Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli: Unique Effects of Plasminogen Activation and α2-Antiplasmin Inactivation

Abstract

Background: In patients with hemodynamically significant pulmonary embolism, physiological fibrinolysis fails to dissolve thrombi acutely and r-tPA (recombinant tissue-type plasminogen activator) therapy may be required, despite its bleeding risk. To examine potential mechanisms, we analyzed the expression of key fibrinolytic molecules in experimental pulmonary emboli, assessed the contribution of α2-antiplasmin to fibrinolytic failure, and compared the effects of plasminogen activation and α2-antiplasmin inactivation on experimental thrombus dissolution and bleeding.

Methods: Pulmonary embolism was induced by jugular vein infusion of 125I-fibrin or fluorescein isothiocyanate-fibrin labeled emboli in anesthetized mice. Thrombus site expression of key fibrinolytic molecules was determined by immunofluorescence staining. The effects of r-tPA and α2-antiplasmin inactivation on fibrinolysis and bleeding were examined in a humanized model of pulmonary embolism.

Results: The plasminogen activation and plasmin inhibition system assembled at the site of acute pulmonary emboli in vivo. Thrombus dissolution was markedly accelerated in mice with normal α2-antiplasmin levels treated with an α2-antiplasmin-inactivating antibody (P<0.0001). Dissolution of pulmonary emboli by α2-antiplasmin inactivation alone was comparable to 3 mg/kg r-tPA. Low-dose r-tPA alone did not dissolve emboli, but was synergistic with α2-antiplasmin inactivation, causing more embolus dissolution than clinical-dose r-tPA alone (P<0.001) or α2-antiplasmin inactivation alone (P<0.001). Despite greater thrombus dissolution, α2-antiplasmin inactivation alone, or in combination with low-dose r-tPA, did not lead to fibrinogen degradation, did not cause bleeding (versus controls), and caused less bleeding than clinical-dose r-tPA (P<0.001).

Conclusions: Although the fibrinolytic system assembles at the site of pulmonary emboli, thrombus dissolution is halted by α2-antiplasmin. Inactivation of α2-antiplasmin was comparable to pharmacological r-tPA for dissolving thrombi. However, α2-antiplasmin inactivation showed a unique pattern of thrombus specificity, because unlike r-tPA, it did not degrade fibrinogen or enhance experimental bleeding. This suggests that modifying the activity of a key regulator of the fibrinolytic system, like α2-antiplasmin, may have unique therapeutic value in pulmonary embolism.

Keywords: fibrinolysis; hemorrhage; pulmonary embolism; thrombosis; α2-antiplasmin.

© 2016 American Heart Association, Inc.

Figures

Figure 1. Expression of plasminogen activators and plasminogen at the site of pulmonary emboli

Lung tissue containing FITC-fibrin labeled (green) pulmonary emboli was immunostained (red) to detect the expression of (A) tPA, (B) uPA and (C) plasminogen (Pg) (D) The total immune-stained area (arbitrary units; a.u) for each protein was measured in a 20× (100 μm) image of an embolized vs non embolized (Control; dashed yellow outline) pulmonary artery in the lungs. n=3, mean ± SEM. **p<0.01; ns, non-significant.

Figure 2. Expression of fibrinolytic inhibitors at the site of pulmonary emboli

Lung tissue containing FITC-fibrin labeled (green) pulmonary emboli was immunostained (red) to detect the expression of (A) α2-antiplasmin (a2AP) and (B) PAI-1. (C) The total immune-stained area (arbitrary units; a.u) for each protein was measured in a 20× (100 μm) image of embolized vs non-embolized (control; outlined yellow) pulmonary artery in the lungs. n=3, mean ± SEM. **p<0.01; ns, non-significant.

Figure 3. Effect of α2-antiplasmin-inactivation on plasmin activity and the dissolution of pulmonary emboli

(A) The dissolution of 125I-fibrin-labeled pulmonary emboli was measured in anesthetized α2-antiplasmin (a2AP)−/− mice (N=16) with physiologic levels of α2-antiplasmin (after intravenous supplementation) with or without (control) treatment with an a2AP-inactivating antibody (a2AP-I). The percent thrombus dissolution was assessed by measuring the residual 125I-fibrin radioactivity in the lungs four hours after embolization. ****p<0.0001 vs. control. (B) The activity of plasmin in the presence of no a2AP or a2AP, with or without an a2AP-inactivating (a2AP-I) or a control monoclonal antibody of the same isotype (MCA334B0). Plasmin activity was monitored by cleavage of S2251, a paranitroanilide substrate, at A405. Experiments were performed in duplicate and the amount of plasmin activity was expressed relative to the amount of plasmin activity in the absence of a2AP or any antibody. (C) The effect of no a2AP (Control, Ctl) or a2AP with or without various monoclonal antibodies on plasmin activity, expressed as percentage of the activity of plasmin in the absence of a2AP. ***p<0.001 (D) Effect of an a2AP-I antibody or buffer alone (control) on dissolution of 125I-fibrinogen-labeled human clots in vitro in the presence of trace amounts of r-tPA (0.2 nM) after 3 h incubation at 37 °C. ***p<0.001

Figure 4. Effects of pharmacologic r-tPA and α2-antiplasmin inactivation on thrombus dissolution in mice with experimental pulmonary emboli

Anesthetized mice were treated thirty minutes after thromboembolism with r-tPA, an α2-antiplasmin-inactivating antibody (a2AP-I) alone, the combination of low dose r-tPA and a2AP-I or nothing (Control) as shown (N=48). Four hours after PE, the experiments were terminated and thrombus dissolution was measured. The number of mice per dose is shown. *** p# p<0.05 vs. r-tPA 3 mg/kg.

Figure 5. Effects of pharmacologic r-tPA and α2-antiplasmin-inactivation on fibrinogen consumption and bleeding dissolution in mice with experimental PE

(A) Fibrinogen consumption in experimental groups given an α2-antiplasmin-inactivating antibody (a2AP-I) or r-tPA or no treatment (Control). Fibrinogen levels were measured by the sodium sulfite method and are expressed relative to fibrinogen levels in normal wild-type mice without PE. (B) Tail bleeding was measured as hemoglobin (Hgb) loss by Drabkin’s reagent in the same experimental groups. N per group is shown. **p<0.01, ***p<0.001 vs. controls.

Figure 6. Comparative effects of plasminogen activation and α2-antiplasmin on thrombus dissolution and associated bleeding

(A) A hypothetical plot of thrombus dissolution and bleeding for potential optimal treatment for PE. (B) Experimental data showing the effects (means ± SE) of r-tPA, α2-antiplasmin-I and the combination of the two agents on thrombus dissolution and bleeding. Higher dose r-tPA (1.2 or 3 mg/kg) is indicated by larger symbols.