damaged area to prevent undue blood loss. After collagen exposure, the start of the intrinsic pathway of coagulation, the fibrinolytic system is ready, and activated in the blood plasma to play its subsequent role in clot dissolution. Fibrinolysis is the physiological process that removes stable fibrin clots through enzymatic digestion . The enzyme responsible for this process is plasmin. As healing occurs,clot should be dissolved for the reestablishment of blood flow to take place. Continuous healing is attributed mainly to the active mitosis of smooth muscle fibers within the walls/ tunics of the blood vessels. The endothelium which lines the vessel walls also fill the gaps in between its squamous cells thus sealing the wound effectively. Plasmin, digests fibrin and fibrinogen by hydrolysis to produce progressively smaller fragments, fibrinogen / fibrin degradation products (FDPs )or sometimes called fibrinogen/ fibrin split products (FSPs). This process is slow acting which gradually dissolves away clot as tissue repair is taking place, with the particulate matter being phagocytized by the cells of the mononuclear phagocytic system (MPS) also referred to as reticulo-endothelial system (RES). Inactive plasminogen circulates in the plasma until an injury (wound) occurs. The activators of plasminogen consists of endogenous and exogenous groups. Plasminogen activation of plasmin is the result of the activity of a number of proteolytic enzymes. These enzymes, the kinases, are referred to as the plasminogen activators. Plasminogen activators are found in various sites such as the vascular endothelium or lysosomal granules and biological fluids. At least 2 forms of tissue activators have been described: those that seem related to urokinase, a urinary activator of plasminogen and those unrelated to urokinase. The activators unrelated to urokinase include thrombin, bacterial products such as streptokinase from beta-hemolytic streptococci, and staphylokinase. Plasma activators of plasminogen include plasma kallekrein, activated plasma thromboplastin antecedent (Factor XI), and activated Hageman Factor (Factor XIIa). Through lysis of fibrin or fibrinogen, plasmin is responsible for forming FSPs/ FDPs, consisting of intermediate fragments X and Y and fragments D and E. These fragments exert an antithrombin effect, inhibit the hemostasic system through interference with fibrin • Fibrinolysis is the physiological process that removes insoluble fibrin deposits by enzymatic digestion of the stabilized fibrin polymers. o Clots are dissolved by plasmin. ▪ Plasmin digests fibrin and fibrinogen by hydrolysis to produce progressively smaller fragments. • Inactive plasminogen circulates in the plasma until an injury occurs. • Plasminogen activation to plasmin is a result of the activity of a number of proteolytic enzymes which are referred to as Plasminogen Activators. o Removes stable fibrin clot o Plasmin also has regulatory mechanism to maintain levels of plasmin and regulate lysis ▪ Irregularities in fibrinolysis PLASMIN • • • • Aka fibrinolysin Originate from the liver; active enzyme from plasminogen Plasminogen (Profibrinolysin) - proenzyme Actions: o Digests fibrinogen – Will lead to formation of fibrinogen degradation products or fibrinogen split products o Digests fibrin – Will lead to formation of fibrin degradation products o Digests stable fibrin clot – Will lead to formation of DDimers o Digests Factors II and XII – Will prevent clot formation o Digests Factors V and VIII – Will prevent clot formation o These will result to a phenomenon called hypocoagulability → hemorrhagic diasthesis (tendency to bleed) • Prothrombinase complex - consists of Xa & Va (Rodak) o Consists of Xa, Ca, PF3, Va (Henry’s) ▪ PF3 - PPL; participates in primary & secondary hemostasis where coagulation will act o Important in coagulation mechanism where prothrombin is converted into thrombin o Thrombin - very potent enzyme; allow conversion of fibrinogen to fibrin ▪ Converts XIII to XIIIa ▪ In the presence of thrombomodulin, it activates Protein C to serve as anticoagulant INHIBITORS • • FIBRINOLYSIS PROCESS 1. 2. 3. 4. 5. 6. 7. 8. Plasminogen and tPA from endothelial cells in blood vessels tPA will be activated by thrombin tPA and Plasminogen will be attracted to clot or fibrin tPA and Plasminogen will combine to clot or fibrin Plasmin will be formed Fibrin will be cleaved into two forming fragment X which is still clottable Fragment X will be cleaved forming fragment D & fragment Y Fragment D is not clottable and fragment Y is clottable which will be cleaved into fragment D and E. These are called dead end degradation Products COMPONENTS OF THE PLASMA FIBRINOLYTIC SYSTEM • • • Plasminogen Activators o Promotes activation of plasminogen to plasmin o Endogenous ▪ Tissue plasminogen activator (t-PA) • From endothelial cells ▪ Urokinase (u-PA) - From urinary tract ▪ XIa, XIIa, Kallikrein o Exogenous ▪ Streptokinase • Exoenzymes of the S. pyogenes (group of toxic/pathogenic organism) ▪ Acyl-plasminogen streptokinase activator complex (APSAC) Plasminogen Inhibitors o Engenders activation of plasminogen to plasmin o Alpha2-antiplasmin o Alpha2-macroglobulin o Thrombomodulin o Plasminogen activator inhibitor 1 & 2 (PAI-1 & PAI-2) ▪ PAI-1 - most important inhibitor Principles: o Increased Activator = Increased fibrinolytic activity = Increased risk of thrombosis o Increased Inhibitor = Decreased fibrinolytic activity = Increased risk of bleeding ACTIVATORS • • Tissue Plasminogen Activator (t-PA) o Main converter of Plasminogen to plasmin o It is secreted by Endothelial cells (ECs) o It is bound with thrombus and PAI-1 Urokinase Plasminogen Activator (u-PA) o It is secreted by , Kidneys, UT epithelial cells, Monocytes and Macrophages o Circulates in plasma • • Plasminogen Activator Inhibitor 1 o Principal inhibitor of plasminogen activation (in tPA and uPA) o Produced by ECs, Megakaryocytes, smooth muscle cells, Fibroblasts, Monocytes, Adipocytes, Hepatocytes o PAI-1 has high concentration than tPA o PAI-1 Deficiency → Chronic Mild Bleeding o It also functions as Acute Phase Reactant α2-Antiplasmin o Primary inhibitor of Free Plasmin o Synthesized in the liver a2-Macroglobulin o Inhibits plasmin and prekallikrein Thrombin Activatable Fibrinolysis Inhibitor o Also synthesized in the liver o Functions as anti-fibrinolytic enzyme o Inhibits the binding of TPA and plasminogen to fibrin o Also plays a role in inflammation and wound healing OTHER INHIBITORS OF COAGULATION • • • • • • Protein C and S o Inhibitors of factor V and VIII o 40% - free o 60% - bound to complement system to aid in immune response o In cases of disease, free protein C decreases o Accelerators Antithrombin III o Inhibitor of thrombin o Becomes 2,000 times more active in the presence of heparin o Aka heparin-cofactor II Heparin o Amplifies the activity of antithrombin III C1 inhibitor o Inactivate/inhibitors kallikrein (activated form of PK) Alpha-1-antitrypsin o Inhibits thrombin, Xa, Xia Thrombin + Thrombomodulin → Thrombin Thrombomodulin Complex PROTEINS OF THE FIBRINOLYSIS PATHWAY D-DIMER TEST • • • • • • Half-life - time taken for the substance to the halved LABORATORY EVALUATION OF FIBRINOLYSIS • Determination of Lysis Time o Whole Blood Clot Lysis Time o Dilute Whole Blood Clot Lysis Time o Plasma Clot Lysis Time o Euglobulin Lysis Time WHOLE BLOOD CLOT LYSIS TIME • • • • • • • Allow to clot in a test tube @ 37 C Place in water bath to 48 hours Form a clot Normal: clot is intact in 24 hours Abnormal: clot is lysed before 24 hours o Faster fibrinolysis due to overactivity of plasmin Hypofibrinolysis - no plasmin activity; cause thrombophilia Similar with single tube method of clot retraction DILUTE WHOLE BLOOD CLOT LYSIS TIME • • • Same as whole blood clot lysis time but blood is diluted. More sensitive NV: Lysis of clot after 6-10 hours EUGLOBULIN LYSIS TIME • • • • • Euglobulin - fraction of plasma that contains fibrinogen, plasminogen, plasminogen activators with traces of antiplasmin Lysis of a fibrin clot formed by the addition of thrombin is a measure of fibrinolytic activity Plasma is treated with an acid to precipitate certain clotting factors in a complex called euglobulin fraction. Euglobulin fraction will be allowed to clot at 37degC and incubated. Normal Value: o Lysis in about 300 mins or longer - normal (5h) o Lysis in 120 mins – increased lytic activity (increased plasmin activity) o Lysis in 60 mins or less – strong lysis. QUANTITATIVE ASSAY OF FIBRIN/FIBRINOGEN DEGRADATION PRODUCTS • • Based on agglutination (Hemagglutination, Staphylococcal, Latex agglutination) o Hemagglutination - uses stand RBC as carrier o Staphylococcal - for cell wall; bacteria as carrier o Latex - uses plastic polystyrene latex as carrier Uses monoclonal antibodies specific for FDPS o FDPS - very anti-thrombin • Superior test; Same principle as FPD TEST but more sensitive Uses monoclonal antibodies specific for D-Dimers Detects intravascular coagulation o May cause vascular irregularities (stroke, kidney problems, MI) Normal coagulation occurs extravascularly Requires 3 phenomena to occur o Coagulation (formation of clot) o Factor XIIIa cross-link to make a stable clot o Fibrinolysis Presence of D-Dimer signify presence of intravascular coagulation o For administration of thrombolytic drugs 2 Types Of Clots • • • Non-Cross-linked - not actioned by factor XIIIa Cross-linked - action by factor XIIIa To differentiate, use 5M urea, 1% monochloroacetic acetic o Upon addition to clot, if clot lyses = it is a non-crosslinked; not affected = cross-linked o Can be used to test for factor XIIIa