Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen-plasmin system.

Ischaemic stroke, accompanied by neuroinflammation, impairs blood-brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood-brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen-plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane.

Interplay between fibrinolysis and complement: plasmin cleavage of iC3b modulates immune responses.

BACKGROUND: The plasmin(ogen) and complement systems are simultaneously activated at sites of tissue injury, participating in hemostasis, wound healing, inflammation and immune surveillance. In particular, the C3 proteolytic fragment, iC3b, and its degradation product C3dg, which is generated by cleavage by factor I (FI) and the cofactor complement receptor CR1, are important in bridging innate and adaptive immunity. Via a thioester (TE) bond, iC3b and C3dg covalently tag pathogens, modulating phagocytosis and adaptive immune responses. OBJECTIVE: To examine plasmin-mediated proteolysis of iC3b, and to evaluate the functional consequences, comparing the effects with products generated by FI/CR1 cleavage of iC3b. METHODS: Dose-dependent and time-dependent plasmin-mediated cleavage of iC3b were characterized by analytical gel electrophoresis. The properties of the resultant TE bond-containing fragments on phagocytosis and induction of pro-inflammatory cytokines were measured in cell culture systems. RESULTS: At low concentrations, plasmin effectively cleaves iC3b, but at numerous previously undescribed sites, giving rise to novel C3c-like and C3dg-like moieties, the latter of which retain the TE bond. When attached to zymosan or erythrocytes and exposed to THP-1 macrophages, the C3dg-like proteins behave almost identically to the bona fide C3dg, yielding less phagocytosis as compared with the opsonin iC3b, and more macrophage secretion of the pro-inflammatory cytokine, IL-12. CONCLUSION: Plasmin cleavage of iC3b provides a complement regulatory pathway that is as efficient as FI/CR1 but does not require a cellular cofactor.

Inhibition of plasmin protects against colitis in mice by suppressing matrix metalloproteinase 9-mediated cytokine release from myeloid cells.

BACKGROUND & AIMS: Activated proteases such as plasmin and matrix metalloproteinases (MMPs) are activated in intestinal tissues of patients with active inflammatory bowel diseases. We investigated the effect of plasmin on the progression of acute colitis. METHODS: Colitis was induced in Mmp9(-/-), Plg(-/-), and C57BL/6 (control) mice by the administration of dextran sulfate sodium, trinitrobenzene sulfonic acid, or CD40 antibody. Plasmin was inhibited in control mice by intraperitoneal injection of YO-2, which blocks its active site. Mucosal and blood samples were collected and analyzed by reverse-transcription polymerase chain reaction and immunohistochemical analyses, as well as for mucosal inflammation and levels of cytokines and chemokines. RESULTS: Circulating levels of plasmin were increased in mice with colitis, compared with controls. Colitis did not develop in control mice injected with YO-2 or in Plg(-/-) mice. Colons from these mice had reduced infiltration of Gr1+ neutrophils and F4/80+ macrophages, and reduced levels of inflammatory cytokines and chemokines. Colonic inflammation and colitis induction required activation of endogenous MMP9. After colitis induction, mice given YO-2, Plg(-/-) mice, and Mmp9(-/-) mice had reduced serum levels of tumor necrosis factor and C-X-C motif chemokine ligand 5, compared with control mice. CONCLUSIONS: In mice, plasmin induces a feedback mechanism in which activation of the fibrinolytic system promotes the development of colitis via activation of MMP9 or proteolytic enzymes. The proteolytic environment stimulates the influx of myeloid cells into the colonic epithelium and the production of tumor necrosis factor and C-X-C motif chemokine ligand 5. In turn, myeloid CD11b+ cells release the urokinase plasminogen activator, which accelerates plasmin production. Disruption of the plasmin-induced chronic inflammatory circuit therefore might be a strategy for colitis treatment.

A central role of plasmin in cardiac injury initiated by fetal exposure to maternal anti-Ro autoantibodies.

OBJECTIVE: Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating ß2-glycoprotein I (ß2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. METHODS: Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. RESULTS: uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 ± 0.1 vs 1.9 ± 0.05 ng/ml (P < 0.0001), 6.6 ± 0.3 vs 2.1 ± 0.2 ng/ml (P < 0.0001) and 435 ± 34 vs 220 ± 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 ± 0.1 vs 1.9 ± 0.05 ng/ml; P = 0.0086, 6.2 ± 1.4 vs 2.2 ± 0.7 ng/ml; P = 0.147 and 412 ± 61 vs 260 ± 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. CONCLUSION: Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.

Streptococcus pneumoniae endopeptidase O (PepO) is a multifunctional plasminogen- and fibronectin-binding protein, facilitating evasion of innate immunity and invasion of host cells.

Streptococcus pneumoniae infections remain a major cause of morbidity and mortality worldwide. Therefore a detailed understanding and characterization of the mechanism of host cell colonization and dissemination is critical to gain control over this versatile pathogen. Here we identified a novel 72-kDa pneumococcal protein endopeptidase O (PepO), as a plasminogen- and fibronectin-binding protein. Using a collection of clinical isolates, representing different serotypes, we found PepO to be ubiquitously present both at the gene and protein level. In addition, PepO protein was secreted in a growth phase-dependent manner to the culture supernatants of the pneumococcal isolates. Recombinant PepO bound human plasminogen and fibronectin in a dose-dependent manner and plasminogen did not compete with fibronectin for binding PepO. PepO bound plasminogen via lysine residues and the interaction was influenced by ionic strength. Moreover, upon activation of PepO-bound plasminogen by urokinase-type plasminogen activator, generated plasmin cleaved complement protein C3b thus assisting in complement control. Furthermore, direct binding assays demonstrated the interaction of PepO with epithelial and endothelial cells that in turn blocked pneumococcal adherence. Moreover, a pepO-mutant strain showed impaired adherence to and invasion of host cells compared with their isogenic wild-type strains. Taken together, the results demonstrated that PepO is a ubiquitously expressed plasminogen- and fibronectin-binding protein, which plays role in pneumococcal invasion of host cells and aids in immune evasion.

Antibody-directed neutralization of annexin II (ANX II) inhibits neoangiogenesis and human breast tumor growth in a xenograft model.

Activation of the fibrinolytic pathway has long been associated with human breast cancer. Plasmin is the major end product of the fibrinolytic pathway and is critical for normal physiological functions. The mechanism by which plasmin is generated in breast cancer is not yet fully described. We previously identified annexin II (ANX II), a fibrinolytic receptor, in human breast tumor tissue samples and observed a strong positive correlation with advanced stage cancer (Sharma et al., 2006a). We further demonstrated that tissue plasminogen activator (tPA) binds to ANX II in invasive breast cancer MDA-MB231cells, which leads to plasmin generation (Sharma et al., 2010). We hypothesize that ANX II-dependent plasmin generation in breast tumor is necessary to trigger the switch to neoangiogenesis, thereby stimulating a more aggressive cancer phenotype. Our immunohistochemical studies of human breast tumor tissues provide compelling evidence of a strong positive correlation between ANX II expression and neoangiogenesis, and suggest that ANX II is a potential target to slow or inhibit breast tumor growth by inhibiting neoangiogenesis. We now report that administration of anti-ANX II antibody potently inhibits the growth of human breast tumor in a xenograft model. Inhibition of tumor growth is at least partly due to attenuation of neoangiogenic activity within the tumor. In vitro studies demonstrate that anti-ANX II antibody inhibits angiogenesis on three dimensional matrigel cultures by eliciting endothelial cell (EC) death likely due to apoptosis. Taken together, these data suggest that selective disruption of the fibrinolytic activity of ANX II may provide a novel strategy for specific inhibition of neoangiogenesis in human breast cancer.

[Preparation and characterization of monoclonal antibodies against human plasmin-alpha 2-antiplasmin complexes].

AIM: To prepare monoclonal antibodies (mAbs) against human plasmin-alpha(2)-antiplasmin complexes (PAP). METHODS: BALB/c mice were immunized with PAP purified from human fresh plasma. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by indirect ELISA with equimolar plasminogen, alpha(2)-antiplasmin and PAP as immobilized antigen, respectively. The specificity and affinity of mAbs in ascitic fluids were characterized by Western blot and ELISA, respectively. RESULTS: Among the 24 specific mAbs obtained, 7 were directly against neoantigens (new emerging epitopes that were different from PAP precursor plasminogen and alpha(2)-antiplasmin, which are formed during PAP generation), 16 against plasmin domain and 1 against modified alpha(2)-antiplasmin in PAP molecule. Titers of all the mAbs to PAP were from 2 x 10(-4) to 1 x 10(-8), and 4 of them could strongly recognize PAP or plasminogen (K(d) from 5.62 x 10(-9) to 3.58 x 10(-11) mol/L). CONCLUSION: The mAbs against PAP neoantigens with high affinity was acquired successfully, which provided a tool for determination of plasma levels of PAP without interferences from plasminogen and alpha(2)-antiplasmin and for research on fibrinolytic status in-vitro.

The membrane attack complex of complement contributes to plasmin-induced synthesis of platelet-activating factor by endothelial cells and neutrophils.

Thrombolytic agents, used to restore blood flow to ischaemic tissues, activate several enzymatic systems with pro-inflammatory effects, thus potentially contributing to the pathogenesis of ischaemia-reperfusion injury. Platelet-activating factor (PAF), a phospholipid mediator of inflammation, has been implicated in the pathogenesis of this process. We previously showed that the infusion of streptokinase (SK) induces the intravascular release of PAF in patients with acute myocardial infarction (AMI), and that cultured human endothelial cells (EC) synthesized PAF in response to SK and plasmin (PLN). In the present study, we investigated the role of the membrane attack complex (MAC) of complement in the PLN-induced synthesis of PAF. In vivo, we showed a correlation between the levels of soluble terminal complement components (sC5b-9) and the concentrations of PAF detected in blood of patients with AMI infused with SK. In vitro both EC and polymorphonuclear neutrophils (PMN), incubated in the presence of PLN and normal human serum, showed an intense staining for the MAC neoepitope, while no staining was detected when they were incubated with PLN in the presence of heat-inactivated normal human serum. Moreover, the insertion of MAC on EC and PMN plasmamembrane elicited the synthesis of PAF. In conclusion, our results elucidate the mechanisms involved in PAF production during the activation of the fibrinolytic system, showing a role for complement products in this setting. The release of PAF may increase the inflammatory response, thus limiting the beneficial effects of thrombolytic therapy. Moreover, it may have a pathogenic role in other pathological conditions, such as transplant rejection, tumoral angiogenesis, and septic shock, where fibrinolysis is activated.

Antibodies against dengue virus E protein peptide bind to human plasminogen and inhibit plasmin activity.

Both mice and rabbits immunized with dengue virus E protein peptide spanning amino acids 100-119 (D4E) produced antibodies that reacted not only with the D4E peptide itself but also with human plasminogen, as shown by ELISA and Western blot. Sera from dengue virus-hyperimmunized mice and dengue patients also contained antibodies against D4E and plasminogen. Furthermore, such sera all contained plasmin inhibitory activity. Using affinity-purified anti-D4E antibodies and free D4E peptide for competitive inhibition, we demonstrated that the inhibition of plasmin activity was due to anti-D4E antibodies rather than other substances in the sera. Taken together, these results suggest dengue virus E protein amino acids 100-119 are a cross-reactive immunogenic region, and antibodies against this region may interfere with human fibrinolysis.

Binding and activation of plasminogen on the surface of osteosarcoma cells.

Plasmin (Pm) is a broad action serine protease implicated in numerous physiological functions. In bone, Pm may play a role in growth, resorption, metastasis, and the activation of growth factors. The various components of the Pm system are known to bind and function on the cell surface of various cell types, but no pertinent data are available describing membrane-bound Pm or its zymogen, plasminogen (Pg), in either normal or neoplastic bone cells. We report here that Pg binds to the surface of the human osteosarcoma cell line MG-63 and is activated to Pm by endogenous urokinase plasminogen activator (uPA). These conclusions are based on experiments utilizing radiolabeled compounds and a cell surface proteolytic assay measuring amidolytic activity of Pm. 125I-Pg binding to cells was time dependent, saturable, reversible, and specific. Binding was characterized by a relatively low affinity (Kd approximately 0.9 microM) and a high capacity (approximately 7.5 x 10(6) sites/cell). The binding of 125I-Pg was associated with lysine binding sites of the plasminogen molecule. Activation of 125I-Pg to 125I-Pm occurred on the cell surface and was dependent upon cell bound uPA, as determined by inhibitory antibodies. Binding of Pg to MG-63 monolayers represented approximately 80% bound specifically to the cell surface and the remainder to the surrounding extra-cellular matrix. Either co-incubation with uPA or pre-incubation with Pm resulted in increased 125I-Pg binding to osteosarcoma cells. Cell surface Pm proteolytic activity was confirmed by an amidolytic chromogenic assay. Both Pm and Pg bound to cells with Pg being activated by endogenous uPA. Plasmin activated on the cell surface was partially protected from inhibition by alpha 2-antiPm (requiring Pm lysine binding site interaction) but inhibited by aprotinin, (interacting directly with the Pm catalytic site). Resistance of cell bound Pm to alpha 2-antiPm inhibition suggests that cell surface proteolysis can occur in the presence of a soluble Pm inhibitor known to exist in the extracellular space. Based on these results, we speculate that the various bone physiological processes implicating Pm may occur at or near the bone cell surface.

Plasmin modulators, aprotinin and anti-catalytic plasmin antibody, efficiently inhibit destruction of bovine vascular endothelial cells by choriocarcinoma cells.

The interaction of human gestational choriocarcinoma cell line, SMT-cc1, with bovine vascular endothelial cells, CPAE, was examined in an in vitro coculture model. SMT-cc1 cells have urokinase-type plasminogen activator (uPA) on their cell surface, and more than half of the cell-associated uPA is enzymatically inactive single-chain uPA (pro-uPA). uPA is bound to a specific surface receptor that is not completely saturated. Also, the plasmin activity is detected on their cell surface. We measured the ability of added SMT-cc1 cells to cause morphological changes in CPAE cells, leading to destruction of CPAE cells. SMT-cc1 cells that adhered to CPAE cell monolayers were capable of causing CPAE cell destruction, followed by detachment, within 6 hr after coculture. Nonspecific serine proteinase inhibitor, aprotinin, and anti-catalytic antibody against plasmin(ogen) effectively inhibited the destruction/detachment in a dose-dependent manner. SMT-cc1 cell-mediated CPAE cell destruction was suppressed for 6 hr in the presence of aprotinin, at a concentration of 20 micrograms/ml, or anti-plasmin antibody, at a concentration of > or = 10 micrograms/ml, suggesting that the destruction is closely related to the proteolytic enzyme, plasmin. We suggest that the destruction of endothelial cells by some tumor cells is associated with tumor cell-associated proteolytic activity, and the uPA-plasmin cascade plays an important role as a critical step during blood-borne metastasis.

Plasminogen activation in vivo upon intravenous infusion of DDAVP. Quantitative assessment of plasmin-alpha 2-antiplasmin complex with a novel monoclonal antibody based radioimmunoassay.

Infusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established. A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor alpha 2-antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated alpha 2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system. Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex. We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

Determination of plasmin-alpha 2-antiplasmin complex in plasma samples by means of a radioimmunoassay.

Immunization of a goat with partially reduced and S-carboxymethylated plasmin B-chain-alpha 2-antiplasmin complex resulted in a large population of antibodies with rather high specificity towards the complex. These antibodies do not react with plasminogen or plasmin in complex with other inhibitors than alpha 2-antiplasmin. However, they react fully with native alpha 2-antiplasmin, but a 200-fold higher concentration, as compared to plasmin-alpha 2-antiplasmin complex, is needed to obtain a similar displacement curve in a double-antibody radioimmunoassay. The results indicate a conformational change in the vicinity of the reactive site in alpha 2-antiplasmin, as a result of complex formation with plasmin. A method for determination of plasmin-alpha 2-antiplasmin complex in plasma has been elaborated using the described radioimmunoassay. About 1.5 mg plasmin-alpha 2-antiplasmin complex/l can be detected, which equals the condition when about 1% of the alpha 2-antiplasmin in plasma is in complex with plasmin. In normal individuals plasmin-alpha 2-antiplasmin complex could be detected only rarely. However, patient with acute processes, as evidenced by high fibrinogen levels, surgical patients postoperatively or patients with malignancy have often detectable levels.

Circulating plasmin-antiplasmin complexes in acute leukaemia.

Thirty consecutive patients presenting with acute leukaemia were studied throughout their hospital course to determine if plasma-alpha 2 antiplasmin (P-AP) complexes could be detected during episodes of increased fibrinolytic activity and to correlate this finding with other more conventional laboratory parameters. Increased fibrinolytic activity was a common finding, it was detected in 19 (63%) patients. This was usually present at diagnosis, but occasionally occurred later as a transient phenomenon. Increased fibrinolytic activity could not clearly be associated with either infection or chemotherapy. P-AP complexes were found in 11 (37%) patients and were almost always accompanied by additional laboratory evidence of increased fibrinolysis. These complexes were present in most instances at diagnosis and disappeared following successful chemotherapy. Forty-five per cent of patients with P-AP complexes had low alpha 2-antiplasmin levels and 36% had low plasminogen levels which returned to normal following successful chemotherapy. At diagnosis six of eight patients with P-AP complexes had major haemorrhagic manifestations.