Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

Abstract

Stasis of venous blood triggers deep vein thrombosis by activating coagulation, yet its effects on the fibrinolytic system are not fully understood. We examined the relationship between stasis, fibrinolysis, and the development of experimental venous thrombosis. Effects of stasis-induced deep vein thrombosis and fibrinolysis on thrombosis were examined by inferior vena cava ligation in congenic mice with and without α2-antiplasmin (α2AP), the primary inhibitor of plasmin. Venous thrombus weights were measured and thrombus composition was determined by Martius scarlet blue and immunofluorescence staining. Venous thrombi from α2AP+/+ mice contained plasminogen activators, plasminogen activator inhibitor-1, plasminogen, and α2AP, which changed with thrombus age. Normal, α2AP+/+ mice developed large, occlusive thrombi within 5 hours after ligation; thrombi were even larger in plasminogen-deficient mice (P < .001). No significant thrombus formation was seen in α2AP-/- mice (P < .0001) or in α2AP+/+ mice treated with an α2AP-inactivating antibody (P < .001). Venous stasis activated fibrinolysis, measured by D-dimer levels, in α2AP-/- mice vs α2AP+/+ mice (P < .05). Inhibition of fibrinolysis by the indirect plasmin inhibitor ε-aminocaproic acid or by α2AP restored thrombosis in α2AP-/- mice. In addition to its effects on acute thrombosis, thrombus formation was also markedly suppressed in α2AP-/- mice vs α2AP+/+ mice (P < .0001) 1, 7, and 14 days after ligation. We conclude that experimental venous stasis activates the fibrinolytic system to block the development of venous thrombosis. Suppression of fibrinolysis by α2AP appears essential for stasis-induced thrombus development, which suggests that targeting α2AP may prove useful for preventing venous thrombosis.

Conflict of interest statement

Conflict-of-interest disclosure: G.L.R is a founder of Translational Sciences. The remaining authors declare no competing financial interests.

© 2019 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Expression of fibrinolytic system components in 24 h and 7 days old venous thrombi. Panels A-F show the expression (red) of (A-B) tPA, (C-D) uPA, (E-F) PAI-1, (G-H) plasminogen (Pg), (I-J) α2AP, and (K-L) their quantification in 8-µm cryosections of 24 hours and 7 days thrombi as labeled. Red color (Alexa Fluor 555) represents each component as labeled and blue color shows 4′,6-diamidino-2-phenylindole–stained nuclei. For comparison, IVC from sham mice were immunostained and quantified for each antibody under the same color range in the histogram mode of Image Pro-Plus software. The total immunostained area (arbitrary units; AU/mm2) for each protein was measured in 500 μm (original magnification ×4) images of an IVC sample. (N = 4-5 per group, mean ± SEM). *P < .05, **P < .01, ***P < .001.

Figure 2.

Effects of α2AP deficiency on early thrombus formation. (A) Representative IVCs in different groups as labeled. (B) Vein thrombus weight milligram/gram of body weight. Each symbol represents an animal used in each group (N = 33). The bar graph shows the statistical significance between different groups by one-way ANOVA and Newman-Keuls post hoc test. ****P < .0001; ***P < .001; ns, nonsignificant. (C-F) Formalin-fixed paraffin-embedded (5 µm) sections of IVC thrombi were stained with Martius scarlet blue kit (scale bar = 500 µm). Representative image of N = 5 for each group. (C) Thrombus formation in the IVC of α2AP+/+ mice 5 hours after ligation (500 µm). (D) The composition of 5-hour-old thrombi (fibrin, red blood cell [RBC], and leukocytes; black arrows, scale bar = 100 µm). The pink/yellow color represents fibrin and nuclei are darkly stained. This panel shows a magnified image of the area in the black box highlighted in panel C. (E-F) Martius scarlet blue-stained representative images of IVC in α2AP−/− mice 5 hours after IVC ligation. The black arrows indicate areas of minimum thrombosis.

Figure 3.

Effects of α2AP deficiency on acute thrombus formation. (A-B) Representative IVCs in α2AP+/+ and α2AP−/− mice (A) 24 hours after IVC ligation and (B) their thrombus weights. Each symbol in the bar graph represents an animal used in each group (N = 12); ****P < .0001, Student t test. (C) Martius scarlet blue (scale bar = 500 µm) staining shows 24-hour thrombi rich in fibrin (pink color). (D) Magnified image (original magnification ×20; scale bar = 100 µm) of panel C showing a fibrin-rich (pink) thrombus area. (E) Minimum thrombus formation (black arrows) in α2AP−/− mice 24 hours after IVC ligation.

Figure 4.

Effects of α2AP deficiency on chronic thrombus formation. (A) Martius scarlet blue-stained 7-day-old thrombi (scale bar = 500 µm). (B) Magnified images (original magnification ×20) of IVC thrombus showing lamellate fibrin-rich (pink) lines of Zahn. (C) Thrombi were not formed in α2AP−/− mice after 7 days of IVC ligation. Arrow indicates the minimum thrombus formation. (D) Martius scarlet blue stained 14 days old thrombi (scale bar = 500 µm) in α2AP+/+ mice. (E) Magnified image (original magnification ×20) of panel D showing a fibrin-rich (pink), porous thrombus. (F) Martius scarlet blue-stained IVC showing minimum thrombus in α2AP−/− mice. (G) Representative images of IVC thrombus formation in different groups as labeled including sham mice (7 days). (H) Vein thrombus weight milligram/gram of body weight. Each symbol represents an animal used in each group (N = 38). One-way ANOVA; ****P < .0001, **P < .01.

Figure 5.

Effects of α2AP supplementation/inhibition. (A) Representative images of IVC thrombus formation in α2AP−/− mice after 5 hours’ ligation after administration of α2AP or α2AP + α2AP-inactivating antibody (α2AP-I) as labeled. (B) Vein thrombus weight in these groups in milligram/gram of body weight as labeled. Each symbol represents a single experiment, N = 14. The horizontal red and blue lines in the graph show, for reference, the mean thrombus weight in α2AP+/+ and α2AP−/− mice, respectively. ****P < .0001; one-way ANOVA (among 4 groups).

Figure 6.

Effects of α2AP on fibrinolysis. (A) Plasma D-dimer levels in α2AP+/+ and α2AP−/− mice after 5 hours of IVC ligation. The mice were supplemented with 10 mg/kg human fibrinogen. *P < .05 (Student t test). (B) Vein thrombus weight milligram/gram of body weight in α2AP+/+ and α2AP−/− mice after 5 hours’ IVC ligation used for D-dimer measurements ***P < .001 (Student t test). (C) Representative images of IVC thrombus formation in EACA-treated α2AP−/− mice 5 hours after ligation as labeled. (D) Vein thrombus weight (milligram/gram of body weight) in α2AP−/− mice with or without EACA treatment. The horizontal red line in the graph shows the mean vein thrombus weight in α2AP+/+ mice for reference. *P < .05, one-way ANOVA (among 3 groups). (E) Vein thrombus weight (milligram/gram of body weight) in Pg−/− mice in comparison with α2AP+/+ mice. (N = 21) ***P < .001, Mann-Whitney test.