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A Practical Guide to Laboratory Haemostasis

 

Tissue-type Plasminogen Activity [T-PA] Assays



Introduction

t-PA (tissue plasminogen activator) is a serine protease and the main activator of plasminogen to plasmin (the other is urinary plasminogen activator) and is, therefore, a vital component of the fibrinolytic system. It is mostly found on endothelial cells where it acts to limit the propagation of clots in areas of intact vasculature. T-PA is released continuously into the blood and is cleared equally rapidly by the liver. The T½ of t-PA is approximately 5 minutes. Activation of plasminogen to plasmin is slow/insignificant in the absence of a fibrin clot but fibrin strands offer binding sites for both plasminogen and t-PA and bring t-PA and its substrate into close proximity at the site of clot formation.
t-PA activity is inhibited by PAI-1 (plasminogen activator inhibitor-1) and most circulating t-PA is bound to this inactivator.
Elevated levels of t-PA have been identified as a risk factor for cardiovascular disease and stroke though the strength of this association is modified by PAI-1 levels.
The activity of t-PA is significantly enhanced through proteolytic cleavage by Plasmin at Arg275-Ile276 to generate a two chain molecule.


Principles

Laboratory evaluation of t-PA levels is complicated by fluctuation of t-PA levels in blood (acute phase reactant), as well as fluctuating levels of PAI-1 (acute phase reactant, diurnal variation), which binds and inactivates t-PA 1..

The ELISA assay for t-PA has the advantage of being unaffected by the interaction of t-PA with its inhibitors e.g. PAI-1 and which may continue following sample collection.

Functional Chromogenic (indirect determination through a plasmin-mediated reaction) or bio-immunoassay.

Method

1. Immunological Assays - Commercial kits are readily available for measuring t-PA antigen.

Functional assay general points: Fibrinolytic activity is influenced by multiple factors and accurate results of functional assays require maximal standardisation of blood collection. Typically samples should be taken from a rested, fasting subject in the morning with no consumption of cigarettes or alcohol in the hour beforehand. The sample should be taken into a tube containing a stabilisation medium which prevents t-PA and PAI-1 complex formation in vitro.

Bioimmunoassay (BIA): t-PA is complexed onto an immobilised monoclonal anti-t-PA at ph6. After washing any inhibitors are removed and the remainder of the assay proceeds at pH 7-8. Human plasminogen and a chromogenic plasmin substrate are added and then incubated at 37°C. The amount of colour change is measured photometrically and is proportional to the amount of t-PA present.

Acidification of the plasma prevent interference from any PAI-1 present.

Chromogenic assay: These are similar in to the bioimmunoassay except that the t-PA is not antibody immobilised and a fibrin-like stimulator is usually added to augment plasminogen conversion to plasmin by t-PA .

In producing a standard curve for calibration of the bioimmunoassay and functional chromogenic assays pooled human plasma and not recombinant t-PA should not be used as the latter does not give calibration curves suitable for assaying human t-PA .

Interpretation

ELISA: Provides a very reliable quantitation of total t-PA though very high heparin levels may affect the assay.

Functional assay: The presence of other agents which lyse plasminogen will interfere with the test. Heparin binds to t-PA and increases its activity. These factors must be considered when interpreting results. Abnormal tests should always be verified by repeat and abnormal levels should not be diagnosed on the basis of a single abnormal result.

t-PA activity levels are decreased during pregnancy but rise rapidly following delivery.
Anabolic steroids will increase t-PA activity. Similarly t-PA activity is elevated in patients with liver cirrhosis.

interpretation of t-PA assays

Overestimation of t-PA levels Reduced t-PA activity Reduced t-PA antigen  Elevated t-PA activity
Streptokinase, rt-PA and other lytic agents

Heparin [the heparin-tPA complex has increased plasminogen activation activity]
Alcohol [increases PAI-1 levels]

Smoking [increases PAI-1 levels]
Some dietary factors Alcohol [rebound increase in activity ~12 hours later]
DDAVP Exercise Insect venom in patients allergic to venom
Physiological and psychological stress
Oral contraceptives Venous occlusion
Pregnancy 
Anabolic steroids
Liver cirrhosis


Reference Ranges

Local normal ranges should be determined in general are in the region of:
t-PA antigen 1-20 ng/ml
t-PA activity 0.2-2 IU/ml

 

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.