The Natural Anticoagulants and the Fibrinolytic System

D-dimer assay Lab Testing for Fibrinolysis

1) D-dimer solution is added to wells with antibodies attached to the sides of the wells. 2) If D-dimer is present they will attach to the antibodies. 3) Detector antibody with an enzyme is then added, and will attach to the other side of the D-dimer. 4) A substrate is then added and will turn a color if the antibody--D-dimer--detector enzyme antibody complex is present. ~ ELISA assay is based on an enzyme immunoassay. The patient D-dimer is bound by D-dimer monoclonal antibody covalently bound to the microtiter well. The wells are washed and a detector antibody conjugated to horseradish peroxidase is added. Color production is proportional to the concentration patient of D-dimer.

Summary Points

1) Physiologic anticoagulants including antithrombin, heparin cofactor II, protein C and protein S are plasma proteins 2) AT is produced in the liver. It inhibits factos 2, 9, 10, 11, and 12. Heparin increases the inhibitory action of AT. 3) Protein C is a vitamin K dependent protein. Protein S is a cofactor for protein C activation. Activated protein C deactivates factors 5 and 8. 4) Plasminogen is converted to plasmin and then destroys the fibrin clot 5) Naturally occurring inhibitors of fibrinolysis are plasminogen activator inhibitor 1 and alpha 2 antiplasmin

Fibrinolytic System

Includes prekallikrein, HMWK, factor 12 and protein C and S

Hemostatic Balance

~ Activation of coagulation ----- Thrombin --> fibrinogen --> fibrin --> stabilized clot ~ Activation of fibrinolysis ----- Plasminogen --> plasmin (anticoagulant) --> degradation of stabilized clot ~ Find a balance between these two, if balance is off it results in problems (deficiencies, mutations)

Antiphospholipid Syndrome and Lupus Anticoagulants

~ An acquired disorder that produces antibodies to phospholipid-binding proteins. ~ Antibodies bind to phospholipids and prolong phospholipid dependent tests (aPTT) ~ Symptoms include thrombosis and fetal loss (miscarriage) ~ The most common form is lupus anticoagulant (LA) and anticardiolipin antibody (ACA) ~ Lab Tests: ----- aPTT (prolonged) ----- dilute Russell viper venom (DRVVT) ----- Mixing study (mix normal plasma 50/50 with patient, will be elevated if inhibitor present which means it is not a factor deficiency) ----- platelet neutralization test (PNP) or hexagonal phase phospholipids

Acquired Thrombotic Disorders

~ Antiphospholipid syndrome and Lupus anticoagulants ----- produce antibody to phospholipid which inceases clotting time and increases the number of clots ~ Heparin induced thrombocytopenia (HIT) ----- produce antibody to platelets, end up with clots

Anticoagulants

~ Antithrombin III ----- MAJOR INHIBITOR OF COAGULATION ----- Manufactured in the liver ----- Binds and neutralizes thrombin (factor 2) and factors 2, 9, 10, 11, 12 and kallikrein ~ HEPARIN COFACTOR ----- Inhibits thrombin ~ ALPHA 2 MACROGLOBULIN ----- Inhibits thrombin and kallikrein ~ ALPHA 1 ANTITRYPSIN ----- Inhibits 11a, weakly inhibits thrombin ~ PROTEIN C AND S ----- Vitamin K dependent, synthesized in the liver ----- Activated by thrombin in the presence of a substance called thrombomodulin ----- Inhibits 5a and 8a (protein C in the presence of its cofactor protein S)

Inactivation of clotting enzymes by heparin

~ Antithrombin III is a slow inhibitor without heparin ~ Heparin binds to AT-III through high affinity pentasaccharide and induces a conformational change in AT-III, thereby converting AT-III form a slow to a very rapid inhibitor ~ AT-III binds covalently to the clotting enzyme, and the heparin dissociates from the complex and can be reused

Inherited Thrombotic Disorders (excessive clotting)

~ Decreased inhibitors: Antithrombin, protein C or protein S, Factor 12, prekallikrein and HMWK ~ Increased clotting factors (prothrombin G20210A) and elevated factor 8 ~ Others: Factor 5 Leiden (won't listen and won't break down clot), hypercysteinemia (causes damage in endothelial --> increases platelets)

Fibrinolysis

~ Fibrinolysis is the process that removes insoluble fibrin deposits (clots). ~ Is mediated by activation of PLASMINOGEN TO PLASMIN. ~ Plasmin digests fibrin and fibrinogen to produce smaller fragments. This is a slow-acting process that dissolves the clot as tissue repair takes place. ~ When fibrinogen or fibrin degradation by plasmin occurs ----- Fragments are produced called fibrin degradation products (FDP) of fibrinogen split products (FSP) ----- Plasmin degrades both fibrinogen and fibrin = produces fragments called X, Y, D (D-dimer) and E.

Heparin-induced thrombocytopenia

~ Immune-mediated complication associated with heparin therapy ~ HIT may develop in 3-5% of patients in 5 to 14 days after heparin therapy ~ Can be life threatening (plt decreases extremely) ~ Antibodies are produced against HEPARIN-PLATELET FACTOR 4 COMPLEX; causing platelet activation, thrombocytopenia and a hypercoaguable state

Fibrin(ogen) Split Products Lab Testing Fibrinolysis

~ Latex particles are coated with antibodies to FSP's ~ If increased levels of FSP are in the serum, agglutination occurs.

Complications of Fibrinolysis

~ PRIMARY Fibrinolysis = breakdown of fibrinogen, not fibrin ----- fibrinogenolysis ----- no fibrin deposition ----- fibrinogen is degraded ----- result of trauma, surgery or malignancy ~ SECONDARY Fibrinolysis = breakdown of fibrin (deposited) ----- fibrin has been deposited ----- D-dimers are produced ----- results from pulmonary embolism or DIC or deep vein thrombosis

Fibrinolysis Inhibitors

~ Plasminogen Activator Inhibitor-1 ----- Inhibits activation of plasminogen which breaks down clot ~ Alpha 2 antiplasmin ----- Inhibits activation of plasmin which breaks down clot

D-Dimer

~ Presence of D-dimer is a specific indicator of in vivo lysis of fibrin rather than fibrinogen. ----- clotting has taken place ~ Increased D-dimer levels are seen in patients with venous thrombosis, pulmonary embolism (PE), deep vein thrombosis (DVT), and disseminated intravascular coagulation (DIC) ~ The test is used to rule out thrombosis, eg. a low result with low clinical indications, would indicate no increased lysing of a clot, suggesting no clot. (ED patients)

Fibrin degradation or split products FDP/FSP

~ Products of fibrinolysis, the result of plasmin digestion of both crosslinked and non-crosslinked fibrin or fibrinogen. ~ Fragments X and Y are early split products from fibrinogen and non-crosslinked fibrin. ~ Fragments D and E are late split products (crosslinked fibrin clot) ~ FDPs that are normally cleared rapidly by liver ~ Large quantities of FDPs can act as anticoagulants prohibiting fibrin polymerization and interfering with normal platelet function.

Coagulation Regulatory Mechanisms

~ The delicate balance between the coagulation and fibrinolytic processes is maintained by a group of inhibitors that help regulate the system. ~ The in vivo existence of natural anticoagulant systems is essential to prevent thrombosis (inhibits coagulation in body) ----- Antithrombin III ----- Protein C and protein S ----- Heparin cofactor II ----- Tissue factor pathway inhibitor (thromboplastin) ----- Alpha 1-antitrypsin ----- Alpha 2-macroglobulin

Anticoagulant Therapy

~ Thromboembolic diseases are treated with antithrombotic drugs, which include: ----- Antiplatelet drugs = prevent platelet activation and aggregation ----- Anticoagulant drugs = inhibit thrombin and fibrin formation (unfractionated and low molecular weight heparin, warfarin (coumarin), direct thrombin inhibitors (heparin) ----- Thrombolytic drugs = break down fibrin clots, restore vascular function

Russell's Viper Venom

~ thromboplastin like substance ~ Activates factor 10 without factor 7 ~ If factor 7 is deficient, Russell's viper venom will produce a normal time ~ But if factor 10 or any other factor in the common pathway is deficient, the time will be prolonged