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Coriandrum sativum (coriander) is an annual herb in the Apiaceae family. Its leaves and seeds are used for cooking. Coriander has several beneficial functions such as anti-inflammatory, analgesic and anti-cancer effects. Although anti-carcinogenic potential of coriander has been known well, the effects of coriander on cancer metastasis have not yet been fully elucidated. In the present study, the effects of coriander on migration and invasion were investigated in vitro and in vivo by using human hepatocellular carcinoma cell line (HepG2) and mouse melanoma cell line (B16F10). The migration and invasion abilities of cancer cells had been evaluated by trans-well double chamber and these abilities were significantly impaired by treatment of cancer cells with coriander extract whose concentration did not affect proliferation. The treatment of cancer cells with coriander extract significantly reduced both matrix metalloproteinase 2 (MMP-2) and urokinase-type plasminogen activator (u-PA) activities, which were involved in cell migration and invasion, in their conditioned media. Furthermore, coriander extract suppressed the phosphorylation of Erk 1 or IkB in B16F10 cells, and inhibited the expression of MMP-2 or u-PA mRNA. After injection of B16F10 cells into the tail vein of C57BL/6J mice, the number of metastatic regions in lungs were counted. Mice fed with diet containing coriander possessed a smaller number of metastatic regions than those fed with control diet. It was suggested that coriander extract might have the abilities to suppress cancer cell migration and invasion, indicating that coriander provides the improvement of cancer prognosis.

Dysfunction of vascular endothelial cells causes the risk of thrombosis. Aim of this study is to evaluate the antithrombotic effect of Okinawa mozuku (brown seaweed, Cladosiphon okamuranus) extract by using cultured vascular endothelial cells and rat carotid arterial thrombosis model induced by ferric chloride (FeCl3). The cell line (TKM-33) established from human umbilical vein endothelial cells were cultured with or without Okinawa mozuku extract. After incubation for 24 h, the conditioned medium was collected to evaluate urokinase-type plasminogen activator (u-PA) activity. Next, rats were fed with water or water containing 5% of Okinawa mozuku extract for 8 wk. After 8 wk of treatments, the rats were provided for the carotid arterial thrombosis model, and fibrinolytic factor and coagulation factor in blood were measured. Okinawa mozuku extract significantly augmented u-PA activity in the conditioned medium. The decrease of carotid artery blood flow induced by 40% FeCl3 injury in rats fed with Okinawa mozuku extract was less than that in control rats. Thus, oral administration of Okinawa mozuku extract prevented thrombus formation in this model. Oral administration of Okinawa mozuku extract significantly increased u-PA activity in euglobulin fraction, compared with control group. On the other hand, platelet aggregation activity, activated partial thromboplastin time, and active PAI-1 level in plasma exhibited no significant differences between control and Okinawa mozuku groups. These results indicate that oral administration of Okinawa mozuku enhances fibrinolytic activity in plasma and prevents thrombus formation which is induced by injury of vascular endothelial cells.

Coriandrum sativum (coriander) is an annual herb in the Apiaceae family. Its leaves and seeds are used for cooking. Coriander has several beneficial functions such as anti-inflammatory, analgesic and anti-cancer effects. Although anti-carcinogenic potential of coriander has been known well, the effects of coriander on cancer metastasis have not yet been fully elucidated. In the present study, the effects of coriander on migration and invasion were investigated in vitro and in vivo by using human hepatocellular carcinoma cell line (HepG2) and mouse melanoma cell line (B16F10). The migration and invasion abilities of cancer cells had been evaluated by trans-well double chamber and these abilities were significantly impaired by treatment of cancer cells with coriander extract whose concentration did not affect proliferation. The treatment of cancer cells with coriander extract significantly reduced both matrix metalloproteinase 2 (MMP-2) and urokinase-type plasminogen activator (u-PA) activities, which were involved in cell migration and invasion, in their conditioned media. Furthermore, coriander extract suppressed the phosphorylation of Erk 1 or IkB in B16F10 cells, and inhibited the expression of MMP-2 or u-PA mRNA. After injection of B16F10 cells into the tail vein of C57BL/6J mice, the number of metastatic regions in lungs were counted. Mice fed with diet containing coriander possessed a smaller number of metastatic regions than those fed with control diet. It was suggested that coriander extract might have the abilities to suppress cancer cell migration and invasion, indicating that coriander provides the improvement of cancer prognosis.

Diabetes-induced podocyte apoptosis is considered to play a critical role in the pathogenesis of diabetic kidney disease (DKD). We proposed that hyperglycaemia can induce podocyte apoptosis by inhibiting the action of podocyte survival factors, thus inactivating the cellular effects of insulin signalling. In this study, we aimed to determine the effects of linagliptin on high glucose-induced podocyte apoptosis. Linagliptin reduced the increase in DNA fragmentation as well as the increase in TUNEL-positive cells in podocytes induced by high-glucose condition. Furthermore, linagliptin improved insulin-induced phosphorylation of insulin receptor substrate 1 (IRS1) and Akt, which was inhibited in high-glucose conditions. Adenoviral vector-mediated IRS1 overexpression in podocytes partially normalised DNA fragmentation in high-glucose conditions, while downregulation of IRS1 expression using small interfering RNA increased DNA fragmentation even in low-glucose conditions. Because reactive oxygen species inhibit glomerular insulin signalling in diabetes and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is one of the most important intrinsic antioxidative systems, we evaluated whether linagliptin increased Nrf2 in podocytes. High-glucose condition and linagliptin addition increased Nrf2 levels compared to low-glucose conditions. In summary, linagliptin offers protection against DKD by enhancing IRS1/Akt insulin signalling in podocytes and partially via the Keap1/Nrf2 pathway. Our findings suggest that linagliptin may induce protective effects in patients with DKD, and increasing IRS1 levels could be a potential therapeutic target in DKD.

The efficacy of objective structured skill examination (OSSE) was evaluated as a new and useful examination. The second grade students at Kinki University Faculty of Medicine were prospectively eligible. Students took problem-based learning (PBL) tutorials in addition to classroom lectures and laboratory practice in each session. The sessions were on: cardiac, circulation, hematological, and renal systems. At the end of each session, students took the paper examination as a part of conventional evaluation. After all sessions, OSSE was performed to evaluate the students’ performance. The technical skills evaluated in OSSE were as follows: electrocardiogram recording, blood pressure measuring, use of centrifuge, pipette technique, hematocrit measurement, urinary specific gravity determination, and measurement of urine volume. One order sheet and the experimental apparatus for OSSE were positioned at each station and students terminate the maneuver in 2 min. The score from OSSE was compared to that from the conventional evaluation. Significant positive correlation was not observed. More than half of the students considered OSSE to be meaningful. In conclusion, we developed a new structured examination to objectively assess the students' technical skills in laboratory practice. Key words: curriculum reform, laboratory practice, objective structured skill examination, physiology.

Objective Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs. Patients with SSc have enhanced plasma levels of the plasmin–α2-antiplasmin (α2AP) complex, and we recently implicated α2AP in the development of fibrosis through transforming growth factor β (TGFβ) production. This study was undertaken to clarify how α2AP induces TGFβ production and the development of fibrosis. Methods To clarify the detailed mechanism by which α2AP induces TGFβ production, we focused on adipose triglyceride lipase (ATGL)/calcium-independent phospholipase A2 (iPLA2) and examined whether ATGL/ iPLA2 is associated with α2AP-induced TGFβ production. The mouse model of bleomycin-induced SSc was used to evaluate the role of α2AP in the development of fibrosis. Dermal thickness and collagen content were determined in mouse skin treated with phosphate buffered saline or bleomycin. Moreover, we cultured SSc-like fibroblasts from the bleomycin-treated mouse skin and examined the production of TGFβ and prostaglandin F2α (PGF2α). Results We found that α2AP binding to ATGL promoted PGF2α synthesis through iPLA2 in fibroblasts, and the PGF2α synthesis that was promoted by α2AP induced TGFβ production in fibroblasts. In addition, the neutralization of α2AP attenuated the production of TGFβ and PGF2α in SSc-like fibroblasts from mice. The α2AP deficiency attenuated bleomycin-induced fibrosis and PGF2α synthesis, while the administration of PGF2α to α2AP-deficient mice facilitated α2AP deficiency–attenuated fibrosis. Conclusion These findings suggest that α2AP regulates the development of fibrosis by PGF2α synthesis through ATGL/iPLA2. The inhibition of α2AP-initiated pathways might provide a novel therapeutic approach to fibrotic diseases.

Oxidative stress is implicated in the pathologies of vascular endothelial cells. However, the importance of specific antioxidant enzymes in vascular endothelial cells is not fully understood. The purpose of this study was to elucidate the importance of Glutathione peroxidase 4 (GPx4), and the involvement of ferroptosis on cell death induced by GPx4 loss in human vascular endothelial cells. In addition, we examined the compensatory activity of brown rice on GPx4 ablation condition. Human umbilical vein endothelial cells were transfected with GPx4 or scramble control siRNA. GPx4 knockdown caused the increase in the levels of lipid oxidation, and induced cytotoxicity. On the other hand, α-tocopherol (vitamin E) and extract of brown rice, ameliorated lipid peroxidation, cytotoxicity, and delay of proliferation induced by GPx4 knockdown. Furthermore, ferrostatin-1, inhibitor of ferroptosis, also prevented cytotoxicity and delay of proliferation. In conclusion, our data demonstrated that GPx4 is an essential antioxidant enzyme for protecting lipid peroxidation, and is a regulator of ferroptosis in vascular endothelial cells. Furthermore, vitamin E rich food, such as brown rice, can compensate for GPx4 loss by protecting cells against lipid peroxidation.

SummaryUrokinase-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-reperfu-sion 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.

To cite this article: Okada K, Ueshima S, Matsuno H, Nagai N, Kawao N, Tanaka M, Matsuo O. A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue-type plasminogen activator. J Thromb Haemost 2011; 9: 997–1006. Summary. 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-typeplasminogenactivator(t-PA). Methods: Theeffectsof 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 (kcat/ Km) 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 mousemodelofthrombosis,earlyrecanalizationwasobservedin a dose-dependent manner. However, SP did not cause recanalization int-PAgene-deficientmice. Conclusions: SPboundtothe B region and promoted the activation of plasminogen by t-PA, and then induced effective thrombolysis.

SummaryUrokinase-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.

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.

As stem cells can regenerate damaged tissue, their therapeutic potential on brain damage has been investigated. In this study, the effects of embryonic stem cell transplantation on brain damage were investigated by using a photochemically induced thrombotic brain damage model. Mice with systemic transplantation of embryonic stem cells expressing enhanced green fluorescence protein on day 1 showed a smaller brain lesion size on day 8 than the control mice. The smaller lesion was accompanied by the increase in the number of microvessels at the border of the damaged area. Inside and around the damaged lesion, no EGFP-positive cells were observed. These findings suggested that embryonic stem cell transplantation reduced the brain lesion through the acceleration of angiogenesis by endogenous endothelial cells.

Introduction Although the involvement of plasminogen in liver repair has been reported, its roles are still poorly understood. Here, we investigated the role of plasminogen in accumulations of macrophages and neutrophils after liver injury in mice with gene deficient of plasminogen ( Plg −/− ) or its wild type ( Plg +/+ ). Materials and Methods Mice received traumatic liver injury caused by stabbing on the lobe or hepatic ischemia-reperfusion, and the damaged sites were histologically analyzed. Results After the traumatic liver injury, both the stab wound and the damaged tissue were decreased until day 7 in the Plg +/+ mice. In contrast, both the stab wound and the damaged tissue were still remained until day 7 in the Plg −/− mice. On day 4 after traumatic liver injury, macrophages were abundant at the surrounding area of the damaged site in the Plg +/+ mice. However, the macrophage accumulation was impaired in the Plg −/− mice. After hepatic ischemia-reperfusion injury, macrophage accumulation and decrease in the damaged tissue were also observed in the Plg +/+ mice until day 7. In contrast, these responses were also impaired in the Plg −/− mice. Furthermore, neutrophil accumulation at the surrounding area of the damaged site was also impaired in the Plg −/− mice on day 4 after both liver traumatic liver injury and hepatic ischemia-reperfusion injury. Conclusions Our data indicate that plasminogen plays a crucial role in macrophage accumulation together with the neutrophil accumulation after liver injury in both models, which may be essential for triggering the subsequent healing responses including decrease in the damaged tissue.

SummaryUrokinase-type plasminogen activator receptor (uPAR) plays a role in cellular responses which include cellular adhesion, differentiation, proliferation and migration. The aim of this study was to clarify the role of uPAR on the development of adipose tissue. To clarify the role of uPAR on adipogenesis, we examined the effect of uPAR overexpression and uPAR deficiency on the adipocyte differentiation. Adipocyte differentiation was induced by incubation of 3T3-L1 cells with differentiation media containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthin. uPAR overexpression by transfection of uPAR expression vector induced adipocyte differentiation. In addition, we examined the difference in adipocyte differentiation of mesenchymal stem cells from wild-type mice and uPAR knockout (uPAR-/-) mice. The uPAR deficiency attenuated differentiation media-induced adipocyte differentiation. Moreover, we found that the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway attenuated uPAR overexpression-induced adipocyte differentiation, and uPAR overexpression induced the activation of Akt. We also found that an increase of the adipose tissue mass in uPAR-/- mice was less than that observed in wild-type mice. The present results suggest that uPAR plays a pivotal role in the development of adipose tissue through PI3K/Akt pathway.

Systemic sclerosis results in tissue fibrosis due to the activation of fibroblasts and the ensuing overproduction of the extracellular matrix. We previously reported that the absence of alpha2-antiplasmin (alpha2AP) attenuated the process of dermal fibrosis; however, the detailed mechanism of how alpha2AP affects the progression of fibrosis remained unclear. The goal of the present study was to examine the role of alpha2AP in fibrotic change. We observed significantly higher levels of alpha2AP expression in the skin of bleomycin-injected systemic sclerosis model mice in comparison with the levels seen in control mice. We also demonstrated that alpha2AP induced myofibroblast differentiation, and the absence of alpha2AP attenuated the induction of myofibroblast differentiation. Moreover, we found that connective tissue growth factor induced the expression of alpha2AP through both the extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways in fibroblasts. Interestingly, alpha2AP also induced transforming growth factor-beta expression through the same pathways, and the inhibition of ERK1/2 and JNK slowed the progression of bleomycin-induced fibrosis. Our findings suggest that alpha2AP is associated with the progression of fibrosis, and regulation of alpha2AP expression by the ERK1/2 and JNK pathways may be an effective antifibrotic therapy for the treatment of systemic sclerosis.

Plasmin is an essential enzyme located in the pericellular microenvironment of liver cells during liver regeneration. Previously, we reported that liver regeneration ability was significantly increased in alpha2-antiplasmin gene knockout mice as compared to wild-type mice, but it was significantly decreased in plasminogen knockout mice, or Plg/alpha2-antiplasmin gene knockout mice. The present study aimed to demonstrate direct interaction between plasminogen and mouse hepatocytes in the process of liver regeneration. Using the isolated hepatocytes from mice we analyzed following subjects: binding capacity of plasminogen to hepatocytes, plasminogen activation in the presence of hepatocytes, and proliferation ability of hepatocytes cocultured with liver nonparenchymal cells. The isolated hepatocytes from plasminogen wild-type mice bound to immobilized plasminogen. The mouse hepatocytes enhanced plasminogen activation, and impaired the inhibitory effect of alpha2-antiplasmin. The proliferation ability of hepatocytes after liver injury was studied. In plasminogen wild-type and plasminogen knockout mice, the hepatocytes cocultured with nonparenchymal cells, which were obtained from mice without CCl4 injection, showed similar proliferation abilities. On the contrary, the proliferation ability of hepatocytes cocultured with nonparenchymal cells, which were obtained from CCl4-treated plasminogen knockout mice, was significantly impaired as compared to wild-type mice. These results indicate that the plasminogen-plasmin system on the surface of mouse hepatocytes plays an important role in liver regeneration.

Objective—Alpha2-antiplasmin (α2-AP) is the major circulating inhibitor of plasmin, which plays a determining role in the regulation of intravascular fibrinolysis. We investigated the role of α2-AP on vascular remodeling in response to angiotensin II (Ang II).Methods and Results—α2-AP–deficient mice were performed. Ang II andNω-nitro- L-arginine methyl ester (L-NAME)–induced perivascular fibrosis was significantly decreased in α2-AP−/−mice compared with wild-type mice. In situ gelatinolytic activity analysis shows that perivascular gelatinolytic activity was increased in α2-AP−/−mice, which was responsible for decreased perivascular fibrosis in response to Ang II and L-NAME. Ang II–induced arterial wall thickening, vascular cell proliferation, apoptosis, c-Myc, and collagen Ι expression were significantly decreased in α2-AP−/−mice compared with wild-type mice. Further analysis shows that increased p53 and p21 expression were responsible for inhibition of Ang II–induced vascular remodeling in α2-AP−/−mice.Conclusion—The results show that α2-AP is a critical regulator for vascular remodeling by inhibiting p53/p21 pathway, suggesting that α2-AP is proposed to be a potential therapeutic target for vascular remodeling.

Urokinase-type plasminogen activator receptor (uPAR) is a key component of the plasminogen activation system at the cell surface. Recent studies showed that uPAR is expressed in the ischemic damaged brain, suggesting its involvement in brain damage. In this study, we evaluated the role of uPAR in ischemic brain damage induced by permanent middle cerebral artery (MCA) occlusion in mice with genetic deficiency of uPAR (uPAR(-/-)) or of uPA (uPA(-/-)). Brain damage at 3 days was smaller in uPAR(-/-) mice (4.5+/-1.0mm(3)) than in littermate wild-type mice (uPAR(+/+)) (9.1+/-1.8mm(3), p

Objectives The purpose of this study was to investigate whether c-Myc regulates expression of urokinase plasminogen activator (uPA) on hypoxia-induced vascular smooth muscle cells (VSMCs). Methods VSMCs were isolated from thoracic aorta of wild-type (WT), tissue plasminogen activator (tPA) and uPA-deficient mice. Gene and protein expression levels were examined by reverse-transcription PCR and Western blotting, respectively. c-Myc and uPA transcriptional activity were determined by luciferase analysis. Zymography analysis was used to test the activity of matrix metalloproteinases (MMPs), tPA, and uPA. Results Hypoxia significantly promoted WT and tPA−/− VSMC migration and invasion. However, uPA−/− severely decreased hypoxia-induced VSMC migration and invasion. Hypoxia increased uPA and MMP-2 activity, while uPA−/− decreased hypoxia-induced MMP-2 activity. c-Myc expression and transcriptional activity were increased in response to hypoxia, and silenced c-Myc abolished hypoxia-induced uPA and MMP-2 activity. In addition, hypoxia-induced Bcl2 expression and Bcl2 binding to c-Myc led to enhanced c-Myc-mediated uPA and MMP-2 activity in response to hypoxia. Conclusions The results show that c-Myc was essential for hypoxia-induced uPA expression and activity, resulting in VSMC migration and invasion. In addition, Bcl2 enhanced the c-Myc-mediated uPA/MMP-2 pathway.

SummaryStaphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin. The interaction between the plasmin-SAK complex and plasminogen was investigated using synthesized peptides, which were constructed according to the amino acid sequence of the SAK molecule.A synthetic nonadecapeptide (SAK22–40) corresponding to Glu22-Leu40 by the SAK molecule enhanced the activation of Glu-plasminogen by the plasmin-SAK complex.Analysis of IAsys resonant mirror biosensor showed that SAK22–40 bound to Glu-plasminogen.This binding was completely inhibited by IgG against the B-chain in the plasminogen molecule. But, this binding was not inhibited by IgG against lysine-binding sites (LBS) of the A-chain in the plasminogen molecule. The substitution of Lys35 with Ala in SAK22–40 did not enhance the activation of Glu-plasminogen by the plasmin-SAK complex. When SAK22–40 was administrated in a mouse thrombosis model, earlier recanalization was observed than in mice with vehicle administration. Thus, a newly synthesized peptide, SAK22–40 enhanced Glu-plasminogen activation and induced effective thrombolysis.

Since the activation of coagulation system and platelets triggers the thrombosis, the agents possessing anticoagulation or anti-platelet function have been used for the antithrombotic procession actions. However, in the physiological condition, the fibrinolytic system serves as antithrombotic, which removes the thrombus from the circulation. The fibrinolytic system plays an important role in the dissolution of fibrin, which is the main component of thrombus. So that, the balance between the coagulation and fibrinolytic system regulates the formation of thrombus. Now, the new agents that activate the fibrinolytic system have been clinically applied for the thrombolytic therapy. The main factor in fibrinolytic system is the plasmin, which is activated from the plasminogen by the plasminogen activator (PA). The plasminogen activator is mainly used for the thrombolytic therapy. There are two types of PA. One is the non fibrin-specific PA such as streptokinase (SK) and two-chain urokinase-type PA (tcu-PA, urokinase), and another is the fibrin-specific PA such as tissue-type PA (t-PA) and single-chain urokinase-type PA (scu-PA). Recently, some derivatives of t-PA have been developed to obtain the longer half-life than native t-PA and allowed to administrate as the single-bolus. Further, the new fibrin-specific PA such as staphylokinase and bat-PA has been developed. Many attempts have been made to develop the agents that would induce the release of t-PA from endothelial cells. In this review, the development of new fibrinolytic agents is summarized.

This study was performed to clarify the relationship between fibrinolytic components and the pathology of allergy, particularly that during the development of nasal allergy and nasal tissue changes. Intranasal OVA challenge after sensitization by i.p. administration of OVA induced a higher level of excess subepithelial collagen deposition in wild-type (WT) C57BL/6J mice than in plasminogen activator inhibitor (PAI)-1-deficient (PAI-1−/−) mice. The excess PAI-1 induction in the nasal mucosa and higher level of active PAI-1 in the nasal lavage fluid of WT-OVA mice compared with those in WT-control mice suggested that the decrease of proteolytic activity inhibits the removal of subepithelial collagen. The frequency of sneezing, nasal rubbing, nasal hyperresponsiveness, production of specific IgG1 and IgE in the serum, and production of IL-4 and IL-5 in splenocyte culture supernatant increased significantly in WT-OVA mice. In PAI-1−/− mice, these reactions were absent, and specific IgG2a in serum and IFN-γ in splenocyte culture medium increased significantly. Histopathologically, there were marked goblet cell hyperplasia and eosinophil infiltration into the nasal mucosa in WT-OVA mice, but these were absent in PAI-1−/− mice. These results indicate that the immune response in WT-OVA mice can be classified as a dominant Th2 response, which would promote collagen deposition. In contrast, the Th2 response in PAI-1−/− mice was down-regulated, and the immune response shifted from Th2-dominant reaction to a Th1-dominant one. Taken together, these findings suggest that PAI-1 plays an important role not only in thrombolysis but also in immune response.

The growth rate of vascular smooth muscle cells (VSMCs), which were derived from aorta of mice deficient in the fibrinolytic factors tissue-type plasminogen activator (t-PA(-/-)), urokinase (u-PA(-/-)), u-PA receptor (u-PAR(-/-)) and type 1 plasminogen activator inhibitor (PAI-1(-/-)), as well as wild-type (WT) mice, was investigated in the presence of mouse melanoma cells (B16). In the VSMCs cultured with a basal medium supplemented with 10% fetal calf serum (FCS), there was no difference in the growth rate among the gene-lacking VSMCs and WT VSMCs, indicating that these fibrinolytic factors were not involved in the FCS-mediated cell proliferation. On the other hand, when these VSMCs were cultured with B16 cells in either the mixed culture or a double-chamber, only u-PAR(-/-) VSMCs showed a significantly lower growth rate. In addition, these suppressive effects on u-PAR(-/-) VSMCs were also observed in the presence of B16-derived conditioned medium (B16/CM). The growth rate of all the VSMCs except u-PAR(-/-) VSMCs was increased in the presence of B16/CM. The degree of the increase in cell number was comparable to that obtained with FCS. These effects on growth activity were partially associated with the levels of mitogen-activated protein kinase (MAPK, p42/p44) activity. The findings suggest that u-PAR plays an important role in the proliferative response of VSMCs and that without u-PAR, there is no intracellular signaling for cell proliferation.

We here report that the arterial blood flow after endothelial injury in mice deficient in α2-antiplasmin (α2-AP-/- mice) was well maintained compared with that of wild-type mice. Moreover, the development of neointima 4 weeks after injury in α2-AP-/- mice was significantly decreased. Histologic observations showed a prompt recovery of endothelial cells with a much higher proliferating index in repaired endothelium in α2-AP-/- mice. The amount of secreted vascular endothelial growth factor (VEGF) by explanted vascular smooth muscle cells (SMCs) from α2-AP-/- mice was significantly increased. In separate experiments using a human endothelial cell (EC) line, we could demonstrate that plasminogen binds to ECs and that this binding can be prevented by α2-AP. Finally, an injection of either an anti-VEGF receptor-1 antibody or α2-AP reduced the prompt endothelial healing. α2-AP is the main inactivator of plasmin, which cleaves extracellular matrix-bound VEGF to release a diffusible proteolytic fragment. Lack of α2-AP, therefore, could lead to a local over-release of VEGF by the continuously active plasmin in the injured area, which could result in a prompt re-endothelialization after vascular injury. Our results provide new insight into the role of α2-AP and VEGF in the pathogenesis of re-endothelialization following vascular injury. (Blood. 2003;102: 3621-3628)

The effect of deep sea water on the fibrinolytic properties of human vascular endothelial cells was investigated. There was no difference in the growth ratio between human umbilical vein endothelial cells (HUVECs) cultured with growth medium (RPMI-1640 containing 20% fetal calf serum) prepared with Hawaii deep sea water (HDSW medium) and those with medium prepared with normal distilled water (control medium). The secretion of type 1 plasminogen activator inhibitor (PAI-1) from HUVECs was significantly reduced by about twofold. However, the levels of PAI-1 mRNA in HUVECs cultured with HDSW medium did not change when compared with those cultured with control medium. Though HDSW medium also reduced the secretion of tissue-type plasminogen activator, the suppressive effect was more prominent for PAI-1. Thus, the balance of fibrinolytic activity was turned toward anti-thrombotic in HUVECs. This was evidenced by the lysis of 125I-fibrin clot in the presence of plasminogen. That is, HUVECs cultured with HDSW medium degraded 125I-fibrin more efficiently than HUVECs with control medium. Such enhanced clot lysis was maintained as long as HDSW medium was present. The accelerated effect of HDSW medium on clot lysis disappeared after the exchange of HDSW medium to control medium. These findings suggest that HDSW may specifically and predominantly affect the process of molecular transfer of PAI-1 after its transcription, resulting in an enhanced fibrinolytic activity of HUVECs. Since HDSW reduces the thrombotic potential of cultured HUVECs, it is speculated that the materials contained in HDSW may prevent the incidence of thrombotic disorders.

Identification of a novel therapy for prevention of sudden death by ischemic cardiac infarction is an area of intensive investigation. We here report that the mortality due to an experimental acute myocardial infarction (AMI) was markedly increased in mice deficient in alpha2-antiplasmin (alpha2-AP(-/-) mice) but not in mice deficient in other components acting in fibrinolysis (tissue-type PA, urokinase type PA, or plasminogen activator inhibitor-1) even if the infarct area in alpha2-AP(-/-) mice was not different from those in the other mice. Echocardiography showed in alpha2-AP(-/-) mice after AMI an overload of the right ventricle and that pulmonary permeability was increased. According to the experiments using explanted myocytes and vascular smooth muscle cells, it was found that the amount of secreted vascular endothelial cell growth factor (VEGF) in alpha2-AP(-/-) mice was markedly increased compared with that in wild-type mice. Finally, an injection of an anti-VEGF antibody decreased the mortality after AMI in alpha2-AP(-/-) mice. Plasmin cleaves extracellular matrix-bound VEGF to release a diffusible proteolytic fragment and is inactivated mainly by alpha2-AP. Therefore, lack of alpha2-AP could markedly result in overrelease of VEGF by the continuous activation of plasmin because of AMI and could result in an acute cor pulmonale. Our results provide new aspects on the role of alpha2-AP and VEGF in the pathogenesis of cardiac events.

Plasminogen activator inhibitor-1 (PAI-1) and alpha2-anti-plasmin (alpha2-AP) may contribute to arterial thrombolysis resistance. The role of these components on thrombus evolution in vivo was investigated in mice deficient for PAI-1 (PAI-1(-/-)) or alpha2-AP (alpha2-AP(-/-)) or their wild-type counterparts (PAI-1(+/+), alpha2-AP(+/+)). Moreover, the influence of either PAI-1 or alpha2-AP deficiency on the results of pharmacologic inhibition of glycoprotein IIb/IIIa of platelets or thrombin was also investigated. A thrombus was induced in the murine carotid artery by endothelial injury. The alpha2-AP(-/-) mice were indistinguishable from wild-type, whereas the time to occlusion in PAI-1(-/-) was significantly prolonged to 24.9 +/- 3.7 min. Vascular patency was markedly increased in both PAI-1- and alpha2-AP-deficient mice. In separate animals, either a glycoprotein IIb/IIIa antagonist or a thrombin inhibitor was applied. The time required to occlusion was prolonged in a dose-dependent manner in all types of mice. When each compound was administered to PAI-1(-/-) mice, significant changes were observed. In conclusion, lack of PAI-1 prolongs the time to occlusion and accelerates clot lysis, whereas alpha2-AP only has an effect on spontaneous reperfusion. Consequently, the inhibition of PAI-1, but not of alpha2-AP, could enhance the effects of anti-thrombotic therapy.

Background: The role of α2-antiplasmin (α2-AP) on platelet aggregation was investigated using mice deficient in α2-AP (α2-AP−/−) or using wild type mice (α2-AP+/+).

Renal fibrosis is the final common pathway of a wide variety of chronic kidney diseases. Myofibroblast formation via the differentiation of from tissue-resident fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs) and epithelial-to-mesenchymal transition (EMT) is known to play a pivotal role in the development of renal fibrosis. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated the role of alpha 2-antiplasmin (α2AP) in myofibroblast formation and the development of renal fibrosis. We observed the development of renal fibrosis using unilateral ureteral obstruction (UUO). α2AP had accumulated in the UUO-induced obstructed kidneys and α2AP deficiency attenuated UUO-induced renal fibrosis in mice. The degree of myofibroblast formation in the obstructed kidneys of α2AP−/− mice was less than that in α2AP+/+ mice. In vitro, α2AP induced myofibroblast formation in renal tubular epithelial cells (RTECs), renal fibrosblasts and bone marrow-derived mesenchymal stem cells (MSCs). α2AP also induced the production of TGF-β, which is known to be a key regulator of myofibroblast formation and fibrosis. α2AP-induced the TGF-β production was significantly reduced by SP600125, c-Jun N-terminal kinase (JNK) specific inhibitor. Our findings suggest that α2AP induces myofibroblast formation in the obstructed kidneys and mediates the development of renal fibrosis.

Aim: Effects of Enzamin, which is a product from Bacillus subtilis AK and Lactobacillus, on obesity-related metabolic disorders in obese db/db mice were examined to explore a novel agent for the pr...

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.

Liver regeneration is regulated by variety of growth factors. Release and activation of these growth factors are deeply related to degradation of extracellular matrix (ECM), which is regulated by plasminogen-activating system. Since plasminogen is assum ed to be essential in these functions in liver regeneration, partial 70% hepatectomy (PH) was performed on plasminogen deficient (Plg−/−) mice and wild-type (Plg+/+) mice. The sequential recovery of the liver weight after 70% PH gradually increased over 1 to 14 days in Plg+/+ mice. However, in Plg−/− mice, it increased over 1 to 7 days and thereafter increased no more. Thus, the recovery in Plg−/− mice was significantly impaired at 10 and 14 days compared with Plg+/+ mice. Plg+/+ mice exhibited an increase in DNA synthesis over5 days, and then a decrease thereafter; however, in Plg−/− mice, it increased over 3 days, and decreased thereafter, with the significant decrease at 14 days. The apoptotic nuclei labelling index slightly increased over 3 days, and decreased thereafter in Plg+/+ mice. In Plg−/− mice, it was few detected over 3 days, and gradually increased over 7 to 14 days. The apoptotic index in Plg−/− mice was higher than Plg+/+ mice at 14 days. In the histological examination of liver, focal area of cellular loss with fibrin deposition were detected in Plg−/− mice after PH, which were rarely detected in Plg+/+ mice. In Plg−/− mice, expressions of t-PA, u-PA and pro-MMP-9 were increased in the liver at 14 days after PH compared with Plg+/+ mice. These findings suggest that plasminogen plays an important role in liver regeneration after PH.

Background Deep venous thrombosis (DVT), which is often associated with pulmonary embolism (PE), is a serious complication after total knee arthroplasty (TKA). In the present study, we examined the overall thrombotic and thrombolytic status using Global Thrombosis Test (GTT) in non-anticoagulated blood of patients undergoing TKA to develop the predictable marker for the incidence of DVT. Methods DVT was diagnosed using doppler ultrasonography a day after the surgery in 31 patients with osteoarthritis (n = 24), rheumatoid arthritis (n = 6) and ankylosing spondylitis (n = 1) by the well-trained operator. We measured overall thrombotic and thrombolytic status using GTT and other biomarkers, which is associated with blood coagulation and fibrinolysis, before and immediately after the surgery. Results Newly-generated DVT during the operation was detected in 11 of 31 patients (35.4%) 1 day after TKA. There were no differences in markers of coagulation (PT and APTT), platelet activity (platelet aggregation-induced by ADP and collagen) and fibrinolysis (FDP and D-dimer) between non-DVT and DVT group both before and after the surgery. Both Pre- and Post-operative GTT-occlusion times (OT), an index of platelet reactivity, were tended to be shorter, but not significant, in DVT group compared with non-DVT group. Pre-operative GTT-lysis time (LT), an index of thrombolytic activity, was significantly shorter in DVT group compared with non-DVT group, while there were no differences in post-operative value of this index between DVT group and non-DVT group, suggesting overall thrombolytic activity was enhanced in DVT group before surgery. Conclusions Our data suggest that enhancement of pre-operative thrombolytic activity assessed by GTT may be a predictable marker for the incidence of DVT after TKA.

Urokinase type plasminogen activator (uPA) may influence brain pathophysiology after injury. We studied disruption of the blood-brain barrier (BBB) and changes in the vasculature after a brain stab wound in uPA-deficient, uPA receptor-deficient, and PA inhibitor-1 (PAI-1) deficient mice. The extravasation of immunoglobulin was greater in PAI-1 deficient mice; less pronounced in uPA-deficient mice; similar to controls in uPA receptor-deficient mice. Vasculatures in the wound proliferated in PAI-1 deficient mice. Our study shows that uPA affects BBB disruption. PA enhances angiogenesis after brain injury.

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

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.

We investigated the involvement of fibrinolytic system in the process of wound healing as well as angiogenesis in rats using a model system, in which polystyrene capsules with 100 perforated pores filled without (empty capsule) or with fibrin gel (fibrin capsule) were subcutaneously implanted into the dorsal of rats. The intracapsular tissue was accompanied by neovascularization and the tissue weight in the fibrin capsule was three times that of the empty capsule on day 5 after implantation. Both plasminogen activation and amidolytic activities of urokinase-type plasminogen activator (u-PA) in the tissue extracts and intracapsular fluid obtained from the fibrin capsule were higher than those in the empty capsule on day 5 after implantation. Both u-PA and its specific receptor (u-PAR) mRNAs were detected in the intracapsular tissues formed in both capsules. On day 5 after implantation, though the levels of u-PA mRNA in the tissue of the empty capsule and those of the fibrin capsule were not significantly different, more u-PAR mRNA was expressed in the tissue of the fibrin capsule than in the empty capsule. Furthermore, when the cultured endothelial cells were incubated with intracapsular fluids, the proliferation of the cells was significantly enhanced. These results indicate that fibrinolytic activities increase in the presence of fibrin gel, possibly by up-regulation of the u-PA/u-PAR system in the early stage of rat wound healing.

We demonstrated previously that tissue-type plasminogen activator (t-PA) bound to its specific receptor (t-PAR) on human umbilical vein endothelial cells (HUVEC) in suspension and that t-PAR of mol wt. 20 kDa interacted only with t-PA to form 90 kDa complex (Fukao, H., Hagiya, Y., Nonaka, T., Okada, K., and Matsuo, O. (1992) Biochem. Biophys. Res. Commun. 187, 956–962). In the present study, 20 kDa t-PAR was purified from HUVEC and the function of the t-PAR was investigated by analyzing its effect on plasminogen activation by t-PA. About 2.2 μg t-PAR protein was purified from cell lysate of 1.0×109 HUVEC as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by gel filtration with TSK-3000SW and reversed phase separation with high performance liquid chromatography (HPLC). 125I-t-PA but not 125I-plasminogen specifically bound to the purified t-PAR in ligand blot assay. Plasminogen activation by t-PA in the presence of purified t-PAR in solution was increased. Furthermore, t-PA bound to immobilized t-PAR efficiently expressed its plasminogen activation activity. Kinetic analysis revealed that t-PA in the presence of soluble t-PAR and t-PA bound to immobilized t-PAR exhibited 34- and 90-fold increase in plasminogen activation, respectively. The t-PAR did not interact with anti-annexin II antibody. These findings indicate that the 20 kDa t-PAR is a novel molecule which immobilizes t-PA and enhances its proteolytic activity on the cell surface of endothelial cells.

The α2-Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule-associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP⁻/⁻ mice than in the neurons from α2AP⁺/⁺ mice. Exogenous treatment with α2AP enhanced the microtubule-associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP-induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen⁻/⁻ mice. The activation of p38 MAPK was involved in the α2AP-induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.

Staphylokinase obtains plasminogen activating activity by forming a complex with plasminogen. Although the enzymatic activity of staphylokinase is enhanced by fibrin, how fibrin enhances enzymatic activity has not been determined yet. The effects of fibrin, or fibrinogen fragments, on the activation of plasminogen by staphylokinase was investigated using CNBr-digested fibrinogen fragments (FCB-2 and FCB-5) and plasmin-degraded cross-linked fibrin fragments ((DD)E complex, DD fragments and E fragments). Kinetic analysis of the activity of staphylokinase revealed that its plasminogen activating activity, which was expressed as kcatlKm, was enhanced by FCB-2 (10-fold) and FCB-5 (5-fold). These fibrin fragments caused 38-, 30-, and 8.5-fold increases in activity for the DD fragment, (DD)E complex and E fragment, respectively. Although a,-antiplasmin inhibited the activation of plasminogen by staphylokinase, FCB-2 abolished its inhibitory effects, and the plasminogen activating activity of staphylokinase was restored. The inhibitory effects of a,-antiplasmin on the activation of mini-plasminogen by staphylokinase were less than for Glu- or Lys-plasminogen, and the inhibitory effect of a,-antiplasmin was not altered by fibrin or EACA. These findings indicate that the staphylokinaselplasmin(ogen) complex reacts with fibrin even in the presence of a,-antiplasmin, and efficient plasminogen activation takes place on the surface of fibrin.

Tissue-type plasminogen activator (t-PA) can occur in two different forms, single- and two-chain t-PA. Such a difference in structure of the t-PA molecule may induce different biological functions. We compared the thrombolytic efficacy of single-chain t-PA with that of two-chain t-PA and urokinase in a canine thrombosis model. The thrombolytic properties of single- and two-chain recombinant t-PA and urokinase with equivalent activity in vitro after intravenous infusion were examined in 24 anesthetized, open-chest dogs with intracoronary thrombi. Repeated coronary angiography showed that the time to reperfusion was 29.0 +/- 4.8, 32.5 +/- 6.3, and 39.2 +/- 6.8 min, respectively. One dog in the single-chain t-PA group and all dogs in the vehicle group showed no recanalization. Plasma t-PA antigen reached similar plateau levels within 15. min in both the single- and two-chain t-PA groups. The infusion of single- and two-chain t-PA was not associated with systemic fibrinolytic activation, except for a decrease in alpha 2-plasmin inhibitor in single-chain t-PA. However, the infusion of urokinase was associated with significant depletion of fibrinogen. These findings suggest that single-chain t-PA has potent thrombolytic effect without systemic fibrinolytic activity similar to that of two-chain t-PA in a canine coronary artery thrombosis model.