Termination of bleeding by a specific, anticatalytic antibody against plasmin

Abstract

Background: Excessive, plasmin-mediated fibrinolysis augments bleeding and contributes to death in some patients. Current therapies for fibrinolytic bleeding are limited by modest efficacy, low potency, and off-target effects.

Objectives: To determine whether an antibody directed against unique loop structures of the plasmin protease domain may have enhanced specificity and potency for blocking plasmin activity, fibrinolysis, and experimental hemorrhage.

Methods: The binding specificity, affinity, protease cross-reactivity and antifibrinolytic properties of a monoclonal plasmin inhibitor antibody (Pi) were examined and compared with those of epsilon aminocaproic acid (EACA), which is a clinically used fibrinolysis inhibitor.

Results: Pi specifically recognized loop 5 of the protease domain, and did not bind to other serine proteases or nine other non-primate plasminogens. Pi was ~7 logs more potent in neutralizing plasmin cleavage of small-molecule substrates and >3 logs more potent in quenching fibrinolysis than EACA. Pi was similarly effective in blocking catalysis of a small-molecule substrate as α₂ -antiplasmin, which is the most potent covalent inhibitor of plasmin, and was a more potent fibrinolysis inhibitor. Fab or chimerized Fab fragments of Pi were equivalently effective. In vivo, in a humanized model of fibrinolytic surgical bleeding, Pi significantly reduced bleeding to a greater extent than a clinical dose of EACA.

Conclusions: A mAb directed against unique loop sequences in the protease domain is a highly specific, potent, competitive plasmin inhibitor that significantly reduces experimental surgical bleeding in vivo.

Keywords: antifibrinolytic agents; fibrinolysis; hemorrhage; plasmin; α2-antiplasmin.

Fig. 1.. Binding specificity and avidity of Pi for human plasmin.

A) Ribbon diagram of microplasminogen showing the loops of the plasmin protease domain (PDB entry 1QRZ) visualized with iCN3D (https://www.ncbi.nlm.nih.gov/Structure/icn3d/icn3d.html). B) Pi binds specifically to the protease domain of reduced human plasmin by immunoblotting. Relative migration of molecular standards (kDa) is shown. C) Binding of Pi to various plasmin protease loop mutants. Loop amino acid sequences in the plasmin protease domain were chimerized with the corresponding loop sequences of factor D as indicated (loop 3: TRFGQ changed to LNGA; loop 5: AHCLEKSPRPSSY changed to AHCLEDAADGKV; loop 6: AHQEVNLEPHV changed to AHSLSQPEPSK; loop 7: EPTRKD changed to HPDSQPDTIDHD). Wells of a microtiter plate were coated with native (wild-type, WT) or various microplasmin loop mutants (5 ug/ml). After blocking, Pi, a polyclonal rabbit anti-plasmin (plasmin Ab) or a non-reactive mouse (anti-digoxin, control) antibody (1 ug/ml) were added to wells. The bound antibodies (Pi, control) were detected by a secondary anti-mouse peroxidase (1:10, 000) and the polyclonal plasmin antibodies were detected by anti-rabbit peroxidase antibody (1:5, 000) followed by TMB substrate with monitoring at A370.

Fig. 2.. Comparative inhibition of plasmin activity by Pi EACA and a2AP.

A). Human plasmin (2.8 nM) was mixed with S2251 substrate and Pi (1x10⁻⁹ to 3.3x10⁻⁸M) or EACA (3.9x10⁻³ to 0.25x10⁻¹M) or no inhibitor, and the rate of substrate cleavage was monitored at A405 nm for 20 min. The percent residual activity of plasmin as a function of the log concentration of inhibitor is shown. B). Effect of Pi on velocity of substrate cleavage by human plasmin. Human plasmin (100 nM, Hematologic Technologies, Inc.) was mixed with or without Pi (50 nM) in the presence of the substrate S2251 (0.1 to 4 mM). The initial rate of substrate cleavage (6 min.) was monitored at A405. Data were plotted and analyzed using Michaelis-Menten kinetics by the Graphpad Prism Program. Means (duplicate observations) are shown and are representative of 4 separate experiments. C). Comparative inhibition of human plasmin by a2AP and P. Human plasmin (200 nM) was mixed with a2AP (12.5 to 200 nM) or Pi (15.625 to 500 nM) or no inhibitor and the cleavage of S2251 was monitored at A405. The means of duplicate observations are shown. D). Fab and chimeric Fab forms of Pi efficiently inhibit plasmin enzymatic activity. Plasmin (25 nM) was mixed with various concentrations (0 – 200 nM) of mouse Fab or chimeric Fab (cFab) and S2251 (0.5 mM) in TBS buffer pH 7.4. Plasmin activity was measured by the release of paranitroanilide product at A405 over time. E) Saturation binding of Pi. Wells of microtiter plate were coated with human plasminogen (2ug/ml, Pg) or nothing, followed by washing and blocking with bovine serum albumin (BSA, control). After washing, various concentrations of purified Pi (shown) were added for 1 h. After washing the amount of antibody bound was detected by a secondary anti-mouse antibody followed by TMB substrate with monitoring at A370 nm. Data shown the means ± SE of triplicate observations, experiments repeated at least twice. The data were analyzed with Graphpad Prism, r=0.95.

Fig. 3.. Binding and inhibitory specificity of the Pi.

A) Binding of Pi to various serine proteases. The binding of Pi or a control monoclonal antibody (anti-digoxin, Ctl) to human (h) plasminogen (Pg), mouse (m) Pg, tissue plasminogen activator (tPA), trypsin (tryps.), chymotrypsin (chym.) or bovine serum albumin (was assessed in microtiter plates by an ELISA as described in Methods. B) Effect of Pi or no Pi on plasmin activity generated in various animal plasmas after the addition of urokinase. Plasmin activity in human (Hu), dog, guinea pig (GP), bovine (Bov), cat (Cat), gerbil (Ger), hamster (Ham), pig, rabbit (Rab) and rat plasmas was assessed after addition of urokinase (UK) by the cleavage of S2251 substrate as described in Methods. C) Dose-related inhibition of plasmin activity in cynomolgus plasma. Streptokinase (15 U, 10 uL) was added to cynomolgus plasma (10 ul) containing S2251 (10 ul, final 0.5mM), TBS buffer (10 ul, pH 7.4), Pi (0-2 uM, 10 ul). The cleavage of S2251 at 37° C was measured for 20 min. and the percent of activity was determined by comparison to samples without Pi.

Fig. 4.. Comparative inhibition of plasmin mediated factor V cleavage and human clot fibrinolysis (dissolution) by Pi, EACA and a2AP.

A) Effect of EACA and Pi on plasmin-mediated cleavage of factor V. Human factor V (2 ug) was mixed with EACA (1 mM), a control monoclonal antibody (CTL, anti-digoxin, 5 ug) or Pi (5 ug) for 1 hr at room temperature, followed by SDS-PAGE on 7.5% gels. The relative migration of molecular weight standards is shown (kDa). Closed arrow heads indicate uncleaved factor V, open arrowheads indicated cleaved factor V. B). Comparative, dose-related effects of EACA and Pi on human clot lysis. Human plasma was clotted with CaCl2 (10 mM) and thrombin (1 U/ml) in the presence of trace amounts of ¹²⁵I fibrinogen for 1 h at 37° C. After washing in 1 ml of TBS pH 7.4, tPA (5 nM) was added in the presence of various amounts of Pi (62.5 to 1000 nM), EACA (0.125 mM to 2mM) or no inhibitor. After 2.5 h the amount of fibrinolysis was determined by measuring the amount of ¹²⁵I-fibrin dissolved in the supernatant. Means of duplicate observations are shown. C) Comparative effects of a2AP vs. Pi on fibrinolysis. Clots were formed as above in the presence of human plasminogen (2 uM) and a2AP (1 uM) or Pi (1 uM) and various amounts of tPA (0-10 nM). Clot dissolution was monitored in duplicate over 2 h in microtiter plate wells at A405 nm. The amount of fibrinolysis was determined by the relative decrease in A405 as described in Methods. D) Comparative effect of different Pi molecular forms on dissolution of human clots. Human plasma clots were mixed with various concentrations of Pi IgG, Pi Fab and chimeric Pi Fab (25nM-400nM) and plasmin was activated by urokinase. The amount of clot lysis was determined as described in Methods.

Fig. 5.. Pi suppresses fibrinolytic bleeding in vivo. A) Duration of tail bleeding. B) Blood loss from bleeding.

Anesthetized mice were treated in a randomized, blinded fashion with saline (Control), Pi (100 nmole/kg) or EACA (510 micromole/kg) intravenously. Human plasminogen (100 nmole/kg) and plasminogen activator (80,000 IU/kg) were given over 60 min. Tail bleeding was initiated 40 min. after the infusion. Tails were pre-warmed for 5 mins in 3 mL of 37°C saline and bleeding was monitored as described [23, 43]. N=5-6, mean bleeding time ± SE and mean bleeding volume ± SE are shown. *p<0.05, **p<0.01; ns, not significant. One-way ANOVA, Neuman Keul’s corrections.