Tissue plasminogen activator worsens experimental autoimmune encephalomyelitis by complementary actions on lymphoid and myeloid cell responses.

BACKGROUND: Tissue plasminogen activator (tPA) is a serine protease involved in fibrinolysis. It is released by endothelial cells, but also expressed by neurons and glial cells in the central nervous system (CNS). Interestingly, this enzyme also contributes to pathological processes in the CNS such as neuroinflammation by activating microglia and increasing blood-brain barrier permeability. Nevertheless, its role in the control of adaptive and innate immune response remains poorly understood. METHODS: tPA effects on myeloid and lymphoid cell response were studied in vivo in the mouse model of multiple sclerosis experimental autoimmune encephalomyelitis and in vitro in splenocytes. RESULTS: tPA-/- animals exhibited less severe experimental autoimmune encephalomyelitis than their wild-type counterparts. This was accompanied by a reduction in both lymphoid and myeloid cell populations in the spinal cord parenchyma. In parallel, tPA increased T cell activation and proliferation, as well as cytokine production by a protease-dependent mechanism and via plasmin generation. In addition, tPA directly raised the expression of MHC-II and the co-stimulatory molecules CD80 and CD86 at the surface of dendritic cells and macrophages by a direct action dependent of the activation of epidermal growth factor receptor. CONCLUSIONS: Our study provides new insights into the mechanisms responsible for the harmful functions of tPA in multiple sclerosis and its animal models: tPA promotes the proliferation and activation of both lymphoid and myeloid populations by distinct, though complementary, mechanisms.

Increased brain plasmin levels following experimental ischemic stroke in male mice.

Many coagulation factor proteases are increased in the brain during ischemic stroke. One of these proteases is plasmin. In this study we established a novel method for direct quantitative measurement of plasmin activity in male mouse brain slices using a sensitive fluorescent substrate in the presence of specific protease inhibitors. In both the ischemic and contralateral hemispheres, plasmin activity increased 3, 6, and 24 hr following stroke in comparison to healthy mice (F(3, 72) = 39.5, p < 0.0001, repeated measures ANOVA) after the induction of permanent middle cerebral artery occlusion (PMCAo). Plasmin activity was higher in the ischemic hemisphere (F(1,36) = 9.1, p = 0.005) and there was a significant interaction between time and ischemic hemisphere (F(3,36) = 4.4, p = 0.009). Plasmin activity was correlated with infarct volume (R2  = 0.5289, p = 0.0009 by Spearman). The specificity of the assay was verified utilizing tissue-type plasminogen activator (tPA)-deficient mice which, as expected, had significantly lower levels of plasmin 24 hr following ischemia compared to wild-type mice (ischemic (0.6 ± 0.23 and 1.94 ± 0.5, respectively), p = 0.049 and contralateral hemispheres (0.13 ± 0.14 and 0.75 ± 0.10, respectively), p = 0.018 by t test). There is a time-dependent increase in plasmin levels and an association of higher levels of plasmin with larger infarct volumes in an experimental stroke model. This suggests caution in the use of recombinant tPA (rtPA) and that plasmin inhibition in the brain may be a therapeutic target in acute ischemic stroke.

tPA Deficiency Underlies Neurovascular Coupling Dysfunction by Amyloid-ß.

The amyloid-ß (Aß) peptide, a key pathogenic factor in Alzheimer's disease, attenuates the increase in cerebral blood flow (CBF) evoked by neural activity (functional hyperemia), a vital homeostatic response in which NMDA receptors (NMDARs) play a role through nitric oxide, and the CBF increase produced by endothelial factors. Tissue plasminogen activator (tPA), which is reduced in Alzheimer's disease and in mouse models of Aß accumulation, is required for the full expression of the NMDAR-dependent component of functional hyperemia. Therefore, we investigated whether tPA is involved in the neurovascular dysfunction of Aß. tPA activity was reduced, and the tPA inhibitor plasminogen inhibitor-1 (PAI-1) was increased in male mice expressing the Swedish mutation of the amyloid precursor protein (tg2576). Counteracting the tPA reduction with exogenous tPA or with pharmacological inhibition or genetic deletion of PAI-1 completely reversed the attenuation of the CBF increase evoked by whisker stimulation but did not ameliorate the response to the endothelium-dependent vasodilator acetylcholine. The tPA deficit attenuated functional hyperemia by suppressing NMDAR-dependent nitric oxide production during neural activity. Pharmacological inhibition of PAI-1 increased tPA activity, prevented neurovascular uncoupling, and ameliorated cognition in 11- to 12-month-old tg2576 mice, effects associated with a reduction of cerebral amyloid angiopathy but not amyloid plaques. The data unveil a selective role of the tPA in the suppression of functional hyperemia induced by Aß and in the mechanisms of cerebral amyloid angiopathy, and support the possibility that modulation of the PAI-1-tPA pathway may be beneficial in diseases associated with amyloid accumulation.SIGNIFICANCE STATEMENT Amyloid-ß (Aß) peptides have profound neurovascular effects that may contribute to cognitive impairment in Alzheimer's disease. We found that Aß attenuates the increases in blood flow evoked by neural activation through a reduction in tissue plasminogen activator (tPA) caused by upregulation of its endogenous inhibitor plasminogen inhibitor-1 (PAI-1). tPA deficiency prevents NMDA receptors from triggering nitric oxide production, thereby attenuating the flow increase evoked by neural activity. PAI-1 inhibition restores tPA activity, rescues neurovascular coupling, reduces amyloid deposition around blood vessels, and improves cognition in a mouse model of Aß accumulation. The findings demonstrate a previously unappreciated role of tPA in Aß-related neurovascular dysfunction and in vascular amyloid deposition. Restoration of tPA activity could be of therapeutic value in diseases associated with amyloid accumulation.

Abrogating fibrinolysis does not improve bleeding or rFVIIa/rFVIII treatment in a non-mucosal venous injury model in haemophilic rodents.

Essentials The efficacy of systemic antifibrinolytics for hemophilic non-mucosal bleeding is undetermined. The effect of systemically inhibiting fibrinolysis in hemophilic mice and rats was explored. Neither bleeding nor the response to factor treatment was improved after inhibiting fibrinolysis. The non-mucosal bleeding phenotype in hemophilia A appears largely unaffected by fibrinolysis. SUMMARY: Background Fibrinolysis may exacerbate bleeding in patients with hemophilia A (HA). Accordingly, antifibrinolytics have been used to help maintain hemostatic control. Although antifibrinolytic drugs have been proven to be effective in the treatment of mucosal bleeds in the oral cavity, their efficacy in non-mucosal tissues remain an open question of significant clinical interest. Objective To determine whether inhibiting fibrinolysis improves the outcome in non-mucosal hemophilic tail vein transection (TVT) bleeding models, and to determine whether a standard ex vivo clotting/fibrinolysis assay can be used as a predictive surrogate for in vivo efficacy. Methods A highly sensitive TVT model was employed in hemophilic rodents with a suppressed fibrinolytic system to examine the effect of inhibiting fibrinolysis on bleeding in non-mucosal tissue. In mice, induced and congenital hemophilia models were combined with fibrinolytic attenuation achieved either genetically or pharmacologically (tranexamic acid [TXA]). In hemophilic rats, tail bleeding was followed by whole blood rotational thromboelastometry evaluation of the same animals to gauge the predictive value of such assays. Results The beneficial effect of systemic TXA therapy observed ex vivo could not be confirmed in vivo in hemophilic rats. Furthermore, neither intravenously administered TXA nor congenital knockout of the fibrinolytic genes encoding plasminogen or tissue-type plasminogen activator markedly improved the TVT bleeding phenotype or response to factor therapy in hemophilic mice. Conclusions The findings here suggest that inhibition of fibrinolysis is not effective in limiting the TVT bleeding phenotype of HA rodents in non-mucosal tissues.

Distant Space Processing is Controlled by tPA-dependent NMDA Receptor Signaling in the Entorhinal Cortex.

In humans, spatial cognition and navigation impairments are a frequent situation during physiological and pathological aging, leading to a dramatic deterioration in the quality of life. Despite the discovery of neurons with location-specific activity in rodents, that is, place cells in the hippocampus and later on grid cells in the entorhinal cortex (EC), the molecular mechanisms underlying spatial cognition are still poorly known. Our present data bring together in an unusual combination 2 molecules of primary biological importance: a major neuronal excitatory receptor, N-methyl-D-aspartate receptor (NMDAR), and an extracellular protease, tissue plasminogen activator (tPA), in the control of spatial navigation. By using tPA-deficient mice and a structure-selective pharmacological approach, we demonstrate that the tPA-dependent NMDAR signaling potentiation in the EC plays a key and selective role in the encoding and the subsequent use of distant landmarks during spatial learning. We also demonstrate that this novel function of tPA in the EC is reduced during aging. Overall, these results argue for the concept that encoding of proximal versus distal landmarks is mediated not only by different anatomical pathways but also by different molecular mechanisms, with the tPA-dependent potentiation of NMDAR signaling in the EC that plays an important role.

Tissue plasminogen activator deficiency preserves neurological function and protects against murine acute ischemic stroke.

BACKGROUND: We tested the hypothesis that tissue plasminogen activator (tPA) deficiency protected against acute ischemic stroke (AIS)-induced brain injury. METHODS AND RESULTS: Wild-type mice (n=54) were categorized into group 1 (sham control, n=18) and group 3 [AIS by permanent ligation of left common carotid artery (CCA) and cramping right CCA for 1h and then reperfusion followed by hypoxia (11% of oxygen supply for 2h), n=36]. Similarly, tPA knockout (tPA(-/-)) mice (n=54) were randomized into group 2 (sham control, n=18) and group 4 (AIS, n=36). By day 28 after AIS procedure, mortality rate was higher in group 3 (77.8%) than in group 4 (38.9%) and lowest in groups 1 (0%) and 2 (0%) (p<0.001). By days 3 and 28, MRI demonstrated a pattern of changes in brain-infarct volume identical to that of mortality among four groups (p<0.001). By day 28, protein expressions of inflammatory (MMP-9, TNF-α, NF-κB, iNOS, PAI-1, RANTES), oxidative (NOX-1, NOX-2, oxidized protein), apoptotic (cleaved caspase-3 & PARP, Bax), and fibrotic (Smad3, TGF-ß) biomarkers and cellular expressions of inflammation (CD11, F4/80, GFAP), DNA-damage (γ-H2AX) and brain-edema (AQP4) markers exhibited an identical pattern compared to that of mortality (all p<0.001), whereas protein expressions of endothelial (eNOS, CD31), anti-fibrotic (Smad1/5, BMP-2) biomarkers, and number of small vessels displayed an opposite pattern (all p<0.001) among four groups. Expressions of protein and cellular angiogenesis markers (VEGF, SDF-1α, CXCR4) were progressively increased from groups 1 and 2 to group 4 (all p<0.0001). CONCLUSION: tPA deficiency protected the brain from AIS injury.

Retina Is Protected by Neuroserpin from Ischemic/Reperfusion-Induced Injury Independent of Tissue-Type Plasminogen Activator.

The purpose of the present study was to investigate the potential neuroprotective effect of neuroserpin (NSP) on acute retinal ischemic/reperfusion-induced (IR) injury. An IR injury model was established by elevating intraocular pressure (IOP) for 60 minutes in wild type and tPA-deficient (tPA-/-) mice. Prior to IR injury, 1 µL of 20 µmol/L NSP or an equal volume of bovine serum albumin (BSA) was intravitreally administered. Retinal function was evaluated by electroretinograph (ERG) and the number of apoptotic neurons was determined via TUNEL labeling. Caspase-3, -8, -9,poly (ADP-ribose) polymerase (PARP)and their cleaved forms were subsequently analyzed. It was found that IR injury significantly damaged retinal function, inducing apoptosis in the retina, while NSP attenuated the loss of retinal function and significantly reduced the number of apoptotic neurons in both wild type and tPA-/- mice. The levels of cleaved caspase-3, cleaved PARP (the substrate of caspase-3) and caspase-9 (the modulator of the caspase-3), which had increased following IR injury, were significantly inhibited by NSP in both wild type and tPA-/- mice. NSP increased ischemic tolerance in the retina at least partially by inhibiting the intrinsic cell death signaling pathway of caspase-3. It was therefore concluded that the protective effect of neuroserpin maybe independent from its canonical interaction with a tissue-type plasminogen activator.

Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

The Tissue Fibrinolytic System Contributes to the Induction of Macrophage Function and CCL3 during Bone Repair in Mice.

Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg-/-), urokinase-type plasminogen activator (uPA-/-) or tissue-type plasminogen activator (tPA-/-) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA-/- mice until day 6, compared with wild-type (uPA+/+) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA-/- mice at the bone injury site on day 4, compared with those in uPA+/+ mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA-/- and Plg-/-, but not in tPA-/- mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-α, IL-1ß, IL-6, IL-4 and IFN-γ mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA-/- and Plg-/-, but not in tPA-/- mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA+/+, but not in uPA-/- mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.

Tissue plasminogen activator modulates emotion in a social context.

Tissue plasminogen activator (tPA) is known to play physiologically and pathologically crucial roles in the central nervous system. However, it is still obscure whether it affects social behavior. We investigated social behavior and neuronal activation after social stimulation in tPA knockout (KO) mice. In a resident-intruder test, the latency to the first contact was significantly delayed in tPA KO mice compared with that in tPA heterogenic (Het) mice. However, tPA KO mice spent significantly more time undertaking active behavior than tPA Het mice did. In a sociability test, tPA KO mice significantly spent more time and walked a greater distance in the chamber containing an empty cage than tPA Het mice. Furthermore, tPA KO mice approached an empty cage more frequently than tPA Het mice did. In a social novelty test, there was no difference in the duration spent sniffing a stimulator mouse between genotypes. However, tPA KO mice approached even a familiar mouse more frequently than tPA Het mice did. tPA KO mice spent similar durations in a chamber containing a familiar mouse and that containing an unfamiliar mouse, and tPA KO mice walked a significantly greater distance in the former chamber than tPA Het mice did. Furthermore, at the cingulate and prelimbic cortices, the number of cFos-positive cells was significantly increased in tPA KO mice compared with that in tPA Het mice after social stimulation. Our results suggest that tPA modulates emotion in a social context through the function of the prefrontal cortex.

Tissue-type plasminogen activator deficiency delays bone repair: roles of osteoblastic proliferation and vascular endothelial growth factor.

Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA(-/-)) and uPA-deficient (uPA(-/-)) mice. Bone repair of the femur was delayed in tPA(-/-) mice, unlike that in wild-type (tPA(+/+)) mice. Conversely, the bone repair was comparable between wild-type (uPA(+/+)) and uPA(-/-) mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA(-/-) mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA(-/-) mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were decreased at the damaged site in tPA(-/-) mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1α at the site of bone damage.

Tissue plasminogen activator promotes postischemic neutrophil recruitment via its proteolytic and nonproteolytic properties.

OBJECTIVE: Neutrophil infiltration of the postischemic tissue considerably contributes to organ dysfunction on ischemia/reperfusion injury. Beyond its established role in fibrinolysis, tissue-type plasminogen activator (tPA) has recently been implicated in nonfibrinolytic processes. The role of this serine protease in the recruitment process of neutrophils remains largely obscure. APPROACH AND RESULTS: Using in vivo microscopy on the postischemic cremaster muscle, neutrophil recruitment and microvascular leakage, but not fibrinogen deposition at the vessel wall, were significantly diminished in tPA(-/-) mice. Using cell transfer techniques, leukocyte and nonleukocyte tPA were found to mediate ischemia/reperfusion-elicited neutrophil responses. Intrascrotal but not intra-arterial application of recombinant tPA induced a dose-dependent increase in the recruitment of neutrophils, which was significantly higher compared with stimulation with a tPA mutant lacking catalytic activity. Whereas tPA-dependent transmigration of neutrophils was selectively reduced on the inhibition of plasmin or gelatinases, neutrophil intravascular adherence was significantly diminished on the blockade of mast cell activation or lipid mediator synthesis. Moreover, stimulation with tPA caused a significant elevation in the leakage of fluorescein isothiocyanate dextran to the perivascular tissue, which was completely abolished on neutrophil depletion. In vitro, tPA-elicited macromolecular leakage of endothelial cell layers was abrogated on the inhibition of its proteolytic activity. CONCLUSIONS: Endogenously released tPA promotes neutrophil transmigration to reperfused tissue via proteolytic activation of plasmin and gelatinases. As a consequence, tPA on transmigrating neutrophils disrupts endothelial junctions allowing circulating tPA to extravasate to the perivascular tissue, which, in turn, amplifies neutrophil recruitment through the activation of mast cells and release of lipid mediators.

Retention of endothelial progenitor cells in bone marrow in a murine model of endogenous tissue plasminogen activator (tPA) deficiency in response to critical limb ischemia.

BACKGROUND: This study tested the hypothesis that tissue plasminogen activator (tPA) is crucial for regulating endothelial progenitor cell (EPC) mobilization from bone marrow to circulation in murine critical limb ischemia (CLI) by ligating the left femoral artery. METHODS: Wild-type (C57BL/6) (n=40) mice were equally divided into group 1A (sham control), group 2A (CLI), group 3A [control-tPA (4.0 mg/kg)] and group 4A [CLI-tPA (intravenously at 3 h after CLI)]. Similarly, tPA knock-out (tPA(-/-)) mice (n=40) were equally divided into group 1B (sham control), group 2B (CLI), group 3B [control-tPA (4.0 mg/kg)], and group 4B (CLI-tPA). RESULTS: The circulating levels of EPCs (C-kit/CD31+, Sca-1/KDR+, CXCR4/CD34+) were lower in groups 1B and 2B than in groups 1A and 2A, respectively (all p<0.01), and were reversed after tPA treatment (3B vs. 3A or 4B vs. 4A, p>0.05) at 6 h and 18 h post-CLI. Levels of these biomarkers decreased again 14 days after CLI in tPA(-/-) mice compared to those in wild-type between the respective groups (all p<0.01). Laser Doppler flowmetry showed a higher ratio of ischemic-to-normal blood flow in 2A than in 2B and in 4A than in 4B by day 14 after CLI (all p<0.05). Angiogenesis at protein (CXCR4, SDF-1α, VEGF) and cellular (CXCR4+, SDF-1α+, and CD31+ cells) levels was highest in animals with CLI-tPA, significantly higher in mice with CLI only than in sham controls for both wild-type and tPA(-/-) mice (p<0.01). CONCLUSION: tPA played an essential role in augmenting circulating EPCs, angiogenesis, and blood flow in the ischemic limb in a murine model.

Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1ß and ERK signaling in the spinal cord of mice.

Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1ß) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA(-/-)) mice and astrocyte cultures. tPA(-/-) mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10mg/kg/day, s.c.), is abrogated in tPA(-/-) mice. Chronic morphine induces tPA expression in glial fibrillary acidic protein (GFAP)-expressing spinal cord astrocytes. Chronic morphine also increases IL-1ß expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1ß, and pERK expression, and the increased IL-1ß and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1ß and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of ionized calcium binding adapter molecule 1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance.

Tissue plasminogen activator regulates Purkinje neuron development and survival.

The cerebellar cortex is centrally involved in motor coordination and learning, and its sole output is provided by Purkinje neurons (PNs). Growth of PN dendrites and their major synaptic input from granule cell parallel fiber axons takes place almost entirely in the first several postnatal weeks. PNs are more vulnerable to cell death than most other neurons, but the mechanisms remain unclear. We find that the homozygous nervous (nr) mutant mouse's 10-fold-increased cerebellar tissue plasminogen activator (tPA), a part of the tPA/plasmin proteolytic system, influences several different molecular mechanisms, each regulating a key aspect of postnatal PN development, followed by selective PN necrosis, as follows. (i) Excess endogenous or exogenous tPA inhibits dendritic growth in vivo and in vitro by activating protein kinase Cγ and phosphorylation of microtubule-associated protein 2. (ii) tPA/plasmin proteolysis impairs parallel fiber-PN synaptogenesis by blocking brain-derived neurotrophic factor/tyrosine kinase receptor B signaling. (iii) Voltage-dependent anion channel 1 (a mitochondrial and plasma membrane protein) bound with kringle 5 (a peptide derived from the excess plasminogen) promotes pathological enlargement and rounding of PN mitochondria, reduces mitochondrial membrane potential, and damages plasma membranes. These abnormalities culminate in young nr PN necrosis that can be mimicked in wild-type PNs by exogenous tPA injection into cerebellum or prevented by endogenous tPA deletion in nr:tPA-knockout double mutants. In sum, excess tPA/plasmin, through separate downstream molecular mechanisms, regulates postnatal PN dendritogenesis, synaptogenesis, mitochondrial structure and function, and selective PN viability.

Plasmin-dependent proteolysis of tissue factor pathway inhibitor in a mouse model of endotoxemia.

BACKGROUND: The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. OBJECTIVE: To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. METHODS: Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue-type plasminogen (Plg) activator (t-PA) or Plg, at 2 h after LPS or saline injection. RESULTS: The sharp loss of lung-associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t-PA(-/-) and Plg(-/-) mice, which are unable to generate plasmin. CONCLUSION: The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications.

Mice deficient in urokinase-type plasminogen activator have delayed healing of tympanic membrane perforations.

Mice deficient in plasminogen, the precursor of plasmin, show completely arrested healing of tympanic membrane (TM) perforations, indicating that plasmin plays an essential role in TM healing. The activation of plasminogen to plasmin is performed by two plasminogen activators (PAs), urokinase-type PA (uPA) and tissue-type PA (tPA). To elucidate the functional roles of PAs in the healing of TM perforations, we investigated the phenotypes of single gene-deficient mice lacking uPA (uPA(-/-)) or tPA (tPA(-/-)) after TM perforation. Delayed healing of TM perforations was observed in uPA(-/-) mice but not tPA(-/-) mice. The migration of keratinocytes was clearly delayed and seemed to be misoriented in uPA(-/-) mice. Furthermore, fibrin deposition and the inflammatory response were persistent in these mice. Our findings demonstrate that uPA plays a role in the healing of TM perforations. The observed phenotypes in uPA(-/-) mice are most likely due to the reduced generation of plasmin.

Morphological assessment of the luminal surface of olfactory epithelium in mice deficient in tissue plasminogen activator following bulbectomy.

OBJECTIVE: This study aimed to investigate the function of tissue plasminogen activator in the olfactory epithelium of mice following neural injury. METHOD: Transmission electron microscopy was used to study the changes in the morphology of the olfactory epithelium 1-7 days after surgical ablation of the olfactory bulb (bulbectomy). RESULTS: Prior to bulbectomy, a uniformly fine material was observed within some regions of the olfactory epithelium of mice deficient in tissue plasminogen activator. At 2-3 days after bulbectomy, there were degenerative changes in the olfactory epithelium. At 5-7 days after bulbectomy, we noted drastic differences in olfactory epithelium morphology between mice deficient in tissue plasminogen activator and wild-type mice (comparisons were made using findings from a previous study). The microvilli seemed to be normal and olfactory vesicles and receptor neuron dendrites were largely intact in the olfactory epithelium of mice deficient in tissue plasminogen activator. CONCLUSION: The tissue plasminogen activator plasmin system may inhibit the regeneration of the olfactory epithelium in the early stages following neural injury.

Tissue plasminogen activator does not alter development of acquired epilepsy.

PURPOSE: Tissue plasminogen activator (t-PA), a proven therapy for acute ischemic stroke, is an endogenous serine protease associated with neuronal activity and synaptic plasticity in the brain. Its expression is enhanced after seizures, and is involved in seizure propagation throughout the brain. Therefore, the increased use of t-PA to treat stroke may have important implications for the development of poststroke epilepsy. Using experimental and clinical approaches, we investigated the role of t-PA in the development of epilepsy. METHODS: Mice deficient in t-PA (t-PA(-/-) ) or mice transgenically modified to overexpress neuronal t-PA (T4) underwent amygdala kindling, and seizure threshold and rates of kindling were compared to those in wild-type mice. For the clinical study, we recruited acute ischemic stroke patients who either received intravenous t-PA treatment on admission to hospital (n = 177; cases) or did not (n = 158; controls). We then assessed the incidence of early and late onset seizures and epilepsy in these patients. KEY FINDINGS: T4 mice were more seizure-prone than wild-type mice, exhibiting lower seizure thresholds (p = 0.002), but there were no significant differences observed in the rate of kindling development when comparing either T4 mice, or t-PA(-/-) mice, to their wild-type controls. Furthermore, we found no significant differences between the proportion of poststroke patients experiencing early or late seizures, or developing epilepsy, between those who received t-PA and those who did not. SIGNIFICANCE: Overexpression of endogenous t-PA lowers seizure threshold but does not influence kindling epileptogenesis. Moreover, the therapeutic administration of t-PA in humans does not influence the development of acquired poststroke epilepsy.

Endogenous tissue-type plasminogen activator impairs host defense during severe experimental Gram-negative sepsis (melioidosis)*.

OBJECTIVE: Melioidosis is a frequent cause of severe sepsis in Southeast Asia caused by the gram-negative bacterium Burkholderia pseudomallei. Patients with melioidosis have elevated circulating levels of tissue-type plasminogen activator, an important regulator of fibrinolysis. In this study, we aimed to investigate the role of tissue-type plasminogen activator during melioidosis. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Wild-type and tissue-type plasminogen activator-deficient C57BL/6 mice. INTERVENTIONS: Mice were intranasally infected with viable Burkholderia pseudomallei and killed after 24, 48, or 72 hrs for harvesting of lungs, liver, and blood. Additionally, survival studies were performed. MEASUREMENTS AND MAIN RESULTS: Experimentally induced melioidosis was associated with elevated levels of tissue-type plasminogen activator in lungs of infected wild-type mice. During infection with Burkholderia pseudomallei, tissue-type plasminogen activator-deficient mice were protected when compared to wild-type mice as demonstrated by a strongly decreased mortality (62% vs. 100% amongst wild-type mice, p < .0001), together with decreased pulmonary bacterial loads, less severe histopathological scores, and decreased fibrinolysis. These results were accompanied with an early increase in cytokine levels in tissue-type plasminogen activator-deficient mice. CONCLUSIONS: During severe gram-negative sepsis caused by Burkholderia pseudomallei, endogenous tissue-type plasminogen activator has harmful effects with respect to survival and pulmonary bacterial growth. These effects are related to tissue-type plasminogen activator-associated plasmin-induced fibrinolysis and/or a tissue-type plasminogen activator-associated decrease in proinflammatory cytokine production.