Laboratory Evaluation of the Fibrinolytic System

Laboratory Evaluation of the Fibrinolytic System Dorothy M. Adcock, MD

Volume 18, Number 2 March-April 2004 Objective: The reader will be able to identify the components of the fibrinolytic system and the appropriate assays to identify abnormalities of this system.

Hemostasis represents a balance between fibrin clot formation and dissolution. The procoagulant factors function in concert to generate a fibrin clot while lysis of the fibrin clot is dependent on the fibrinolytic system. Also referred to as the fluid phase of the plasminogen system, this fibrinolytic pathway is responsible for fibrin degradation primarily within the intravascular space. Degradation of extracellular matrix proteins within tissues is a function of the tissue phase of the plasminogen system (see the Jan-Feb issue of CHR). The components of the fibrinolytic system include the primary proteolytic enzyme of this system, plasmin, plasminogen activators, a plasminogen activator inhibitor and an inhibitor of plasmin. To provide regulated action of this pathway, this system is under tight control at a variety of points through a number of different mechanisms. Plasmin is a powerful trypsin-like enzyme and can degrade a variety of proteins other than fibrin, such as procoagulant factors and complement proteins. Due to its broad and potent actions, plasmin must be carefully regulated. It has a very short half-life, in the realm of 0.1 seconds. The protein does not circulate in its active state, but rather is generated in the liver as a zymogen or inactive precursor called plasminogen. Plasminogen is converted to plasmin through the action of a plasminogen activator, either tissue plasminogen activator (tPA) or urokinase-like plasminogen activator (uPA). It is ironical that the primary plasminogen activator in the intravascular space is tPA. The basis of this localization is dependent on the requirement for tPA to function most effectively in physical proximity to fibrin. tPA must bind fibrin in order to efficiently convert plasminogen to plasmin. tPA is stored within the endothelial cell and is released in response to a number of stimuli, such as venous occlusion, thrombin generation, bradykinin and desmopressin. Plasminogen activators are under the control of plasminogen activator inhibitors (PAI) of which PAI-1 is the most important physiologically. PAI-1 circulates in excess of tPA. PAI-1 is found in plasma as well as within platelets. PAI-1 is stored in the alpha granules of platelets and is released upon their activation.

CLINICAL HEMOSTASIS REVIEW 3176 S. Peoria Ct, Aurora, CO www.esoterix.com CLINICAL ADVISORS Dorothy M. Adcock, MD CONTRIBUTOR Monica Thibault

Once plasmin is generated, it is rapidly bound and inactivated by alpha-2 antiplasmin. Another means in which the fibrinolytic system is modulated is through the action of thrombin activatable fibrinolysis inhibitor (TAFI). TAFI is produced in the liver as an inactive precursor and is activated by thrombin as well as by trypsin and plasmin. Once activated, TAFIa protects the fibrin clot from lysis by diminishing the action of tPA. This occurs through TAFIa induced cleavage of C-terminal lysine residues on fibrin that are necessary to enhance the rate of plasmin formation. Physiologically, the amount of thrombin generated plays a pivotal role in TAFI activation because high concentrations of thrombin are required to activate TAFI and attenuate fibrinolysis. In situations where thrombin generation is reduced, such as in deficiencies of factors VIII, IX or XI, there may be insufficient thrombin generation to adequately catalyze TAFI. Diminished TAFIa could result in decreased stabilization of the fibrin clot and an enhanced bleeding potential. On the other hand, sustained thrombin generation as may occur with increased levels of the intrinsic clotting factors may lead to prolonged down-regulation of fibrinolysis, resulting in impaired clot lysis and increased thrombotic risk.

CLINICAL IMPLICATIONS OF ABNORMALITIES OF THE FIBRINOLYTIC SYSTEM Clinical Hemostasis Review is published by Esoterix and is circulated to selected physicians Copyright 2004. Esoterix is a leading laboratory services company providing esoteric testing in numerous disease corridors. The opinions expressed in the articles are those of the author(s) and do not necessarily reflect the opinions or recommendations of the advertisers, editors, or publisher. The publisher reserves copyright and renewal on all published material and such material may not be reproduced in whole or in partwithout written permission from the publisher. Consult the full prescribing information on any drug or devices discussed. All correspondence should be directed to the attention of the Editor, Clinical Hemostasis Review, 3176 S. Peoria Ct, Aurora, CO 80014

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Abnormalities of the Fibrinolytic System Abnormalities of the fibrinolytic system can lead to either a bleeding or thrombotic tendency. Impaired fibrinolysis due to insufficient release of tPA, deficient plasminogen, increased PAI-1 or TAFI levels may lead to a thrombotic tendency manifested as venous or arterial thrombosis. Enhanced fibrinolysis due to a deficiency of TAFI, PAI-1 or alpha-2 antiplasmin may lead to a hemorrhagic diathesis.

Plasminogen Hereditary plasminogen deficiency is a rare disorder that occurs most commonly in individuals of Japanese ancestry. It is estimated that approximately 50% of plasminogen deficiencies are type I and the remainder are type II. Homozygous deficiency may be associated with an increased risk of venous thrombosis, while it is not yet certain that a heterozygous plasminogen deficiency increases risk. Due to the inability to generate adequate plasmin, severe plasminogen deficiency may be associated with abnormal deposition of fibrin within tissues. When fibrin deposition occurs, it localizes to the subepithelial space. These fibrin deposits incite an inflammatory reaction and this reaction, in concert with the deposited fibrin, forms wood-like (ligneous) pseudomembranes. The most commonly described clinical condition associated with severe plasminogen deficiency is ligneous conjunctivitis. In this condition, fibrin deposits form on the palpebral surfaces (eyelids) and this can ultimately lead to blindness. Some patients with ligneous conjunctivitis have been successfully treated with topical plasminogen. While some patients with severe plasminogen deficiency develop deposition of fibrin in the eyes only, fibrin deposition can also occur in the mucosa of the oropharynx and nasopharynx, the ear and in the female genital tract. Ligneous cervicitis is an uncommon cause of infertility. Ligneous deposits have been described in severe type I plasminogen deficiency only and not in type II deficiencies. Clinically significant excess in plasma plasminogen concentration has not been described.

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PAI-1 Deficiency PAI-1 deficiency is a rare disorder associated with a lifelong bleeding diathesis in the homozygous individual. Rather than spontaneous bleeding, hemorrhage tends to occur only following trauma or surgery. Heterozygous PAI-1 deficient individuals are asymptomatic. Homozygous deficiency of PAI-1 can reflect a quantitative or qualitative disorder. A quantitative deficiency can be detected with an antigen assay whereas determination of a qualitative deficiency requires an activity assay. The problem detecting a qualitative PAI-1 deficiency is that normal individuals can have near zero PAI-1 levels. These levels are generally below the assay’s lower limit of quantitation. For this reason, it can be difficult to distinguish normal PAI-1 activity levels from PAI-1 deficiency. PAI-1 exists in plasma in three forms; active, latent (inactive) and complexed to tPA. The transition between active and latent forms occurs spontaneously and may, in part, explain why normal individuals may have near zero PAI-1 activity levels. In order to diagnose a deficiency of PAI-1 activity, it may be necessary to perform a vascular challenge study by either applying venous occlusion or administering desmopressin and evaluating PAI-1 activity levels at baseline and following the challenge. In individuals who are PAI-1 deficient, the euglobulin lysis time is frequently abnormally decreased.

PAI-1 Excess PAI-1 is a critical regulatory protein of the fibrinolytic system as it rapidly binds and inhibits both tPA and uPA. PAI-1, therefore, is an important inhibitor of clot lysis. PAI-1 is synthesized and released by a variety of cell types including hepatocytes, endothelial cells, vascular smooth muscle cells and adipocytes. Inflammatory cytokines, as well as thrombin and insulin, can stimulate the release of PAI-1. PAI-1 levels may be increased for a number of reasons. PAI-1 reportedly elevates as an acute phase reactant protein. PAI-1 levels may be elevated in individuals with a polymorphism of the PAI-1 gene, namely the 4G/5G polymorphism. Plasma PAI-1 levels have been shown to be higher in individuals with the 4G/4G genotype. PAI-1 levels are typically increased in individuals with metabolic X syndrome and likely correlate with the degree of hyperinsulinemia. Elevated PAI-1 levels have been associated with an increased incidence of both venous and arterial thrombotic events including deep venous thrombosis and myocardial infarction.

Tissue Plasminogen activator tPA is the primary intravascular plasmin activator and is the major initiator of intravascular fibrinolysis. Synthesized in endothelial cells, tPA is released in response to physical exercise, venous occlusion, bradykinin, catecholamines and desmopressin. tPA shows circadian variation in plasma levels with lowest values in the early morning hours. This may in part explain why most myocardial infarctions occur during this time of day. Severe deficiency of tPA has not been described in humans. Impaired tPA release following appropriate stimuli has, however, been reported to increase the risk of venous thrombosis. Diagnosis can be made only after comparing baseline tPA levels with levels following some sort of stimulation such as venous occlusion or administration of desmopressin. Excess of endogenous tPA has not been described as a cause of bleeding.

Alpha–2 Antiplasmin Alpha-2 antiplasmin regulates and inactivates plasmin activity by forming a 1:1 complex. Congenital deficiency of alpha 2-antiplasmin is rare and expressed as a severe bleeding diathesis in the homozygous state. In these individuals, alpha-2 antiplasmin levels are typically less than 15%. Patients typically present with bleeding from birth, usually from the umbilical stump. Other manifestations include hemarthrosis, muscle bleeding, soft tissue hematomas and prolonged bleeding from wounds. An unusual manifestation associated with severe alpha- 2 antiplasmin deficiency is bleeding into the intramedullary space of the long bones (Miyauchi’s syndrome). Heterozygous deficients usually have levels in the range of 35 to 70% and are asymptomatic or suffer only a mild bleeding disorder. This may manifest as menorrhagia, easy bruising or excessive post-surgical bleeding. Acquired deficiency can occur in systemic amyloidosis, severe liver disease and in those with nephrotic syndrome. Deficiency of alpha-2 antiplasmin may occur as a quantitative abnormality and in this instance can be diagnosed with either an antigen or activity assay. Because severe qualitative deficiencies of alpha-2 antiplasmin have been described, screening should be performed with an activity assay. Elevated levels of alpha-2 antiplasmin have not been reported clinically as a cause of thrombosis.

TAFI The physiological importance of TAFI in humans is not fully realized. Decreased levels of functional TAFI or decreased TAFI activation due to diminished thrombin generation may be a cause of a hemorrhagic tendency or enhance the severity of bleeding in patients with inherited or acquired bleeding disorders. Increased levels of TAFI may be a risk factor for thrombosis. Elevated TAFI levels have been associated with an increased risk of venous thrombosis while the relationship with arterial clot formation is controversial. The Leiden Thrombophilia Study

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reported a 2-fold increase in the risk for developing venous thrombosis in those with TAFI levels greater than 2 standard deviations above normal.

LABORATORY ASSAYS Global Assays for Fibrinolysis Many global assays that measure fibrinolytic activity have been described over the years. All these methods measure clot lysis kinetics using differing formats and sample types. For example, clot lysis methods have been described using whole blood, diluted whole blood, euglobulin fractions, fibrin plates and plasma clots. All clot lysis procedures require many hours for a measurement. Of these assays, euglobulin lysis, fibrin plate lysis and plasma clot lysis are those used most often and perhaps are the most informative. The measurement of clot lysis can be monitored using automated turbidimteric measurement, or as in the past, visually. In the euglobulin clot lysis assay, the test plasma is acidified to precipitate the euglobulin fraction of plasma. This fraction contains fibrinogen, plasminogen, plasmin and plasminogen activators. Importantly, antiplasmin is not part of the precipitate. The precipitate is redissolved and recalcified to produce a clot. The clot lysis kinetics is a measure of the fibrinolytic activity of the sample. Recently, it has been found that variable amounts of plasminogen inhibitors and PAI-1 are also precipitated, complicating the interpretation of euglobulin lysis times. Lysis time is prolonged by decrease in tPA, increase in PAI or by a plasminogen defect. Lysis is abnormally short with PAI-1 deficiency, alpha-2 antiplasmin deficiency, excess plasmin or excess tPA. Samples with very high or very low fibrinogen levels must be interpreted cautiously. The fibrin plate method, measures the ability of plasma or the euglobulin fraction to lyse a fibrin layer in a plastic plate. The fibrin plates are preformed using added commercial fibrinogen and plasminogen. The advantage of fibrin plates is that the clots formed are uniform in composition, especially with respect to plasminogen and fibrinogen levels. Conversely, the consistency of results using fibrin plates is very dependent on the purity of reagents used to form the fibrin clots; for example, fXIII, which promotes clot strength, is present at varying levels in commercial fibrinogen. Differences in factor XIII levels may vary the lysis time of fibrin plates. Plasma clot lysis assays have been used to assess the fibrinolytic activity of a sample. In this procedure, diluted plasma, supplemented with fibrinogen and plasminogen is clotted using thrombin and calcium. tPA is included in the clot and the clotting and lysis kinetics are followed using turbidimetric measurements. Amongst the assay types described, the plasma clot lysis assay may be the most precise and reproducible assay. This version of the assay has been used to test manufactured lots of therapeutic thrombolytic agents such as streptokinase and recombinant tPA. As discussed above, TAFI is an important regulator of fibrinolysis and is activated by increased levels of thrombin. Hence, thrombin generation may be an important determinant of fibrinolytic resistance. The plasma clot lysis assay has also been used in studying the effects of anti-fXa inhibitors and differences have been noted in the extent of clot lysis obtained using various anti-fXa compounds.

Alpha-2 Antiplasmin Activity - Assay Principle Diluted plasma is incubated with a precise excess of plasmin, resulting in rapid complex formation between plasmin and functional alpha-2 antiplasmin in the sample. The complex formed, plasmin-antiplasmin (PAP), is inactive. The extent of plasmin inhibition is directly proportional to antiplasmin levels in the sample. The residual active plasmin hydrolyzes the plasmin chromogenic substrate and liberates a pNA chromophore which is then read spectrophotometrically at 405 nm. Color intensity is inversely proportional to alpha-2 antiplasmin activity in the sample. The reference range by chromogenic assay for alpha-2 antiplasmin activity in plasma is 80 - 150%.

Plasminogen Activity - Assay Principle Plasminogen in the test sample is activated by the addition of excess streptokinase to form a plasminstreptokinase complex. This complex reacts with a chromogenic substrate much like in the alpha-2 antiplasmin assay for plasmin resulting in color production. The amount of color formation correlates to the plasminogen activity in the sample. The reference range for plasminogen activity in plasma is 70 – 130%.

Plasminogen Antigen - Assay Principle Plasminogen in the test plasma is combined with antibody to plasmin that also cross reacts with plasminogen. The immune complexes that form, scatter a beam of light passed through the sample. The intensity of the scattered light is proportional to the concentration of plasminogen antigen in the sample. The reference range for plasminogen antigen is 7.5 – 18.5 mg/dL.

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Plasminogen Activator Inhibitor-1 Activity Assay Principle PAI-1 present in the test sample complexes with active t-PA bound to the surface of a microtiter plate test well. The bound PAI-1 is then quantitated using horseradish peroxidase (HRP)-conjugated monoclonal anti-PAI-1. A HRP substrate is then added and color development is proportional to the concentration of PAI-1 in the sample. The reference range for PAI-1 activity in plasma is