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.

Urokinase-type plasminogen activator and plasminogen mediate activation of macrophage phagocytosis during liver repair in vivo.

Urokinase-type plasminogen activator (u-PA) and plasminogen play a primary role in liver repair through the accumulation of macrophages and alteration of their phenotype. However, it is still unclear whether u-PA and plasminogen mediate the activation of macrophage phagocytosis during liver repair. Herein, we investigated the morphological changes in macrophages that accumulated at the edge of damaged tissue induced by a photochemical reaction or hepatic ischaemia-reperfusion in mice with u-PA ( u-PA-/- ) or plasminogen ( Plg-/- ) gene deficiency by using transmission electron and fluorescence microscopy. In wild-type mice, the macrophages aligned at the edge of the damaged tissue and extended a large number of long pseudopodia. These macrophages clearly engulfed cellular debris and showed well-developed organelles, including lysosome-like vacuoles, nuclei, and Golgi complexes. In wild-type mice, the distribution of the Golgi complex in these macrophages was biased towards the direction of the damaged tissue, indicating the extension of their pseudopodia in this direction. Conversely, in u-PA-/- and Plg-/- mice, the macrophages located at the edge of the damaged tissue had few pseudopodia and less developed organelles. The Golgi complex was randomly distributed in these macrophages in u-PA-/- mice. Furthermore, interferon γ and IL-4 were expressed at a low level at the border region of the damaged tissue in u-PA-/- mice. Our data provide novel evidence that u-PA and plasminogen are essential for the phagocytosis of cellular debris by macrophages during liver repair. Furthermore, u-PA plays a critical role in the induction of macrophage polarity by affecting the microenvironment at the edge of damaged tissue.

A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue-type plasminogen activator.

BACKGROUND: A synthetic nonadecapeptide (SP; GPYLMVNVTGVDGKGNELL) previously enhanced the activation of plasminogen by the SAK/plasmin complex. OBJECTIVES: To identify the binding site for SP on plasminogen and elucidate the effects of SP on plasminogen activation by the tissue-type plasminogen activator (t-PA). METHODS: The effects of SP on plasminogen activation were estimated using a chromogenic substrate and from the cleavage of plasmin on SDS-PAGE under reduced conditions. The binding to SP of various peptides derived from the amino acid sequence of plasminogen was analyzed with an IAsys biosensor. The SP-mediated structural change to plasminogen was analyzed by circular dichroism (CD) spectroscopy. The thrombolytic effects of SP were examined using a mouse model of thrombosis. RESULTS: SP enhanced the activation of plasminogen by t-PA. The catalytic efficiency (k(cat)/K(m)) of Glu-plasminogen activation by t-PA was 11.4-fold higher in the presence than absence of SP. The binding of SP to plasminogen was greatly inhibited by a synthetic peptide, FEKDKYILQGVTSWGLG, located close to the C-terminal of the plasminogen B region. Near-ultraviolet CD spectra of the complex between SP and Glu-plasminogen significantly differed from those of Glu-plasminogen. When SP was administered in a mouse model of thrombosis, early recanalization was observed in a dose-dependent manner. However, SP did not cause recanalization in t-PA gene-deficient mice. CONCLUSIONS: SP bound to the B region and promoted the activation of plasminogen by t-PA, and then induced effective thrombolysis.

Urokinase-type plasminogen activator contributes to heterogeneity of macrophages at the border of damaged site during liver repair in mice.

Urokinase-type plasminogen activator (u-PA) plays an important role in tissue remodelling through the activation of plasminogen in the liver, but its mechanisms are less well known. Here, we investigated the involvement of u-PA in the accumulation and phenotypic heterogeneity of macrophages at the damaged site during liver repair. After induction of liver injury by photochemical reaction in mice, the subsequent pathological responses and expression of phenotypic markers in activated macrophages were analysed histologically. Fibrinolytic activity at the damaged site was also examined by fibrin zymography. In wild-type mice, the extent of damage decreased gradually until day 14 and was associated with an accumulation of macrophages at the border of the damaged site. In addition, the macrophages that accumulated near the damaged tissue expressed CD206, a marker of highly phagocytic macrophages, on day 7. Further, macrophages that were adjacent to CD206-positive cells expressed inducible nitric oxide synthase (iNOS), a pro-inflammatory marker. u-PA activity increased at the damaged site on days 4 and 7, which distributed primarily at the border region. In contrast, in u-PA-deficient mice, the decrease in damage size and the accumulation of macrophages were impaired. Further, neither CD206 nor iNOS was expressed in the macrophages that accumulated at the border region in u-PA-deficient mice. Mice deficient for the gene encoding either u-PA receptor (u-PAR) or tissue-type plasminogen activator experienced normal recovery during liver repair. These data indicate that u-PA mediates the accumulation of macrophages and their phenotypic heterogeneity at the border of damaged sites through u-PAR-independent mechanisms.

Plasminogen is essential for granulation tissue formation during the recovery process after liver injury in mice.

SUMMARY BACKGROUND: The involvement of plasminogen in liver repair has been reported, but its exact role in promoting this process is unknown. OBJECTIVE: To elucidate the underlying mechanism, we examined the dynamics of liver repair by using a reproducible liver injury model in plasminogen gene-deficient mice and their wild-type littermates. METHODS: Liver injury was induced by photochemical reaction and the subsequent responses were histologically analyzed. RESULTS: In wild-type animals, the area of the damage successively decreased, and the repair process was associated with macrophage accumulation at its border. Neutrophils were also attracted to the damaged region on day 1 and were evident only at its border by day 4, which spatially and temporally coincided with the expression of macrophage chemoattractant protein-1 (MCP-1). Neutrophil depletion suppressed recruitment of macrophages at the border between the damaged and the normal tissues. These changes were followed by activated hepatic stellate cell accumulation, collagen fiber deposition and angiogenesis at the boundaries of the injured zone. In contrast, in plasminogen gene-deficient mice, the decrease in the area of damage, macrophage accumulation, late-phase neutrophil recruitment, hepatic stellate cell accumulation, collagen fiber deposition and angiogenesis were all impaired. CONCLUSION: Our data suggest that accumulated neutrophils at the border of the damaged area may contribute to macrophage accumulation at granulation tissue via the production of MCP-1 after liver injury. The plasminogen system is critical for liver repair by facilitating macrophage accumulation and triggering a cascade of subsequent repair events.

Alpha2-antiplasmin is involved in the production of transforming growth factor beta1 and fibrosis.

BACKGROUND: Fibrotic disease occurs in most tissues. Transforming growth factor (TGF)-beta is the major inducer of fibrosis. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices. However, the detailed mechanism of how this system affects fibrosis remains unclear. METHODS AND RESULTS: We examined experimental fibrosis in mice with a deficiency of alpha(2)-antiplasmin (alpha2AP), which is a potent and specific plasmin inhibitor. We found that the lack of alpha2AP attenuated bleomycin-induced TGF-beta(1) synthesis and fibrosis. In addition, the production of TGF-beta(1) from the explanted fibroblasts of alpha2AP(-/-) mice decreased dramatically as compared to that in wild-type mice. Moreover, we found that alpha2AP specifically induces the production of TGF-beta(1) in fibroblasts. CONCLUSION: The lack of alpha2AP attenuated TGF-beta(1) synthesis, thereby resulting in attenuated fibrosis. This is the first report to describe the crucial role that alpha2AP plays in TGF-beta(1) synthesis during the process of fibrosis. Our results provide new insights into the role of alpha2AP in fibrosis.

A guide to murine fibrinolytic factor structure, function, assays, and genetic alterations.

The components and functions of the murine fibrinolytic system are quite similar to those of humans. Because of these similarities and the adaptability of mice to genetic manipulation, murine fibrinolysis has been studied extensively. These studies have yielded important information regarding the function of the several components of fibrinolysis. This review presents information on the structure, function and assay of mouse fibrinolytic parameters and it discusses the results of the extensive studies of genetically modified mice. It is intended to be a convenient reference resource for investigators of fibrinolysis.

Lack of alpha2-antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions.

BACKGROUND: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. METHODS AND RESULTS: We performed experimental cutaneous wounding in mice with a deficiency of alpha(2)-antiplasmin (alpha(2)AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in alpha(2)AP-deficient (alpha(2)AP-/-) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in alpha(2)AP-/- mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in alpha(2)AP-/- mice than in WT mice. In addition, the amount of released-VEGF from the explanted fibroblasts of alpha(2)AP-/- mice increased dramatically more than in the WT mice. Finally, the intra-jugular administration of anti-VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of alpha(2)AP-/- mice. CONCLUSION: The lack of alpha(2)AP markedly causes an over-release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated-wound closure. CONCLUSIONS: This is the first report to describe the crucial role that alpha(2)AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of alpha(2)AP on cutaneous wound healing.

Alpha2-antiplasmin plays a significant role in acute pulmonary embolism.

The importance of pulmonary embolism (PE) due to venous thrombosis is recognized in the treatment of vascular diseases. We have investigated the physiological effects of plasmin generation in experimental acute PE using mice deficient in plasminogen (Plg-/-) or alpha2-antiplasmin (alpha2-AP-/-). PE was induced by continuous induction of venous thrombus in the left jugular vein by endothelial injury due to photochemical reaction. The mortality of wild-type mice was 68.8% at 2 h after the initiation of venous thrombosis and it was significantly reduced in alpha2-AP-/- mice (41.7%). In contrast, Plg-/- mice did not survive. Histological evidence of thromboembolism in the lung was obtained in all mice. However, whereas a strict thromboembolism was observed in Plg-/- mice, only a few thrombi were detected in the lungs of alpha2-AP-/- mice. Plasma fibrinogen levels measured in mice were not different. When alpha2-AP was infused in alpha2-AP-/- mice, the mortality was indistinguishable from wild-type mice. Tissue-type plasminogen activator (tPA) did not reduce the mortality due to acute PE in wild-type mice. However, in alpha2-AP-/- mice, tPA (0.52 mg x kg-1) significantly decreased the mortality compared with that of alpha2-AP-/- mice without tPA. The bleeding time was not significantly prolonged in either type of mice treated with tPA. The lack of plasminogen increases the mortality due to acute PE while a lack of alpha2-AP decreases the mortality rate, which can be further reduced by tPA administration. Therefore, the combination of inhibition of alpha2-AP with thrombolytic therapy could be beneficial in the treatment of acute PE.

Mechanism of retarded liver regeneration in plasminogen activator-deficient mice: impaired activation of hepatocyte growth factor after Fas-mediated massive hepatic apoptosis.

Urokinase-type plasminogen activator (uPA) is implicated in the regulation of hepatic regeneration by activating hepatocyte growth factor (HGF). Here, we investigated its role in the hepatic regeneration after Fas-mediated massive hepatocyte death employing mice deficient in either uPA or its inhibitor, plasminogen activator inhibitor-1 (PAI-1). We measured kinetics of hepatic levels of proliferating cell nuclear antigen (PCNA)-labeling index, plasmin activity, mature HGF, and its phosphorylated receptor, c-Met. In the genetically targeted and wild-type mice, hepatocytes fell into the same extent of apoptosis 6 to 12 hours after an intraperitoneal injection with anti-Fas antibody, as judged from histologic analysis and a histon-DNA enzyme-linked immunosorbent assay (ELISA). In the wild-type mice, mature HGF emerged in the liver 6 hours following anti-Fas injection, and hepatic PCNA-labeling index started to increase following 24 hours and peaked at 48 hours. In the uPA(-/-) mice, emergence of mature HGF was delayed 12 hours and hepatic regeneration peaked at 96 hours. Supplementation with the uPA gene to the uPA(-/-) mice by in vivo lipofection restored hepatic plasmin levels, and improved a delay in the expression of both mature HGF and phosphorylated c-Met, accompanying a normal rate of liver regeneration. In contrast, PAI-1(-/-) mice showed accelerated liver regeneration; mature HGF emerged as early as 3 hours, and PCNA-labeling index increased at 24 hours. This accelerated regeneration was abolished by administration with anti-HGF antibody. These results strongly suggest a physiologic role of uPA in the proteolytic maturation of HGF, and thereby in hepatic regeneration after Fas-mediated massive hepatocyte death.

tPA, but not uPA, significantly affects antithrombotic therapy by a glycoprotein IIb/IIIa antagonist, but not by a factor Xa inhibitor.

To define the interaction of fibrinolytic components with platelets or coagulation factors on thrombus formation, we investigated mouse deficient in tissue plasminogen activator (tPA -/-) or urokinase plasminogen activator (uPA -/-) and in their wild-type control (tPA +/+, uPA +/+). A thrombus was induced in the murine carotid artery using photochemical reaction. Blood flow was monitored and the time needed before the vessel became completely obstructed was within 12 min in all types of mice. When DX-9065a, a selective factor Xa inhibitor, or GR144053, a platelet glycoprotein (GP) complex IIb/IIIa antagonist was applied, the time required to occlusion was prolonged in a dose-dependent manner in all types of mice. When a factor Xa inhibitor was injected in tPA -/- mice, the estimated ED50 was not changed. However, when GR144053 was injected in tPA -/- mice, the most significant changes were observed: the estimated ED51 was 19.6 times higher than the one in tPA +/+ mice. Platelet aggregation, hemostasis tests, and bleeding times were not significantly different among the different types of mice. In conclusion, the antithrombotic effect of platelet inhibition by a GPIIb/IIIa antagonist, is severely affected by the absence or presence of tPA production. On the contrary, the inhibition of factor Xa shows a stable antithrombotic effect with or without tPA. Thus the lack of tPA, but not of uPA, significantly affects antithrombotic efficacy.

The effect of argatroban on injured endothelial cells by thrombin.

When endothelial cells are exposed to thrombin, they become perturbed and acquire thrombogenic properties. Argatroban is an arginine derivative, synthetic small molecule that binds to the active site of thrombin and inhibits its catalytic activity. Therefore, the effects of argatroban on endothelial cells, which had been injured by thrombin, were investigated. The established endothelial cell line, TKM-33, which had been cloned from human umbilical vein endothelial cells, was used. Endothelial cells produce plasminogen activator (PA) to prevent thrombosis and maintain the blood flow. When the endothelial cells were injured by thrombin, secretion of plasminogen activator inhibitor-1 (PAI-1) increased and then the PA activity proportionally decreased. The treatment of endothelial cells with argatroban after thrombin injury did not restore their reduced PA activity. However, the treatment of endothelial cells with argatroban prior to thrombin injury resulted in inhibiting the induction of PAI-1 secretion. Thus, pretreatment of endothelial cells with argatroban suppresses the inhibition of their PA activity by thrombin. Since the effect of thrombolytic agent may be modified by the fibrinolytic factors produced by the endothelial cells, the activity of staphylokinase (SAK) was measured in the presence of endothelial cells that had been injured by thrombin. SAK is a newly developed thrombolytic agent. SAK activity in the presence of injured endothelial cells by thrombin was lower than that in the presence of endothelial cells without thrombin injury. However, treatment of endothelial cells with argatroban prior to thrombin injury revealed higher SAK activity than that after thrombin injury. These findings indicate that argatroban pretreatment prevents thrombin injury of endothelial cells, which may then maintain their physiological function.

Analysis of plasminogen activation by the plasmin-staphylokinase complex in plasma of alpha2-antiplasmin-deficient mice.

Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin; this PA activity is inhibited by alpha2-antiplasmin (alpha2-AP) in plasma. However, SAK's activity is protected against inhibition by alpha2-AP in the presence of fibrin because the plasmin-SAK complex binds to fibrin. In the present study, the interaction between SAK and murine plasminogen was investigated in the plasma of alpha2-AP-deficient (alpha2-AP-/-) mice or plasminogen-deficient (Plg-/-) mice. Although the human plasmin-SAK complex was formed in equimolar mixtures of plasmin and SAK, the murine plasmin-SAK complex was not formed. Human plasminogen was activated by the human plasmin-SAK complex, although equimolar mixtures of murine plasmin and SAK did not activate murine plasminogen. These findings suggest that SAK does not react with murine plasmin. However, the murine plasminogen was activated by the human plasmin-SAK complex, although this activation was approximately 100-fold weaker than human plasminogen. Human and wild-type mouse plasminogens were not activated by the human plasmin-SAK complex in their plasma. In alpha2-AP-/- mouse plasma, murine plasminogen was activated by the human plasmin-SAK complex. Human or murine plasminogen, which had been added to Plg-/- mouse plasma, was not activated by the human plasmin-SAK complex. However, plasma clot lysis by the human plasmin-SAK complex was observed in both human and murine plasma. These findings indicate that: (1) murine plasmin does not react with SAK, (2) human plasmin-SAK complex activates murine plasminogen, (3) this activation is inhibited by murine alpha2-AP, but (4) this activation is not inhibited by murine alpha2-AP in the presence of fibrin.

The interaction between components of the fibrinolytic system and GPIb/V/IX of platelets thrombus formation in mice.

The interaction of fibrinolytic components with GPIb/V/IX of platelets on thrombus formation, was investigated in mice deficient in tissue type (tPA-/-), urokinase type plasminogen activator (uPA-/-) or plasminogen activator inhibitor-1 (PAI-1-/-) and in their wild type control (tPA+/+, uPA+/+, PAI-1+/+). A thrombus was induced in the murine carotid artery using a photochemical reaction. The times to occlusion after the initiation of endothelial injury in all wild type mice was within 12 min, and no significant changes in occlusion delay were observed in uPA-/- and tPA-/- mice compared to wild type mice, whereas that of PAI-1 mice were significantly prolonged (16.9+/-2.9 min, P<0.05). When high molecular weight aurintricarboxylic acid (ATA), an inhibitor of platelet glycoprotein Ib/V/IX, was administered, the time to occlusion was prolonged in a dose-dependent manner in all types of mice. However, when this compound was injected in tPA-/- mice, the most significant changes were observed: i.e. the estimated ED(50) was 20.2 times higher than that in tPA+/+ mice, but the estimated ED(50) in uPA-/- mice was not changed as compared with that of wild type mice. On the other hand, when ATA was injected in PAI-1-/- mice, the estimated ED(50) was significantly decreased (P<0.05). Platelet aggregation induced by botrocetin was not significantly different among all types of mice. The bleeding time was prolonged in a dose dependent-manner when ATA was injected in all types of mice. In conclusion, the antithrombotic effect of inhibition of platelet GPIb/V/IX is severely affected by the absence or presence of tPA-production on thrombus formation and the inhibition of PAI-1 could enhance this antithrombotic effect.

Binding of mutant tissue-type plasminogen activators to human endothelial cells and their extracellular matrix.

We previously demonstrated that tissue-type plasminogen activator (t-PA) specifically bound to its receptor (t-PAR) on human umbilical vein endothelial cells (HUVEC). In addition to analyses of t-PA binding to plasminogen activator inhibitor-1 (PAI-1) in the extracellular matrix (ECM) and to the t-PAR, we further evaluated the binding of three t-PA mutants, deltaFE1X t-PA lacking finger (F), epidermal growth factor-like (E) domains and one sugar chain at Asn177 thus comprising two kringles (K1 and K2) and protease (P) domains, deltaFE3X t-PA with three glycosylation sites deleted at Asn117, 184, and 448, and deltaFEK1 t-PA comprising K2 and P domains without glycosylation. Wild-type t-PA bound to ECM with high affinity, which was completely blocked by anti-PAI-1 IgG. Wild-type t-PA, deltaFE1X t-PA and deltaFEK1 t-PA bound to two classes of binding sites with high and low affinities on monolayer HUVEC. However, all t-PAs bound to a single class of binding site in the presence of anti-PAI-1 IgG. DeltaFEK1 t-PA bound t-PAR maximally among these t-PAs. These results suggested that the high affinity binding of t-PA mainly occurred with PAI-1 on ECM while the low affinity binding was with t-PAR. The deletion of F, E domains and sugar chains had no effect on binding with t-PAR. However, since only K1-missing t-PA (deltaFEK1) exhibited significantly increased binding sites among these t-PAs, it was suggested that the binding to t-PAR was mediated mainly by K2 domain and that the increase of binding was due to direct exposure of K2 domain.

Antithrombotic regulation in human endothelial cells by fibrinolytic factors.

Vascular endothelial cells (ECs) modulate the blood fibrinolytic system by secreting tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and their inhibitor, type-1 plasminogen activator inhibitor (PAI-1). ECs also express t-PA receptors (t-PAR) and u-PA receptors (u-PAR) on their cell surfaces, assembling both enzymes to regulate the cellular fibrinolytic activity. In addition, ECs modulate these factors in response to several stimuli. Fibrin clots on ECs induce the up- and downregulation of t-PA and PAI-1 production, respectively, thus causing an effective lysis of the fibrin clot. Heat shock (43 degrees C) increases the expression of u-PA, t-PA, PAI-1, and u-PAR by which ECs become more fibrinolytic around the cells. Furthermore, because ECs possess t-PAR and u-PAR on their cell surfaces, the binding of t-PA and u-PA is a critical event, which affords ECs the localized and condensed fibrinolytic potential. Therefore, ECs play a central role in antithrombotic activity by regulating the levels of these fibrinolytic factors.

Effect of hyperthermia on the viability and the fibrinolytic potential of human cancer cell lines.

The effects of heat treatment on the viability and fibrinolytic potential of four cultured human carcinoma cell lines, fibrosarcoma cells (HT-1080), lung adenocarcinoma cells with highly metastatic potential (HAL-8), melanoma cells (Bowes) and osteosarcoma cells (NY), determined by measuring their levels of urokinase-type plasminogen activator (u-PA) and its specific receptor (u-PAR), were investigated by comparing them with those of human umbilical vein endothelial cells (HUVECs). HUVECs incubated at 43 degrees C for 120 min exhibited no decrease in viability but exhibited an increase in both u-PA and u-PAR. HT-1080 and HAL-8 showed a moderately high heat-resistance (viability, 60-90%) that correlated with the reduction of u-PAR but not u-PA. On the other hand, Bowes and NY cells, with poor heat-resistance (viability, 20-50%), exhibited stronger cell-associated u-PA activity when they survived at 43 degrees C for 120 min. Since the u-PA/u-PAR system is directly involved in the invasiveness and metastatic potential of carcinoma cells, hyperthermia would alter the biological activity of these carcinoma cells.