Reduction of thrombus size in murine models of thrombosis following administration of recombinant α1-proteinase inhibitor mutant proteins

Abstract

<jats:title>Summary</jats:title><jats:p>The variant serpin α1-PI M358R inhibits thrombin and other proteases such as activated protein C (APC) and factor XIa. We previously described recombinant proteins HAPI M358R (α1-PI M358R containing an N-terminal extension corresponding to residues 1–75 of heparin cofactor II) and HAPI RCL5 (HAPI M358R with F352-I356 and I360 substituted for the corresponding residues of antithrombin), with enhanced selectivity for thrombin over APC inhibition. We tested the hypotheses that these recombinant proteins would limit thrombosis in three mouse models, and that the HAPI chimeric proteins would be more effective than α1-PI M358R. Recombinant serpins were purified from Escherichia coli by nickel chelate and ion exchange affinity chromatography, and administered to mice intravenously. HAPI RCL5 reduced incorporation of radiolabelled fibrin(ogen) into thrombi in the ferric chloride-injured vena cava in a dose-dependent manner; HAPI M358R was less effective and α1-PI M358R was without effect. In a model of murine endotoxaemia, HAPI RCL5 was more effective than α1-PI M358R in reducing radiolabelled fibrin(ogen) deposition in heart and kidneys; immunohis-tochemistry of tissue sections showed lesser staining with anti-fibrin(ogen) antibodies with both treatments. In the ferric chloride-injured murine carotid artery, administration of both recombinant serpins was equally effective in lengthening the vessel’s time to occlusion. Our results show that the antithrombotic efficacy of the recombinant serpins correlates with their potency as thrombin inhibitors, since HAPI RCL5 inhibits thrombin, but not factors Xa, XIa, XIIa, or neutrophil elastase, more rapidly than α1-PI M358R.</jats:p>

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

“…Others employing different bacterial expression systems, some in which recombinant API was renatured from inclusion bodies, have reported SI values closer to unity [55], [56]. Why the difference arises is not fully understood, but we have demonstrated functionality of API M358R made in this system as an antithrombotic agent in vivo [39], suggesting that it does not have an unnatural fold. Deep sequencing verified that thrombin biopanning enriched many sequences over their abundance in either the naïve library or the mock-panned library, processed through the same five rounds of selection as the thrombin-selected population.…”

“…Recombinant hexahistidine-tagged API proteins expressed by E. coli TOP10 cells transformed with pBAD-H 6 -API plasmids were purified to homogeneity using nickel chelate affinity and ion exchange chromatography as previously described [29], [39]. The rate of reaction of recombinant API proteins was quantified by determining the second order rate constant (k 2 ) of thrombin inhibition in a discontinuous assay, under pseudo first order conditions, as in previous studies from this laboratory [29], [40], [41], as was the stoichiometry of inhibition (SI).…”

The complete N-terminal extension of heparin cofactor II is required for maximal effectiveness as a thrombin exosite 1 ligand

“…In order to express an HCII 1-75-α 1 -PI M358R fusion protein incapable of forming a serpin-enzyme complex with thrombin, the 5153 bp BstXI-EcoRI digestion product of pBAD-H 6 HAPI M358R [19] was combined with the 361 bp fragment formed by digestion of pBAD-API T345R/M358R [24], yielding plasmid pBAD-HAPI T345R/M358R.…”

Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2

“…Mice were anesthetized with 1.5% isoflurane, the vessel exposed, and recombinant protein (12.7 nmol KPI (90 µg/mouse) or KPIHSA (940 µg/mouse, respectively) or saline vehicle was injected intravenously 2 or 60 minutes prior to application of the FeCl 3 -saturated filter paper to the vessel, for 3 minutes. Thirty minutes after removal of the filter paper, clots were excised and g-counted to determine their content of 125 I-fibrin and ( 125 I-fibrinogen ( 125 I-fibrin(ogen)) by g-counting as described above [31, 32]. …”