Effects of Disruption of The Plasminogen Gene on Thrombosis, Growth, and Health in Mice

Abstract

Background Circumstantial evidence suggests that the plasminogen/plasmin system plays a role in many biological processes, including hemostasis, cell migration, and development. Methods and Results The in vivo function of the plasminogen/plasmin system was studied by generation of plasminogen-deficient ( Plg −/− ) mice. Inactivation of the murine plasminogen gene ( Plg ) was achieved by replacing, via homologous recombination in embryonic stem cells, genomic sequences encoding the exons containing the catalytic site amino acids His 605 and Asp 648 with a neomycin phosphotransferase expression cassette. Germline transmission of the mutated allele, as determined by Southern blot hybridization and polymerase chain reaction, was obtained via blastocyst injection. Mendelian inheritance of the inactivated plasminogen allele was observed, and homozygous-deficient mice ( Plg −/− ) displayed normal viability but retarded growth up to at least 12 weeks of age. At 8 weeks of age, body weight was 21.8±1.2 g (n=10) for wild-type ( Plg +/+ ) mice, 21.0±1.1 g (n=16) for heterozygous-deficient ( Plg +/− ) mice, and 17.4±1.3 g (n=12) for Plg −/− mice; P <.05 versus Plg +/+ or Plg +/− . None of 36 Plg +/+ or 65 Plg +/− mice but 7 of 37 Plg −/− mice (19%) developed rectal prolapse at 7.4±0.6 weeks of age ( P =.03 versus Plg +/+ and P =.003 versus Plg +/− ); 4 of 37 Plg −/− mice (11%) became runted and apathic at 5.3±0.3 weeks of age ( P =.041 versus Plg +/− ); and 6 of 37 Plg −/− mice (16%) died prematurely at 8.8±1.7 weeks of age ( P =.057 versus Plg +/+ and P =.029 versus Plg +/− ). Although male and female Plg −/− mice were able to sire offspring, the fertility of Plg −/− female mice was reduced, possibly owing to their impaired health. Levels of plasminogen-related antigen in plasma, measured by ELISA, were 84±8 μg/mL (n=4) in Plg +/+ , 35±2 μg/mL (n=3) in Plg +/− , and 0.076±0.032 μg/mL (n=6) in Plg −/− mice ( P <.001 versus Plg +/− and Plg +/+ ). Plasmin activity generated by urokinase activation was unmeasurable in Plg −/− mice (<5% of Plg +/+ mice). Plasminogen-specific immunoreactivity was observed in hepatocytes from Plg +/+ mice but not from Plg −/− mice (<10% of Plg +/+ mice). Neither native nor variant plasminogen mRNA nor translation products could be identified by Northern or Western blot of liver extracts from Plg −/− mice. Spontaneous lysis within 24 hours of a 125 I-fibrin–labeled pulmonary plasma clot was 85±5% (n=5) in Plg +/+ mice, 62±7% (n=3) in Plg +/− mice, and −2±1% (n=3) in Plg −/− mice ( P <.001 versus Plg +/− and Plg +/+ ). Delayed clot lysis within 72 hours was 33±1% (n=3) in tPA −/− mice and 26±2% (n=3) in Plg −/− mice ( P =.054). Histological examination of several organs revealed fibrin deposition in the liver; lung; and in the stomach, associated with gastric ulcers, in 6- to 12-week-old Plg −/− mice but not in Plg +/+ or Plg +/− littermates. Conclusions Plasminogen-deficient mice survive embryonic development but develop spontaneous fibrin deposition due to impaired thrombolysis and suffer retarded growth and reduced fertility and survival. The Plg −/− phenotype is reminiscent of the combined tPA −/− : uPA −/− phenotype, which suggests that there is no significant additional pathway for physiological plasminogen activation in mice.