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The immature brains of newborns often respond differently from the brains of adults when exposed to similar insults. Previous studies have indicated that although hypoxia-ischemia (HI) induces persistent thrombosis in adult brains, it only modestly impairs blood perfusion in newborn brains. Here, we used the Vannucci model of HI encephalopathy to study age-related responses to cerebral HI in rat pups. We found that HI triggered fibrin deposition and impaired blood perfusion in both neonatal and adult brains. However, these effects were only transient in neonatal brains (<4 hours) and were accompanied by acute induction of both tissue-type and urinary-type plasminogen activators (tPA and uPA), which was not observed in adult brains subjected to the same insult. Interestingly, activation of the plasminogen system persisted up to 24 hours in neonatal brains, long after the clearance of fibrin-rich thrombi. Furthermore, astrocytes and macrophages outside blood vessels expressed tPA after HI, suggesting the possibility of tPA/plasmin-mediated cytotoxicity. Consistent with this hypothesis, injection of alpha2-antiplasmin into cerebral ventricles markedly ameliorated HI-induced damage to neurofilaments and white matter oligodendrocytes, providing a dose-response reduction of brain injury after 7 days of recovery. Conversely, ventricular injection of tPA increased HI-induced brain damage. Together, these results suggest that tPA/plasmin induction, which may contribute to acute fibrinolysis, is a critical component of extravascular proteolytic damage in immature brains, representing a new therapeutic target for the treatment of HI encephalopathy.

Tissue repair requires an adequate cellular proliferation coordinated with the timely proteolysis of matrix elements. Based on the properties of plasminogen activators in liver cell proliferation and tissue proteolysis, we explored the regulatory role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in liver repair. Using carbon tetrachloride (CCl(4)) intoxication as a model of acute liver injury, we found that tPA-deficient mice displayed a mild defect in hepatic repair, whereas livers of uPA-deficient mice had a more substantial delay in repair, with injury of centrilobular hepatocytes persisting up to 14 days after CCl(4). Notably, functional cooperativity between plasminogen activators was strongly inferred from the profound reparative defect in livers of mice lacking tPA and uPA simultaneously, with persistence of centrilobular injury as far out as 35 days. The defective repair was not because of increased susceptibility of experimental mice to the toxin or to inadequate cellular proliferation. Instead, lack of plasminogen activators led to the accumulation of fibrin and fibronectin within injured areas and poor removal of necrotic cells. These data demonstrate that tPA and uPA play a critical role in hepatic repair via proteolysis of matrix elements and clearance of cellular debris from the field of injury.

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.

Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

Neuronal damage in the CNS after excitotoxic injury is correlated with blood-brain barrier (BBB) breakdown. We have used a glutamate analog injection model and genetically altered mice to investigate the relationship between these two processes in the hippocampus. Our results show that BBB dysfunction occurs too late to initiate neurodegeneration. In addition, plasma infused directly into the hippocampus is not toxic and does not affect excitotoxin-induced neuronal death. To test plasma protein recruitment in neuronal degeneration, we used plasminogen-deficient (plg(-/-)) mice, which are resistant to excitotoxin-induced degeneration. Plasminogen is produced in the hippocampus and is also present at high levels in plasma, allowing us to determine the contribution of each source to cell death. Intrahippocampal delivery of plasminogen to plg(-/-) mice restored degeneration to wild-type levels, but intravenous delivery of plasminogen did not. Finally, although the neurons in plg(-/-) mice do not die after excitotoxin injection, BBB breakdown occurs to a similar extent as in wild-type mice, indicating that neuronal death is not necessary for BBB breakdown. These results indicate that excitotoxin-induced neuronal death and BBB breakdown are separable events in the 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.

The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.