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Learning Center
Coagulation Corner
Wednesday, November 5, 2008
The Fibrinolytic System: Testing and Disorders
The one system that gets minimal attention is the fibrinolytic system. This pac man system is responsible for the dissolution of clots. During the appropriate activation of the hemostatic system the outcome expected is the cessation of blood, and the formation of a clot. That clot then must be removed from the system. Spontanous dissolution of clots from living and dead people was noted as early as the time of Hippocratis. It was also noted that once it had liquefied, it could no longer clot again. In 1905 Morawitz concluded that fibrinolysis was probably enzymatic. It wasn't until about 1945 that the terms plasminogen and plasma were utilized, But it wasn't until 1959 that it was discovered in the presence of a clot, the plasminogen is absorbed to the clot so that when the plasminogen converts to plasmin, the plasmin is already in place in the clot.
The fibrinolytic system is responsible for the dissolution of a clot. Fibrin clots are not intended to be permanent, their purpose is to stop the flow of blood until the damaged vessel can be repaired. The presence or absence of hemorrhage or thrombosis depends on a balance between the procoagulant and the fibrinolytic system. The key components of the system are plasminogen, plasminogen activators, plasmin, fibrin, fibrin/FDP and their inhibitors. Fibrinolysis is the process by which the hydrolytic enzyme plasmin digests fibrin and fibrinogen resulting in progressively reduced clots. This system is activated in response to the initiation of the activation of the contact factors. This produces a proteolytic enzyme plasmin. Plasmin is capable of digesting either fibrin or fibrinogen as well as other factors in the cascade (V,VIII,IX and XI). Normal plasma contains the inactive form of plasmin in a precursor called plasminogen. This precursor remains dormant until it is activated by protelolytic enzymes , the kinases, or plasminogen activators. Fibrinolysis is controlled by the plasminogen activator system. Plasmin digests fibrin and fibrinogen to produce smaller fragments. This occurs the same time healing occurs and the cells of the mononuclear phagocytic system phagocytize the products.
An activator tissue-plasminogen activator t-PA results in the activation of plasminogen to plasmin resulting in degrading fibrin. The fibrinolytic system includes several inhibitors. Alpha-2-antiplasmin is a rapid inhibitor of plasmin activity and alpha-2-macroglobulin is an effective slow inhibitor of plasmin activity. This system is in turn controlled by inhibitors to t-PA and plasmin-plasminogen activator inhibitors (PAI-1) and alpha 2-antiplasmin.
Laboratory Testing:
The principle of the Euglobulin Clot Lysis time is used to evaluate increased fibrinolytic activity and is isolated from plasma by precipitation with 1% acetic acid. This euglobulin fraction is relatively free of fibrinolytic inhibitors. The precipitate is redissolved and calcium is added to form a fibrin clot. The resulting clot serves as a substrate for plasmin which is generated from plasminogen by the plasminogen activators. The clot is incubated at 37 degrees C, and examined at 30 minute intervals for evidence of lysis. The euglobulin lysis of the clot is the time required for complete degradation of the clot. Conditions associated with increased fibrinolytic activity are DIC, liver disease, surgery, certain malignancies and women receiving oral contraceptives or during menstruation. A Euglobulin Clot Lysis is decreased in pregnancy due to an increase in fibrinogen, Plasminogen Activator Inhibitor and plasminogen.
Prolongation can be due to a decrease in Plasminogen activator (PA) and an increase in PA- PAI complex, or even a plasminogen defect. Lysis times of less than 30 minutes indicate a hyperfibrinolytic state.
If fibrinogen is greater than 600mg/dl, the fibrin clot formed provides excessive substrate for the formed plasmin and time for full lysis would be increased. However, if fibrinogen values are decreased, the results may be difficult to interpret, since normal fibrinolytic activity may dissolve a small clot somewhat more rapidly than normal.
Clot Lysis time is shortened in Factor XIII deficiency since the fibrin clot formed is poorly crosslinked and dissolution more rapid. Platelets prolong the lysis time due to their antiplasmin activities, therefore, platelet poor plasma must be used.
The lower the pH of the acid-plasma mix, the more prolonged the lysis time is.
The test may be performed on patients receiving heparin since it is removed during the precipitation process. One of the problems with this test is the lack of standardization. The buffers are made from scratch, and determination of the status of the clot is subjective. All persons performing the test should demonstrate concordance in determination of the clot. A normal and abnormal control must also be run when performing the test. Also, since the test is manual, it must be performed in duplicate.
Alpha-2-antiplasmin assay provides a quantitative measure of activity in human plasma by a chromogenic assay. This assay uses excess plasmin that reacts with alpha-2-antiplasmin in the patient sample to from an inactive complex. Residual plasmin activity is then determined by hydrolysis of a plasmin-specific chromogenic substrate which releases p-nitroaniline when cleaved. The absorbance is inversely proportional to the alpha-2-antiplasmin.
Plasminogen is the proenzyme of plasmin, a proteolytic enzyme which lyses fibrinogen/fibrin as part of the fibrinolytic system. Under the influence of tissue or plasma activators, plasminogen is converted into plasmin. The main role of plasmin is to degrade fibrin and secondarily fibrinogen,leading to the production of degradation products of stabilized or non-stabilized fibrin and fibrinogen, respectively.
The test uses a chromogenic substrate to measure biologically active plasmin which can differ from the concentration of immunoreactive plasminogen depending upon the patient population. Measuring the activity as opposed to the antigen, can be an aid in evaluating some fibrinolytic disorders and responses to therapy.
In this assay, plasminogen in the sample forms a complex with the streptokinase . The active plasminogen concentration is determined by measuring proteolytic activity against a synthetic substrate, resulting in an increase in absorbance at 405nm.
This assay is insensitive to plasma inhibitors and fibrin or fibrinogen degradation products. During thrombolytic treatments it is of interest to measure the plasminogen level to monitor the hepatic regeneration level and to control and adjust the perfusion rate if plasminogen is being given to the patient. The plasminogen level in newborn babies is usually low, particularly if they are premature. Falsely low plasminogen activity may be obtained in patients undergoing treatment with Aprotinin.
Congenital Disorders:
Plasminogen Activator Inhibitor-1:
Congenital plasminogen activator inhibitor-1 (PAI-1) deficiency is an extremely rare disorder characterized by a bleeding diathesis that begins in childhood due to hyperfibrinolysis as a result of decreased PAI-1 activity. Deficiencies of PAI-1 inhibitor leads to excessive tissue plasminogen activator activity resulting in excessive plasmin activity. Patients have a history of recurrent episodes of subcutaneous bleeding beginning in early childhood. These episodes are characterized by abnormal prolonged bleeding after trauma, tooth extraction, and surgical procedures, as well as by rebleeding following initial hemostasis. The euglobulin lysis times can be shortened as compared with those in normal control subjects. The Euglobulin Clot Lysis time is used to evaluate increased fibrinolytic activity. The euglobulin fraction of plasma consists of fibrinogen, plasminogen and the activators of plasminogen. This factor is isolated from plasma by precipitation with 1% acetic acid. This euglobulin fraction is relatively free of fibrinolytic inhibitors.
Alpha-2 antiplasmin:
Alpha-2-antiplasmin is also known as alpha-2-plasmin inhibitor or antiplasmin. This is a single-chain glycoprotein with a molecular weight of 70,000 which is produced in the liver. When alpha-2-antiplasmin is absent or present at low concentrations, excessive bleeding may occur. The fibrinolytically active enzyme plasmin with which it extremely rapidly forms an irreversible, inactive complex. Diminished activities of alpha-2 are found in hyperfibrinloysis, which can occur as a complication of disseminated intravascular coagulation (DIC) or in operations on organs with a high content of plasminogen activators. A deficiency may be due to a disturbance in synthesis (for example severe liver cell damage) as well as the additional assessment of problematic cases in fibrinolytic therapy. Homozygotes will have <10% of activity and present with mucosal membrane bleeding particularly in the GI tract, subcutaneous hematomas, spontaneous bruising and severe bleeding in trauma. Heterozygotes are usually asymptomatic but may have mild bleeding tendencies. Inhibiting plasmin results in excess fibrinolysis and causes lysis of fibrin thrombi at sites of vascular injury.
The fibrinolytic system is an important part of hemostasis. This complex process can not be overlooked in it's role regarding the balance between bleeding and clotting.
Donna Castellone
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About the Author
Donna Castellone,
MS, MT(ASCP)SH
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