Absence of functional compensation between coagulation factor VIII and plasminogen in double-knockout mice

Abstract

Plasminogen deficiency is associated with severely compromised fibrinolysis and extravascular deposition of fibrin. In contrast, coagulation factor VIII (FVIII) deficiency leads to prolonged and excessive bleeding. Based on opposing biological functions of plasminogen and FVIII deficiencies, we hypothesized that genetic elimination of FVIII would alleviate the systemic formation of fibrin deposits associated with plasminogen deficiency and, in turn, elimination of plasminogen would limit bleeding symptoms associated with FVIII deficiency. Mice with single and combined deficiencies of FVIII (F8^-/-) and plasminogen (Plg^-/-) were evaluated for phenotypic characteristics of plasminogen deficiency, including wasting disease, shortened lifespan, rectal prolapse, and multiorgan fibrin deposition. Conversely, to specifically examine the role of plasmin-mediated fibrinolysis on bleeding caused by FVIII deficiency, F8^-/- and F8^-/-/Plg^-/- mice were subjected to a bleeding challenge. Mice with a combined deficiency in FVIII and plasminogen displayed no phenotypic differences relative to mice with single FVIII or plasminogen deficiency. Plg^-/- and F8^-/-/Plg^-/- mice exhibited the same penetrance and severity of wasting disease, rectal prolapse, extravascular fibrin deposits, and reduced viability. Furthermore, following a tail vein-bleeding challenge, no significant differences in bleeding times or total blood loss could be detected between F8^-/- and F8^-/-/Plg^-/- mice. Moreover, F8^-/- and F8^-/-/Plg^-/- mice responded similarly to recombinant FVIII (rFVIII) therapy. In summary, the pathological phenotype of Plg^-/- mice developed independently of FVIII-dependent coagulation, and elimination of plasmin-driven fibrinolysis did not play a significant role in a nonmucosal bleeding model in hemophilia A mice.

Graphical abstract

Figure 1.

FVIII deficiency did not correct the pathologic phenotype of Plg^−/−mice. (A) The estimated weight measurements of a cohort of 25 WT, 58 F8^−/−, 21 Plg^−/−, and 61 F8^−/−/Plg^−/− mice that were followed for up to 168 days. Signs above the x-axis indicate where the weight of Plg^−/− (red squares) and F8^−/−/Plg^−/− (purple diamonds) deviated significantly from that of WT and F8^−/− mice, respectively, and where the weight was significantly different between F8^−/−/Plg^−/− and Plg^−/− mice (open green squares) and between F8^−/− and WT mice (blue circles). The weight data were analyzed with a repeated-measurement ANOVA; P < .05 was considered significant. (B) Frequency of rectal prolapse in 31 WT, 51 F8^−/−, 23 Plg^−/−, and 39 F8^−/−/Plg^−/− mice. (C) Survival analysis from a cohort of 30 WT, 43 F8^−/−, 23 Plg^−/−, and 26 F8^−/−/Plg^−/− mice followed for 168 days. Mice were euthanized when reaching predefined humane end points including wasting disease, rectal prolapse, and penile prolapse. The median survival of F8^−/−/Plg^−/− mice was 12.6 weeks, whereas it was 19.6 for Plg^−/− mice. Development of rectal prolapse and survival rate were compared with the log-rank Mantel-Cox test; P < .05 was considered significant. Colored asterisks indicate intergroup comparisons; **P < .01, ****P < .0001.

Figure 2.

FVIII deficiency did not prevent the development of hepatic fibrin deposits in Plg^−/−mice. (A) Representative HE-stained sections and immunohistochemical stains for fibrin and CD11b-expressing inflammatory cells in livers from WT, F8^−/−, Plg^−/−, and F8^−/−/Plg^−/− mice. Note the hepatic fibrin deposits (arrows) and CD11b⁺ cells (arrowheads) in Plg^−/− and F8^−/−/Plg^−/− mice. The anti-CD11b control is a liver section stained with an isotype antibody (Ab). The fibrin-positive and -negative controls are liver sections from WT and fibrinogen^−/− mice subjected to CCl₄-induced liver injury. Scale bar, 100 µm. The number of fibrin lesions (B) was counted manually, whereas the percentage of fibrin-stained tissue area (C) and the percentage of CD11b-stained tissue area (D) were quantified by digital image analysis. Results are shown as individual observations and mean ± standard error of the mean (SEM). Data were analyzed by the Kruskal-Wallis with Dunn multiple comparisons test. P < .05 was considered significant; *P < .05, **P < .01. All stained slides were scanned at 20× magnification on the Nanozoomer slide scanner (Hamamatsu Photonics K.K.), and quantified with Visiopharm Integrator System software (VIS version 6.7.0.2590; Visiopharm).

Figure 3.

FVIII deficiency did not decrease levels of CD11b⁺cells in the spleen of Plg^−/−mice. (A) Representative HE-stained sections and immunohistochemical stains for fibrin and CD11b-expressing cells in spleens from WT, F8^−/−, Plg^−/−, and F8^−/−/Plg^−/− mice. Note the presence of fibrin deposits (black arrows) and CD11b-expressing inflammatory cells (arrowheads). Cells with megakaryocyte morphology were abundant in Plg^−/− and F8^−/−/Plg^−/− spleens (white arrows). The anti-CD11b and anti-fibrin–negative controls were spleen sections stained with an isotype antibody. Scale bar, 100 µm. The percentage of fibrin-stained tissue area (B) and percentage of CD11b-stained tissue area (C) were quantified by digital image analysis. Results are shown as individual observations and mean ± SEM and analyzed with an ANOVA with Bonferroni correction for multiple comparisons. P < .05 was considered significant; *P < .05, ***P < .001, ****P < .0001. All stained slides were scanned at 20× magnification on the Nanozoomer slide scanner (Hamamatsu Photonics K.K.), and quantified with Visiopharm Integrator System software (VIS ver. 6.7.0.2590; Visiopharm).

Figure 4.

Plasminogen deficiency did not alleviate the bleeding phenotype or enhance the response to rFVIII treatment in F8^−/−mice. (A) Graphical representation of individual bleeding profiles of mice treated with Advate buffer or rFVIII at 2.5 and 3.0 U/kg. Each row represents the entire bleeding profile of a single mouse with each bar representing a single bleeding episode for that mouse. The number of spontaneous bleeds (B), total blood loss (C), and bleeding time (D) of F8^−/− and F8^−/−/Plg^−/− mice were quantified. Data are presented as the mean ± SEM and analyzed by 2-way ANOVA applying the Bonferroni test to adjust for multiple comparisons. *P < .05, **P < .01, ***P < .001, ****P < .0001. hgb, hemoglobin.