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A Practical Guide to Laboratory Haemostasis

 

α2-Plasmin Inhibitor [historically known as α2-Antiplasmin]



Introduction

α2-Plasmin Inhibitor [previously known as α2-Antiplasmin [ α2-AP] in conjunction with TAFI and PAI-1 is the primary physiological inhibitor of plasmin and therefore plays a critical role in the regulation of fibrinolysis. α2-PI is synthesised by the liver and also found in the α-granules of platelets and so secreted when platelets are activated.
Two forms of α2-PI circulate in plasma: 1) a 464-residue protein with methionine at the N-terminus [Met-α2-PI] and 2) an N-terminally shortened form [452 amino acids] in which the N-terminal amino acid is an asparagine residue [Asn-α2-PI]. Approximately 30% of the α2-Plasmin Inhibitor in the plasma is found as Met-α2-PI and 70% as Asn-α2-PI. Asn-α2-PI appears to be more physiolocially active than Met-α2-PI.
Fibrinolysis is regulated by α2-Plasmin Inhibitor [α2-PI] in 3 ways:
1. By the formation of a stoichiometric complex with plasmin. α2-PI is much more efficient at inhibiting free plasmin than plasmin bound to the fibrin clot and this allows localised plasmin generation on the fibrin clot while preventing/inhibiting systemic enzyme activation.
2. By the inhibition of plasmin adsorption on the fibrin clot. α2-PI is covalently bound into the fibrin clot by FXIIIa, resulting in increased resistance to fibrinolysis by the fibrin clot.
3. By preventing the binding of plasminogen to the fibrin clot. The C-terminus of α2-Plasmin Inhibitor binds with strong affinity to the lysine binding sites of plasminogen where fibrin also binds. In this way α2-PI prevents plasminogen binding to fibrin.

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The gene for α2-PI [SERPINF2] maps to the short arm of chromosome 17 [17pter-p12] and contains 10 exons and 9 introns. The gene encodes a protein of 51kDa which is synthesised in the liver and circulates either free or bound to plasminogen. The plasma concentration of α2-PI is ~0.7mg/mL with a T½ of 2-6 days.
α2-PI is a member of the SERPIN family of serine protease inhibitors.

Principles & Methodology

α2-Plasmin Inhibitor can be assayed either immunologically or functionally:
1. Immunological α2-PI assays. These are commonly performed using an ELISA assay in which an antibody to α2-PI is immobilised on the microtitre plate and use to capture the α2-PI.
A latex-based method for measuring α2-PI has also been reported.
2. Functional assays:
a. Patient plasma containing α2-PI is incubated with plasmin in excess. The plasmin is rapidly inactivated by the α2-PI and residual plasmin is measured using an amidolytic assay in which the residual plasmin cleaves a chromogenic substrate and the change in colour is detected by measuring the changes in absorption at 405nm. The change in absorption is inversely proportional to the concentration of α2-PI.
b. Plasmin is coated onto a microtitre plate and plasma samples then added to the plate. Functionally active α2-Plasmin Inhibitor binds to and reacts with the plasmin. The wells are washed and an anti-α2-Plasmin Inhibitor added to the wells. The bound antibody is detected using horseradish peroxidase conjugated to a second antibody. The wells are washed and then TMB [a substrate for the horseradish peroxidase] added and the colour change at 405nm is recorded. The amount of colour change is proportional to the the concentration of active α2-PI in the original plasma sample.

Interpretation

Inherited α2-Plasmin Inhibitor deficiency is rare and only a small series of case reports exist in the literature - see references. Its importance lies in the fact that the normal screening tests of coagulation e.g. PT and APTT are normal in cases of α2-Plasmin Inhibitor deficiency.
Patients with homozygous deficiencies of α2-PI usually have values <10% of normal and such cases present with bleeding. Heterozygous individuals with levels of α2-PI in the region of 30-60% are usually asymptomatic.

Reference Ranges

The normal adult reference range is 80-140%. Healthy full-term infants may have borderline-low or mildly decreased levels of α2-PI but these generally reach adult levels by the end of the first week of life.

What Test Next

It is useful to remember that the TEG/ROTEM may be abnormal in cases of suspected α2-PI and may, indeed be a useful screening test for this disorder.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.