Structural and functional characterisation of the serpin; α2-Antiplasmin
thesisposted on 13.01.2017 by Sofian, Trifina
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α2-Antiplasmin (α2-AP) is a member of the serpin family and the principal physiologic inhibitor of plasmin, a proteinase that digests fibrin, the main protein constituent of blood clots. Alignment with other serpins members reveal α2-AP not only possess the conserved serpin core domain, but also consist of unique N- and C-terminal extensions. This study aims to structurally and functionally characterise the α2-AP molecule. A set of recombinant α2-AP proteins were produced using a bacterial expression system. One human recombinant protein; r-hAP and four murine proteins were expressed; full-length (r-FL-mAP), N-terminally truncated Δ1-43 (r-ΔN-mAP), C-terminally truncated Δ410-464 (r-ΔC-mAP) and N- and C-terminally truncated Δ1-43/Δ410-464 (r-ΔN/C-mAP). Crystallographic studies were undertaken and the 2.65Å X-ray crystal structure of r-ΔN-mAP is presented as a published journal article. Biophysical studies using chemical and thermal denaturation experiments reveal truncation of the N-terminal extension resulted in a decrease in TM by ~5°C. This suggests that a disulphide bond constraining the N-terminal extension plays a role in the thermostability of α2-AP. Biochemical studies revealed C-terminally truncated α2-AP (r-ΔC-mAP and r-ΔN/C-mAP) resulted in a ~40-fold decrease in the ka with plasmin. To narrow down the region within the C-terminal extension that was important in the inhibitory reaction with plasmin, overlapping peptides corresponding to the human α2-AP C-terminal extension were produced with an N-terminal cysteine; Peptide 1 (410-433), Peptide 2 (428-447) and Peptide 3 (442-464). Corresponding sheep polyclonal anti-peptide antibodies were produced and purified. The effect of blocking human α2-AP C-terminal extension using these peptides and antibodies were tested in kinetic assays involving r-hAP and human plasmin. Antibody 3 and Peptide 3 showed the greatest effect in slowing down the interaction of r-hAP with plasmin (~10-fold decrease in the ka). However, when they were tested in human plasma clot lysis experiments, Peptide 3 exhibited an anti-fibrinolytic effect. All three C-terminal antibodies produced a pro-fibrinolytic effect, with Antibody 3 showing the most profound effect with ~50% shortening in clot lysis times compared to buffer control. The effect of blocking the extreme C-terminus of Antibody 3 was studied in a thromboplastin-induced murine model of venous thromboembolism. The effect of systemic administration of thromboplastin resulted in massive pulmonary embolism accompanied by sudden death. Treatment with a bolus dose of 1mg/kg of tissue-type plasminogen activator (n=3) failed to rescue the mice from lethal thromboembolism. Treatment with two different doses of Antibody 3 (9mg/kg and 90mg/kg) also failed to improve survival ratios. Taken together, when compared to the human disease this animal model represents the most severe end of the clinical spectrum and may not be suitable in testing the fibrinolytic effects of Antibody 3. This study suggests that the extreme C-terminus of α2-AP (the last 20 amino acids) may be a useful therapeutic target for the treatment of thrombotic diseases. However, future in vivo testing of Antibody 3 still needs to be undertaken in an appropriate animal model of thrombosis whereby the fibrinolytic system can be sufficiently activated.