Plasminogen-deficient Mice Research Articles

Impaired arterial neointima formation in mice with disruption of the plasminogen gene.

To define the role of plasminogen (Plg) in the smooth muscle cell response after arterial wall injury, neointima formation was evaluated after electric injury of the femoral artery in plasminogen-deficient (Plg-/-) mice. The injury destroyed all medial smooth muscle cells, denuded the injured segment of intact endothelium, and induced transient platelet-rich mural thrombosis. In wild-type (Plg+/+) mice, vascular wound healing was characterized by lysis of the thrombus, transient infiltration of inflammatory cells, and progressive removal of necrotic debris and thrombosis. Topographic analysis revealed repopulation of the media and accumulation in the neointima of smooth muscle cells originating from the noninjured borders, which progressed into the necrotic center. In Plg-/- mice, wound healing was significantly impaired with delayed removal of necrotic debris, reduced leucocyte infiltration and smooth muscle cell accumulation, and decreased neointima formation. Smooth muscle cells accumulated at the uninjured borders, but failed to migrate into the necrotic center. Proliferation of smooth muscle cells was not affected by Plg deficiency. Evans blue staining revealed no genotypic differences in reendothelialization. Thus, Plg plays a significant role in vascular wound healing and arterial neointima formation after injury, most likely by affecting cellular migration.

An Extracellular Proteolytic Cascade Promotes Neuronal Degeneration in the Mouse Hippocampus

Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.

Restoration of Thrombolytic Potential in Plasminogen-Deficient Mice by Bolus Administration of Plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue-type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.

Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P &lt; .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P &lt; .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P &lt; .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.

3 Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P &lt; .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P &lt; .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P &lt; .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.

273 Ovulation efficiency and reproduction is reduced in plasminogen deficient mice

273 Ovulation efficiency and reproduction is reduced in plasminogen deficient mice

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

Circumstantial evidence suggests that the plasminogen/plasmin system plays a role in many biological processes, including hemostasis, cell migration, and development. 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 His605 and Asp648 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 micrograms/mL (n = 4) in Plg+/+, 35 +/- 2 micrograms/mL (n = 3) in Plg+/-, and 0.076 +/- 0.032 microgram/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 125I-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. 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.