Fibrinolysis

 
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Fibrinolytic pathway tests measure of clot breakdown products, FDPs and D-dimer

Fibrinolytic pathway tests measure of clot breakdown products, FDPs and D-dimer

Tests for fibrinolysis involve evaluation of the fibrinolytic pathway. Unfortunately, measurement of many components of the fibrinolytic pathway, including plasminogen, tissue plasminogen activator, plasminogen activator inhibitor, have not been developed or validated in animals, or those that have, work poorly. Therefore, these tests are not offered routinely, which is a major shortcoming when evaluating animals for hemostatic or thrombotic disorders. The most common tests of fibrinolytic activity are the tests for fibrin(ogen) degradation products, including D-dimer.

Other tests that are not commonly used are the clot lysis time and the euglobulin lysis time. The clot lysis time is the time required for whole blood clots to lyse at 37 ºC (and can be performed after the clot retraction time) and is dependent on plasmin activity, fibrinogen concentration and the degree of clot retraction. Poor clot retraction will impede the lysis time. The clot is usually lysed within 8 to 20 hours in animals. The euglobulin lysis time is a measure of plasmin activity. In this test, euglobulins (fibrinogen, plasminogen, plasmin, plasminogen activator) precipitate on dilution with water, whereas fibrinolytic inhibitors (plasminogen activator inhibitor, antiplasmin) do not. The removal of these inhibitors allows the plasmin in the sample to lyse the precipitate with the time taken for lysis being the euglobulin lysis time. This test is quite crude and is not offered by most laboratories.

Fibrin(ogen) degradation products (FDP)

The FDPs, fragment D and E are end products of cleavages from fibrinogen/fibrin.

The FDPs, fragment D and E are end products of cleavages from fibrinogen/fibrin.

This assay detects the presence of circulating fragments (FDPs) of fibrinogen and soluble (non-crosslinked) fibrin that are produced by the action of plasmin on these substrates. Plasmin acts on these two substrates similarly, producing an initial cleavage product called fragment X. Plasmin then acts on fragment X, cleaving it into a transient fragment Y, and fragment D. Further cleavage of fragment Y, produces the terminal fibrin(ogen) degradation products, fragment D and E. Thus from one molecule of fibrinogen or soluble fibrin, two terminal FDP fragments D and one terminal fragment E is produced.

There are assays for FDPs in both serum and plasma. Serum FDP assays use polyclonal antibodies that cross-react with intact fibrinogen (that has not been lysed by plasmin), necessitating its removal. This is accomplished by the use of specialized collection tubes containing Botrox atrox venom (Reptilase) and inhibitors of fibrinolysis (aprotonin or soyabean trypsin inhibitor). All serum FDP assays use latex beads that are coated with antibody raised against human fibrin(ogen) degradation products, usually fragments D and E. The antibodies cross-react well enough with the fibrin(ogen) fragments of animals to make the test useful for veterinary patients. 2 mLs (as little as 0.5 mls can be used if the patient is very small) of blood from the patient is put immediately into one of the tubes provided with the test kit.

Serum from the tube is diluted 1:5 and 1:20. A small volume of each dilution is mixed on a plate with an equal volume of the antibody-coated latex beads. Positive and negative control sera are assayed concurrently. Agglutination of the beads at the 1:5 dilution indicates an FDP concentration of 10-40 µl/mL, whereas agglutination at both 1:5 and 1:20 dilutions indicates an FDP concentration >40 µl/mL. Either positive result is abnormal and indicates a higher than normal rate of degradation of fibrinogen and/or fibrin by plasmin or decreased clearance of these products.

Tube used for FDP assay

Tube used for FDP assay

There are newer latex-agglutination kits that are based on monoclonal antibodies that do not cross-react with intact fibrinogen and can thus be used on citrated plasma samples. This is advantageous compared to serum FDP assays, as a specialized collection tube for FDP assay is not required and a single citrated blood sample can be used for all coagulation tests. This is the procedure performed by the Clinical Pathology laboratory at Cornell University and it has only been validated for the dog. The plasma FDP assay is performed similarly to the serum FDP assay, except dilutions of 1:2 and 1:8 are made, with results being reported as < 5 µl/mL, 5-20 µl/mL and > 20 µl/mL. Results > 5 µl/mL are abnormal.

The FDP assay is used with the other coagulation tests to characterize bleeding disorders more completely. The results should never be interpreted alone, without evaluation of clinical signs and results of other coagulation tests. Any cause of pathologic intravascular coagulation (the most common of which is DIC), thrombosis, or severe internal hemorrhage can produce FDP’s. In addition, any condition causing decreased clearance of FDPs by the liver and monocyte-macrophage system, e.g. severe liver disease, can also result in increased FDP values. A negative FDP result does not rule out these processes.

Illustration of a plasma FDP assay

Illustration of a plasma FDP assay

Note that coagulation can also occur in extravascular tissue spaces and high FDPs have been associated with fibrin(ogen)olysis occurring in protein-rich or hemorrhagic effusions into body cavities. Indeed, horses with colic due to gastrointestinal disorders have higher FDPs in blood and peritoneal fluid than healthy control horses. Although the high FDPs in the blood of these colic horses are compatible with DIC (which is often subclinical in horses and results in thrombosis rather than hemorrhage), the FDP values in their peritoneal fluid were higher than in blood, suggesting extravascular production of the FDPs (probably secondary to inflammation-induced tissue factor expression and fibrinolysis in the peritoneal cavity). This could have partly contributed to the high blood FDPs.

The serum FDP assay is of little use when evaluating horses for coagulopathies because of the high frequency of false positive results. Sera from most horses, whether healthy or ill, give positive tests with this assay. A single plasma assay has been evaluated and does not appear to crossreact with equine FDPs (all these latex agglutination assays use antibodies raised against human FDPs).

D-dimer

D-dimer is a specific plasmin-mediated breakdown product of crosslinked fibrin. Thrombin converts fibrinogen into soluble fibrin monomer. This monomer then spontaneously polymerizes to form the soluble fibrin polymer. Thrombin also activates factor XIII, which in the presence of calcium, crosslinks the fibrin polymer, producing crosslinked fibrin. Plasmin cleavage of fibrinogen or soluble fibrin produces the “traditional” FDPs, fragments X, Y, D and E. Plasmin cleavage of crosslinked fibrin produces different degradation products, that vary in molecular weight and are called X-oligomers. D-dimer is a specific neoantigen produced by the factor XIIIa-mediated crosslinking of fibrin and is exposed after plasmin degrades crosslinked fibrin, allowing it to be detected by immunologic-based assays. Note that although plasmin is the main fibrinolytic enzyme, proteolytic enzymes released by neutrophils can also degrade crosslinked fibrin exposing D-dimer.

The process of D dimer production

The process of D dimer production

Thus, D-dimer is more specific for fibrinolysis than FDPs, as its formation requires the action of thrombin (to activate factor XIII) to produce crosslinked fibrin and the cleavage of this fibrin by plasmin. In contrast, traditional FDP assays cannot distinguish between plasmin action on fibrinogen (fibrinogenolysis) and fibrin (fibrinolysis), therefore FDPs can be elevated when there is no clot present (and plasmin is just cleaving fibrinogen).

D-dimer can be detected in human patients with assays using monoclonal antibodies specific for the human D-dimer epitope. Some of these monoclonal antibodies crossreact with some animal species and can be used for veterinary patients. Certain D-dimer latex agglutination assay have been validated in the dog, cat and horse. The test is run similarly to the FDP assays, but the sample can be assayed undiluted (to obtain a positive or negative result) or can be serially diluted to obtain a semi-quantitative D-dimer value. It is far better to get a semi-quantitative D-dimer result, because higher values may be more specific for thrombembolic conditions.

Normal values

Normal dogs and cats have D-dimer values < 250 ng/mL whereas most healthy horses have D-dimer values < 500 ng/ml (but D-dimer can be as high as 1000 ng/mL in the horse).

Increased D-dimer

D-dimer will be increased whenever there is activation of thrombin, to form crosslinked fibrin and fibrinolysis, i.e. thrombosis and fibrinolysis. The prototypical thromboembolic disease is disseminated intravascular coagulation (DIC) and D-dimer is often very high in this disorder (indeed, D-dimer is quite sensitive for DIC and values may increase in early DIC before any other coagulation assays, such as the PT and aPTT, become abnormal). However, any disorder resulting in crosslinked fibrin formation and breakdown can potentially elevate D-dimer (i.e. high D-dimer is not specific for DIC). This includes physiologic (e.g. associated with surgical wound-healing) and pathologic fibrinolysis (associated with thrombosis of any cause, e.g. pulmonary thromboembolism).

Illustration of a latex agglutination D-dimer assay

Illustration of a latex agglutination D-dimer assay

It should be noted that coagulation and fibrinolysis are not restricted to the intravascular space. If thrombin is activated in extravascular tissues in the presence of fibrinogen, e.g. protein-rich or hemorrhagic effusions into body cavities (e.g. joints, thoracic cavity, central nervous system), crosslinked fibrin could conceivably form in these extravascular sites. Activation of plasmin or release of proteolytic enzymes from neutrophils within these sites could degrade this crosslinked fibrin, releasing D-dimer into the fluid. This could potentially be reaborbed into blood, elevating plasma D-dimer levels.

Species-specific interpretation

  • DogsD-dimer values are often very high (> 1000 ng/mL) in dogs with documented thrombo-embolic disease, including DIC. Indeed, D-dimer appears to be a very sensitive indicator of fibrinolysis in dogs with DIC (100% sensitive in one study). However, D-dimer is not specific for DIC or other thrombotic disorders. High values have been reported in dogs secondary to neoplasia, inflammatory disease and hemorrhagic effusions, e.g. hemoperitoneum (although most dogs with hemorrhagic effusions have concurrent disease processes that could independently initiate DIC). Thus, D-dimer indicates fibrinolysis, regardless of whether this is physiologic (i.e. associated with surgical wound healing) or pathologic (associated with disease) and is not specific for DIC. D-dimer has been evaluated in the cerebrospinal fluid (CSF) as a marker of prior hemorrhage into the central nervous system. One study showed that D-dimer values were increased in the CSF of dogs with evidence of hemorrhage, however values were still below the limit of detection of the latex agglutination assays (< 250 ng/mL) limiting the usefulness of this test.
  • Cats: D-dimer has been evaluated in cats with cardiac disease, who are predisposed to aortic thromboembolism. However, D-dimer values were similar between healthy control cats and cats with cardiac disease, arguing against its usefulness to predict thrombosis in cats with cardiac disease. We have seen high D-dimer levels in cats with conditions associated with DIC, e.g. feline infectious peritonitis virus infection, suggesting that D-dimer may still be useful to detect thrombosis in some feline diseases.
  • Horses: D-dimer is increased in plasma in horses with severe colic and is a sensitive diagnostic test for the presence of underlying DIC. In some studies, a high D-dimer was a negative prognostic indicator for outcome.

Related links

  • Further information is available on the D-dimer assay offered by the Animal Health Diagnostic Center’s Comparative Coagulation Laboratory of Cornell University.
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