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A Practical Guide to Laboratory Haemostasis

 

Plasminogen Activator Inhibitor Type 1 [PAI-1] Assays



Introduction

Plasminogen Activator Inhibitor Type 1 (PAI-1) is a serine protease inhibitor, secreted largely by endothelial and adipose cells and the major inhibitor of t-PA (tissue plasminogen activator) and urokinase. During pregnancy Plasminogen Activator Inhibitor Type 2 (PAI-2) is secreted by the placenta with the same function as PAI-1. PAI-1 also plays a role in extracellular matrix remodelling including angiogenesis through its regulation of plasminogen. It may correlate with prognosis in certain forms of cancer, probably because of this role. PAI-1 levels may also be a risk factor for cardiovascular disease and post-operative thrombosis risk. Currently, however, PAI-1 levels do not have clear clinical relevance – i.e. they do not form the basis of therapeutic decision making or useful prognostic information in routine clinical practice. 

Principles & Methods

Laboratory evaluation of PAI-1 is complicated by its role as an acute phase reactant and its’ interaction with t-PA, levels of which may also fluctuate under a wide variety of conditions.

Assay  
ELISA The ELISA assay has the advantage of being unaffected by t-PA–PAI-1 complex formation.

A commonly employed ELISA assay involves the immobilisation of functionally active t-PA to microtitre plates using a monoclonal antibody. PAI-1 contained in the test sample binds to the immobilised t-PA and is then quantified using a peroxidase-la be led monoclonal anti-PAI-1 antibody. Many of these assays are specific for PAI-1 and will not detect PAI-2 e.g. in pregnancy.
Functional Assays These rely on measuring the amount of residual t-PA or urokinase activity in a sample containing a known excess of t-PA or urokinase to which the patient’s plasma has been added. The most common methods use a chromogen to quantitate residual t-PA activity.

1. Latex Agglutination: An automated latex agglutination immunoassay (LPIA) has also been described. Latex microparticles coated with anti-PAI-1 antibody are mixed with patient plasma and agglutination (which is proportional to the amount of PAI-1 antigen present) measured photometrically.

2. Bioimmunoasay [BIA]: A microplate is coated with t-PA The patient sample is incubated with this and any PAI-1 present binds to the immobilised t-PA After washing a solution containing enzyme bound anti-PAI-1 antibody is incubated with the sample. After washing the enzyme substrate is incubated with the sample and the activity measured (e.g. by a chromogenic method).
The amount of enzyme activity detected is proportional to the concentration of PAI-1 in the patient sample. The BIA for t-PA can be performed at acid pH to eliminate PAI-1 interference then again at alkaline pH to permit PAI-1 interference. The ratio of measured t-PA activity reflects the amount of PAI-1 present.

3. Chromogenic assay: This is a two-stage process. In the first stage, a known excess quantity of t-PA is incubated with patient plasma. This permits PAI-1-tPA complexes to form. In the second stage a t-PA stimulator such as poly-D-lysine is added and the sample incubated with a chromogenic plasmin substrate. The colour change is proportional to the amount of t-PA activity in the sample which is, in turn, proportional to the PAI-1 activity in the test sample.

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

A PAI-1 chromogenic detection method with the additional step of using venom from the Bitis arietans viper to eliminate the plasmin inhibitors α2-antiplasmin and α2-macroglobulin has been described with the claim that this improves assay accuracy.
Nonetheless, this step is not used in modern PAI-1 chromogenic detection commercial kits.

Plasma contains a number of anti-plasmins e.g. a2-AP and a2-macroglobulin and these require to be removed from the plasma before a 'true' functional PAI-1 assay can be undertaken.


Interpretation

ELISA Assays: These provide a very reliable quantification of total PAI-1 antigen

Functional assays:
1. BIA: This measures PAI-1 activity in the sample but is less subject to interference from other variables than the standard chromogenic plasmin generation method. This is the most widely used functional assay.

2. Plasmin generation chromogenic assay: The presence of agents which lyse plasminogen will interfere with the test. Heparin binds to t-PA and increases its activity. Agents interfering with plasmin may also interfere with the result. 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.

Reference Ranges

Local normal ranges should be determined.
PAI-1 antigen 5-40 ng/ml
PAI-1activity <15 U/ml
 

Data Interpretation

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