Urokinase-type plasminogen activator modulates mammalian circadian clock phase regulation in tissue-type plasminogen activator knockout mice.

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue-type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA-/- ; B6.129S2-Plattm1Mlg /J) mice, and identify urokinase-type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA-/- mice entrain to a light-dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA-/- mice take significantly longer to entrain than C57BL/6J wild-type (WT) mice. SCN brain slices from tPA-/- mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose-dependency. Pre-treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), inhibits glutamate-induced phase delays in tPA-/- slices. Selective inhibition of uPA with UK122 prevents glutamate-induced phase resetting in tPA-/- but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA-/- slices. Casein-plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA-/- SCN. Finally, tPA-/- SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.

Cytotoxicity of the Urokinase-Plasminogen Activator Inhibitor Carbamimidothioic Acid (4-Boronophenyl) Methyl Ester Hydrobromide (BC-11) on Triple-Negative MDA-MB231 Breast Cancer Cells.

BC-11 is an easily synthesized simple thiouronium-substituted phenylboronic acid, which has been shown to be cytotoxic on triple negative MDA-MB231 breast cancer cells by inducing a perturbation of cell cycle when administered at a concentration equal to its ED50 at 72 h (117 µM). Exposure of cells to BC-11, either pre-absorbed with a soluble preparation of the N-terminal fragment of urokinase-plasminogen activator (uPa), or in co-treatment with two different EGFR inhibitors, indicated that: (i) BC-11 acts via binding to the N-terminus of the enzyme where uPa- and EGF receptor-recognizing sites are present, thereby abrogating the growth-sustaining effect resulting from receptor binding; and (ii) the co-presence of the EGFR inhibitor PD153035 potentiates BC-11's cytotoxicity. Exposure of cells to a higher concentration of BC-11 corresponding to its ED75 at 72 h (250 µM) caused additional impairment of mitochondrial activity, the production of reactive oxygen species and promotion of apoptosis. Therefore, BC-11 treatment appears to show potential for the development of this class of compounds in the prevention and/or therapy of "aggressive" breast carcinoma.

Effect of thyroxine on fibrinolytic system in rat.

Thyroid hormones have many effects on the cardiovascular system. Thyroid dysfunction accelerates atherosclerosis not only through conventional risk factors (dyslipidemia) but they also show a very close relationship with hemodynamic parameters. Thyroxine is determinant of the several components of fibrinolytic system even though the exact relationship is far from clear. Present study was carried out to determine the effect of thyroxine on fibrinolytic parameters such as plasminogen activators (PA) in rat heart, levels of PA and plasminogen activator inhibitor (PAI), glucose in plasma and serum lipid profile. Rats were injected with 50 ug eltroxine/100 gm(-1) body weight intraperitoneally for one week. Compared with controls, thyroxine treatment increased PA activity significantly in rat heart. No changes were seen in PA, PAI and glucose in plasma of two groups of rats. A significant decrease in total cholesterol and LDL-cholesterol levels was seen in serum of treated group resulting in the decrease of LDL/HDL and Total cholesterol/HDL-cholesterol ratios. These results suggest that thyroxine treatment may have considerable clinical significance. It raised PA activity in heart as well as reduced cholesterol content in blood. It is possible that thyroxine treatment may confer a beneficial effect on cardiovascular risk.

The role of SERPIN citrullination in thrombosis.

Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.

The urokinase receptor and its structural homologue C4.4A in human cancer: expression, prognosis and pharmacological inhibition.

The urokinase-type plasminogen activator receptor (uPAR) and its structural homologue C4.4A are multidomain members of the Ly6/uPAR/alpha-neurotoxin protein domain family. Both are glycosylphosphatidylinositol-anchored membrane glycoproteins encoded by neighbouring genes located on chromosome 19q13 in the human genome. The structural relationship between the two proteins is, however, not reflected at the functional level. Whereas uPAR has a well-established role in regulating and focalizing uPA-mediated plasminogen activation to the surface of those cells expressing the receptor, the biological function of C4.4A remains elusive. Nonetheless, both uPAR and C4.4A have been implicated in human pathologies such as wound healing and cancer. A large body of experimental evidence thus demonstrates that high levels of uPAR in resected tumour tissue as well as in plasma are associated with poor prognosis in a number of human cancers including colon adenocarcinoma and pulmonary squamous cell carcinoma. Targeting uPAR in experimental animal models has also provided promising results regarding the interference with pathogenic plasminogen activation. In the case of C4.4A, very recent data have demonstrated that high protein expression in tumour cells of non-small cell pulmonary adenocarcinomas is associated with a particularly severe disease progression. This review will evaluate structural-functional and disease-related aspects of uPAR and C4.4A with a view to possible pharmacological targeting strategies for therapy and for non-invasive bioimaging.

Plasminogen binding inhibitors demonstrate unwanted activities on GABAA and glycine receptors in human iPSC derived neurons.

Plasminogen binding inhibitors (PBIs) reduce the risk of bleeding in hemorrhagic conditions. However, generic PBIs are also associated with an increased risk of seizures, an adverse effect linked to unwanted activities towards inhibitory neuronal receptors. Development of novel PBIs serve to remove compounds with such properties, but progress is limited by a lack of higher throughput methods with human translatability. Herein we apply human induced pluripotent stem cell (hiPSC) derived neurons in combination with dynamic mass redistribution (DMR) technology to demonstrate robust and reproducible modulation of both GABAA and glycine receptors. These cells respond to GABA (EC50 0.33 ±â€¯0.18 µM), glycine (EC50 11.0 ±â€¯3.7 µM) and additional ligands in line with previous reports from patch clamp technologies. Additionally, we identify and characterize a competitive antagonistic behavior of the prototype inhibitor and drug tranexamic acid (TXA). Finally, we demonstrate proof of concept for effective counter-screening of lead series compounds towards unwanted GABAA receptor activities. No activity was observed for a previously identified PBI candidate drug, AZD6564, whereas a discontinued analog, AZ13267257, could be characterized as a potent GABAA receptor agonist.

Extravascular fibrin, plasminogen activator, plasminogen activator inhibitors, and airway hyperresponsiveness.

Mechanisms underlying airway hyperresponsiveness are not yet fully elucidated. One of the manifestations of airway inflammation is leakage of diverse plasma proteins into the airway lumen. They include fibrinogen and thrombin. Thrombin cleaves fibrinogen to form fibrin, a major component of thrombi. Fibrin inactivates surfactant. Surfactant on the airway surface maintains airway patency by lowering surface tension. In this study, immunohistochemically detected fibrin was seen along the luminal surface of distal airways in a patient who died of status asthmaticus and in mice with induced allergic airway inflammation. In addition, we observed altered airway fibrinolytic system protein balance consistent with promotion of fibrin deposition in mice with allergic airway inflammation. The airways of mice were exposed to aerosolized fibrinogen, thrombin, or to fibrinogen followed by thrombin. Only fibrinogen followed by thrombin resulted in airway hyperresponsiveness compared with controls. An aerosolized fibrinolytic agent, tissue-type plasminogen activator, significantly diminished airway hyperresponsiveness in mice with allergic airway inflammation. These results are consistent with the hypothesis that leakage of fibrinogen and thrombin and their accumulation on the airway surface can contribute to the pathogenesis of airway hyperresponsiveness.

Inhibition of plasminogen activators attenuates the death of differentiated retinal ganglion cells and stabilizes their neurite network in vitro.

PURPOSE: Although previous studies have indicated that elevated levels of the tissue plasminogen activator (tPA) and the urokinase plasminogen activator (uPA) associate with the death of retinal ganglion cells (RGCs), it was unclear whether these proteases directly cause cell death. With the use of a transformed and undifferentiated retinal ganglion cell line, RGC-5, which does not express tPA, and by treating this cell line with staurosporine, which induces not only the differentiation of RGC-5 cells but also the expression of uPA and tPA in other neuronal cells, the authors sought to determine whether these proteases regulate the differentiation of RGC-5 cells and whether elevated levels of these proteases directly cause the death of RGC-5 cells. METHODS: Transformed RGC-5 cells were cultured in serum-free medium and were treated with 0.5 muM to 2.0 muM staurosporine to induce their differentiation. Neurite outgrowth was assessed by phase-contrast microscopy and calcein AM staining and quantified with imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by LIVE/DEAD viability assay kit. RESULTS: Compared with untreated RGC-5 cells, cells treated with staurosporine differentiated as early as 1 to 6 hours. However, proteolytic activities of neither tPA nor uPA were observed within this time frame. Differentiated RGC-5 cells expressed detectable levels of uPA proteolytic activity starting at 24 hours and tPA proteolytic activity only at 48 hours. RGC-5 cells synthesized and secreted uPA and tPA into the conditioned medium, depending on staurosporine concentration and treatment time. At lower concentrations of staurosporine, differentiated RGC-5 cells had longer neurites and expressed lower levels of tPA and uPA. At higher concentrations of staurosporine, differentiated RGC-5 cells expressed higher levels of tPA and uPA, had smaller neurites, and most of them died. In contrast, when RGC-5 cells were treated with staurosporine along with inhibitors specific to tPA and uPA, proteolytic activities of both PAs were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived, showed increased neurite outgrowth, and established their neurite network in vitro. CONCLUSIONS: Results presented in this study indicate that RGC-5 cells do not require tPA and tPA for their differentiation. In fact, differentiated RGC-5 cells synthesize elevated levels of tPA and uPA, and elevated levels of these proteases acting in an autocrine-fashion in turn lead to the death of RGC-5 cells.

Inhibition of cancer cell urokinase plasminogen activator by its specific inhibitor PAI-2 and subsequent effects on extracellular matrix degradation.

Isotopically labeled [( 3H]serine, [3H]proline, and [35S]sulfate) subendothelial cell basement membranes were used to determine the role of urokinase plasminogen activator (uPA) and its specific inhibitor plasminogen activator inhibitor 2 (PAI-2) in colon cancer cell extracellular matrix degradation. Recombinant PAI-2 irreversibly inhibited low and high molecular weight purified human uPA in addition to both colon cancer cell-associated and secreted uPA, particularly if pro-uPA had been preactivated. Two selected lines (COLO394 and LIM1215) preferentially degraded differently labeled matrices in a time- and plasminogen-dependent manner. This process was inhibitable by PAI-2 in the medium at levels which suggested that some degree of "shielding" of cell surface uPA from inhibitor occurred. The ability of PAI-2 to regulate the invasive phenotype of cells which express cell surface or receptor-bound uPA is discussed.

Endogenous receptor-bound urokinase mediates tissue invasion of the human lung carcinoma cell lines A549 and Calu-1.

Macrophage colony-stimulating factor (CSF-1) increases the tissue invasive potential of the CSF-1 receptor-bearing lung carcinoma cell lines A549 and Calu-1 by increasing the number of endogenously bound urokinase-type plasminogen activators (u-PA)s on these cells. CSF-1, at concentrations which optimize invasion of A549 and Calu-1 cells into human amnion membranes (250 ng/ml), maximally augments the number of u-PA receptors occupied by endogenously produced urokinase. Preincubation of A549 and Calu-1 cells with the anti-u-PA monoclonal antibody MPW5UK (25 micrograms/ml) or with a 20- to 40-fold stoichiometric excess of fluid phase type 2 plasminogen activator inhibitor abrogates invasiveness, indicating that functionally active cell surface u-PA is essential for tissue invasion. In contrast, fluid phase type 1 plasminogen activator inhibitor (PAI-1, 5-15 units/ml) does not inhibit invasiveness unless preincubated with the amnion membranes. Inhibition of invasion by PAI-1 is abolished by presaturating the amnion membranes with antiitronectin monoclonal antibody (10 micrograms/ml) which prevents binding of PAI-1 to tissue-associated vitronectin. Binding of PAI-1 to tissue vitronectin is therefore a prerequisite for its inhibitory action. Thus, endogenously receptor-bound u-PA is the primary protease mediating CSF-1-induced tissue invasiveness of the lung carcinoma cell lines A549 and Calu-1.

Occupancy of the cancer cell urokinase receptor (uPAR): effects of acid elution and exogenous uPA on cell surface urokinase (uPA).

The development of a simple, sensitive fluorimetric assay for the measurement of cell surface-associated urokinase plasminogen activator (uPA) on viable, adherent HCT116 cells in microtitre plates, after a preincubation with purified human plasminogen is described. The assay determines plasmin activity by the cleavage of H-D-Val-Leu-Lys 4-aminomethyl coumarin under near physiological pH and ionic conditions with a sensitivity in the range of 5-100 mIU uPA/well at excitation 355 nm and emission 460 nm. Plasmin generated during the assay converted all cell-surface sc-uPA to tc-uPA, allowing the determination of total uPA activity. Inhibitor studies (PAI-2, amiloride or Glu-Gly-Arg chloromethylketone) confirmed the specificity of the uPA assay. Removal of these agents prior to assay allowed determination of the cell surface sc-uPA:tc-uPA ratio. Cell surface activity was only partially removed by acid elution. This corresponded with the loss of a number of proteins and uPA-containing species as detected by SDS-PAGE, gelatin enzymography and Western blotting. Although the major protein species eluted had a M(r) of 55 kDa, reacted with a commercial anti-human uPA mAb and correlated with the main lytic zone, other higher M(r) species were also eluted from HCT116 cells. Exogenous uPA increased cell-surface activity markedly on cells previously treated with acid. Following acid elution, cell surface uPA activity was restored after 30h in culture suggesting either de novo synthesis or release of pre-formed uPA with subsequent secretion and binding to uPAR. The assay has enabled studies on adherent cells to address questions about the regulation and expression of cell-surface uPA.

Inhibition of plasminogen activator inhibitor-1 is a potential therapeutic strategy in ovarian cancer.

Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.

Prolactin inhibits plasminogen activator activity in the preovulatory follicles.

The present study was designed to determine the effects of PRL on changes in morphology and plasminogen activator (PA) activity in the preovulatory follicles. Rabbit ovaries were perfused with hCG alone or with hCG plus at 10, 10(2), or 10(3) ng/ml. PRL at 10(3) ng/ml directly inhibited the degeneration and decomposition of surface epithelial cells induced by hCG exposure. The subsurface connective tissue was visualized by treatment with sodium dodecyl sulfate, which removed surface epithelial cells from the ovary, thereby exposing collagen fibrils and the basal lamina. Sodium dodecyl sulfate treatment revealed inhibition of connective tissue disruption at the apex of the follicle wall in PRL-treated ovaries. PA activity in mature follicles in perfused rabbit ovaries exposed to hCG increased from 1.40 +/- 0.08 to 28.4 +/- 4.25 IU/g tissue after 4 h of perfusion. The addition of PRL to the perfusate inhibited the hCG-stimulated increase in intrafollicular PA activity in a dose-dependent fashion. Although at 7 h mature follicles treated by hCG alone showed greater intrafollicular PA activity than those treated with hCG plus PRL, this difference was not significant. These results suggest that PRL may act directly by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles, probably via the inhibition of intrafollicular tissue PA activity.

Human pregnancy serum contains at least two distinct proteolytic activities with the ability to degrade insulin-like growth factor binding protein-3.

The presence of a proteolytic activity in sera from pregnant humans and rodents capable of degrading insulin-like growth factor binding protein-3 (IGFBP-3) has been known for some time. However, the identity of this activity has remained elusive. We have attempted to purify the IGFBP-3 protease activity from pregnant human serum (PHS) using the degradation of 125I-IGFBP-3 as a marker. Following ammonium sulfate precipitation of PHS and further enrichment of active fractions by ion-exchange, protein-A Sepharose, and size-exclusion chromatography, a protease of approximately 70-90 kDa was isolated and subjected to N-terminal analysis. The N-terminal sequence was consistent with plasminogen, a known fibrinolytic enzyme. To further characterize the IGFBP-3 protease activities in both PHS and nonpregnant human serum (NHS), aliquots of serum were first enriched by polyethylene glycol-precipitation and subjected to size-exclusion chromatography. The size-separated fractions were then incubated with 125I-IGFBP-3, and proteolytic activity was measured. PHS contained two separate proteases (>150 kDa and 70-90 kDa), whereas NHS contained only one (70-90 kDa) that had a inhibitor profile similar to plasmin. However, inhibitors of plasmin had no effect on the activity of the >150-kDa protease. Plasminogen activators (PAs) greatly increased the activity of the 70- to 90-kDa protease, but had little effect on the >150-kDa protease activity. Addition of PAs greatly increased the ability of NHS to proteolyze IGFBP-3. In contrast, the ability of plasminogen-depleted plasma to degrade 125I-IGFBP-3 was not affected by the addition of PAs. Both urokinase and tissue-type PA had the ability to proteolyze IGFBP-3 and were, in contrast to the >150-kDa protease activity, inhibited by the specific PA inhibitor D-PHE-PRO-ARG chloromethyl ketone. The present data suggest that sera has the ability to proteolyze IGFBP-3, and that this ability, as demonstrated by NHS, can be regulated by protease inhibitors and PAs. In addition, PHS does indeed contain an unique IGFBP-3 protease activity that is not present in NHS, and its identity is unknown at this time.

Phorbol ester increases plasminogen activator inhibitor accumulation in cultures of human granulosa cells.

Proteolytic enzymes such as plasminogen activators (PAs) and collagenases are implicated in the process of ovarian follicle rupture. Data obtained in rats support the concept that PAs are both hormonally regulated and temporally related to the ovulatory process; however, such data are lacking in the human ovary. The recent identification of a family of PA inhibitors (PAIs) adds a new dimension to the control of PA activity, and in contrast to animal studies, the human preovulatory follicle is characterized by PA inhibitory activity. To initially examine the PA and PAI system in the human ovary, granulosa cells obtained from women undergoing gonadal hyperstimulation for in vitro fertilization were cultured in the presence or absence of the protein kinase-C activator, phorbol 12-myristate 13-acetate. Reverse fibrin autography of conditioned medium from control cells revealed the presence of a putative PAI with an apparent mol wt of 50,000. Phorbol ester stimulated the accumulation of this PAI in a specific and dose-dependent manner. Cumulus cells and noncumulus granulosa cells were similar in terms of presence and regulation of PAI. Immunoprecipitation with specific antisera revealed that this human granulosa cell PAI was immunochemically related to PAI-1. This identification was supported by quantitative analysis revealing a 3.7-fold increase in PAI-1 antigen, as assessed by a specific enzyme-linked immunoabsorbent assay. These findings are the first demonstration of the in vitro regulation of PAI activity in the human ovary.

High-level synthesis of biologically active human plasminogen activator inhibitor type 1 (PAI-1) in Escherichia coli.

Segments of a cDNA encoding human plasminogen activator inhibitor type 1 (PAI-1) were subcloned into a highly regulated and inducible Escherichia coli expression system. A plasmid encoding the mature form of human endothelial PAI-1 produced a functional recombinant molecule, as indicated by its ability to inhibit tissue plasminogen activator's enzymatic activity. In contrast to PAI-1 isolated from human fibrosarcoma cells, the biological activity of the recombinant PAI-1 was not dependent on pretreatment with denaturing agents. A construct encoding a polypeptide lacking the first 80 amino acids of PAI-1 also produced elevated levels of the truncated recombinant protein. However, this truncated product was functionally inactive, indicating that an intact N terminus is required for activity.

Characterization and comparative evaluation of a structurally unique PAI-1 inhibitor exhibiting oral in-vivo efficacy.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 are associated with thrombosis and vascular disease, suggesting that high plasma PAI-1 may promote a hypercoagulable state by disrupting the natural balance between fibrinolysis and coagulation. In this study, we identify WAY-140312 as a structurally novel small molecule inactivator of PAI-1, compare its inhibitory activity with other previously identified small molecule inhibitors, and investigate the mechanism of inactivation of PAI-1 in the presence of both tPA and uPA. In an immunofunctional assay, WAY-140312 inhibited PAI-1 with an estimated inhibitory concentration (IC(50)) of 11.7 micro m, which was the lowest value obtained of the four different PAI-1 inactivators tested. Surface activity profiling indicated that the critical micelle concentration for WAY-140312 was 95.8 micro m, and that each inhibitor exhibited unique physical chemical properties. Using a sensitive direct activity assay, the IC(50) for WAY-140312 was similar when either tPA or uPA was used as the target protease. Immunoblot analysis demonstrated that WAY-140312 near the IC(50) inhibited the complex formation between either tPA or uPA and PAI-1. After oral administration, WAY-140312 exhibited 29% bioavailability with a plasma half-life of approximately 1 h. In an in-vivo model of vascular injury, a 10 mg kg(-1) oral dose of WAY-140312 was associated with improvement in arterial blood flow and reduction in venous thrombosis. Thus, WAY-140312 represents a structurally novel small molecule inhibitor of PAI-1, and is the first such molecule to exhibit efficacy in animal models of vascular disease following oral administration.

Inhibitory spectra of purified protease nexin-II and related proteins towards cellular proteinases.

Amyloid beta protein (beta/A4) is deposited in senile plaques of patients with Alzheimer's disease. This protein is derived from a larger membrane-associated protein, termed amyloid precursor protein (APP). The constitutive processing of APP occurs at the central portion of beta/A4, resulting in the release of large N-terminal peptides. We have purified these peptides from the culture medium of cDNA-transfected COS-1 cells. Some of the isoforms contain the Kunitz-type protease inhibitor (KPI) domain and strongly inhibit trypsin, chymotrypsin and plasmin, but do not inhibit kallikrein, prolyl endopeptidase or granzyme A. The peptides also do not inhibit cysteine proteases such as cathepsin B or calpain. Soluble APPs lacking the KPI domain fail to inhibit any of these proteases. The results indicate that the KPI domain in soluble APPs has protease inhibitory activity against certain serine proteases.

Characterization of a plasminogen activator produced by Acanthamoeba castellanii.

Serine proteases play an important role in a diverse array of biological processes, including embryogenesis, metastasis, angiogenesis, thrombolysis and tissue invasion by certain parasites. The latter observation prompted us to explore the possibility that the tissue-invasive ocular parasite Acanthamoeba castellanii elaborates one or more serine proteases. Acanthamoeba sp. are pathogenic free-living amoebae that can produce an invasive, blinding inflammatory disease of the cornea, termed Acanthamoeba keratitis. The present study reports the preliminary purification and characterization of a novel plasminogen activator from an ocular isolate of A. castellanii. The parasite-derived enzyme has a molecular mass of approx. 40 kDa and produces a single band of lysis on fibrinogen-agarose zymographs. Activity of the enzyme is completely inhibited by treatment with diisopropylfluorophosphate, indicating that it is a serine protease. The parasite-derived serine protease is not inhibited by amiloride which is a strong inhibitor of urokinase-type plasminogen activator. Additionally, the enzyme is not inhibited by plasminogen activator inhibitor-1 which is the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator. It does not cross-react with antibodies specific for human urokinase or tissue-type plasminogen activator. The parasite-derived enzyme activates plasminogen from several mammalian species, including human, cow and pig. Thus, it is possible that this parasite-derived serine protease contributes to the pathogenesis of Acanthamoeba keratitis.

Effect of plasminogen activator inhibitor-1 on tissue-type plasminogen activator-induced fibrinolysis.

The effect of fibrin on the interaction of human recombinant single-chain tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in normal rabbit plasma and in plasma with high levels of native PAI-1. t-PA was added to diluted plasma containing calcium (10 mM) and 125I-fibrinogen at 37 degrees C. Clotting was initiated with human thrombin, and lysis was monitored both turbidimetrically and by release of 125I-fibrin degradation products (fdp). The activity of t-PA (50 IU/ml) was rapidly reduced to 15% of the initial value in plasma containing PAI-1 (23 AU/ml). When thrombin and t-PA were added simultaneously to the plasma, more than 70% of the activity was retained through incorporation of t-PA into the fibrin clot. t-PA-induced fibrinolysis in PAI-1 enriched plasma was further delayed when the temperature was reduced from 37 to 25 degrees C. Turbidimetric and 125I-fdp release data provided complementary information. The former technique traced fiber dissolution, while the latter reflected network integrity. These results indicate that t-PA-induced fibrinolysis in PAI-1 enriched plasma is modulated by the presence of fibrin and by temperature.