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Prion diseases are a group of neurodegenerative diseases, which can progress rapidly. Previous data have demonstrated that prion protein (PrP) stimulates activation of plasminogen (Plg) by tissue plasminogen activator (tPA). In this study, using spectroscopic method, we aimed to determine whether PrP’s role in activating Plg is influenced by metal binding. We also investigated the region in PrP involved in binding to tPA and Plg, and whether PrP in fibrillar form behaves the same way as PrP unbound to any metal ion i.e., apo-PrP. We investigated the effect of recombinant mouse PrP (residues 23-231) refolded with nickel, manganese, copper, and a variant devoid of any metal ions, on tPA-catalyzed Plg activation. Using mutant PrP (H95A, H110A), we also investigated whether histidine residues outside the octarepeat region in PrP, which is known to bind tPA and Plg, are also involved in their binding. We demonstrated that apo-PrP is most effective at stimulating Plg. PrP refolded with nickle or manganese behave similar to apo-PrP, and PrP refolded with copper is least effective. The mutant form of PrP did not stimulate Plg activation to the same degree as apo-PrP indicating that the histidine residues outside the octarepeat region are also involved in binding to tPA and Plg. Similarly, the fibrillar form of PrP was ineffective at stimulating Plg activation. Our data suggest that upon loss of copper specifically, a structural rearrangement of PrP occurs that exposes binding sites to Plg and tPA, enhancing the stimulation of Plg activation.

It has recently been shown that hepatocyte growth factor activator inhibitor-2 (HAI-2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI-2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI-2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non-SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto-cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI-2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI-2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI-2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.

HGF (hepatocyte growth factor) is a pleiotropic cytokine homologous to the serine protease zymogen plasminogen that requires canonical proteolytic cleavage to gain functional activity. The activating proteases are key components of its regulation, but controversy surrounds their identity. Using quantitative analysis we found no evidence for activation by uPA (urokinase plasminogen activator), despite reports that this is a principal activator of pro-HGF. This was unaffected by a wide range of experimental conditions, including the use of various molecular forms of both HGF and uPA, and the presence of uPAR (uPA receptor) or heparin. In contrast the catalytic domains of the TTSPs (type-II transmembrane serine proteases) matriptase and hepsin were highly efficient activators (50% activation at 0.1 and 3.4 nM respectively), at least four orders of magnitude more efficient than uPA. PS-SCL (positional-scanning synthetic combinatorial peptide libraries) were used to identify consensus sequences for the TTSPs, which in the case of hepsin corresponded to the pro-HGF activation sequence, demonstrating a high specificity for this reaction. Both TTSPs were also found to be efficient activators at the cell surface. Activation of pro-HGF by PC3 prostate carcinoma cells was abolished by both protease inhibition and matriptase-targeting siRNA (small interfering RNA), and scattering of MDCK (Madin–Darby canine kidney) cells in the presence of pro-HGF was abolished by inhibition of matriptase. Hepsin-transfected HEK (human embryonic kidney)-293 cells also activated pro-HGF. These observations demonstrate that, in contrast with the uPA/uPAR system, the TTSPs matriptase and hepsin are direct pericellular activators of pro-HGF, and that together these proteins may form a pathway contributing to their involvement in pathological situations, including cancer.

Maspin is a serpin that has multiple effects on cell behavior, including inhibition of migration. How maspin mediates these diverse effects remains unclear, as it is devoid of protease inhibitory activity. We have previously shown that maspin rapidly inhibits the migration of vascular smooth muscle cells (VSMC), suggesting the involvement of direct interactions with cell surface proteins. Here, using immunofluorescence microscopy, we demonstrate that maspin binds specifically to the surface of VSMC in the dedifferentiated, but not the differentiated, phenotype. Ligand blotting of VSMC lysates revealed the presence of several maspin-binding proteins, with a protein of 150 kDa differentially expressed between the two VSMC phenotypes. Western blotting suggested that this protein was the beta1 integrin subunit, and subsequently both alpha3beta1 and alpha5beta1, but not alphavbeta3, were shown to associate with maspin by coimmunoprecipitation. Specific binding of these integrins was also observed using maspin-affinity chromatography, using HT1080 cell lysates. Direct binding of maspin to alpha5beta1 was confirmed using a recombinant alpha5beta1-Fc fusion protein. Using conformation-dependent anti-beta1 antibodies, maspin binding to VSMC was found to lead to a decrease in the activation status of the integrin. The functional involvement of alpha5beta1 in mediating the effect of maspin was established by the inhibition of migration of CHO cells overexpressing human alpha5 integrin, but not those lacking alpha5 expression. Our observations suggest that maspin engages in specific interactions with a limited number of integrins on VSMC, leading to their inactivation, and that these interactions are responsible for the effects of maspin in the pericellular environment.

The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored cellular receptor (uPAR) promotes plasminogen activation and the efficient generation of pericellular proteolytic activity. We demonstrate here that expression of the tetraspanin CD82/KAI1 (a tumor metastasis suppressor) leads to a profound effect on uPAR function. Pericellular plasminogen activation was reduced by approximately 50-fold in the presence of CD82, although levels of components of the plasminogen activation system were unchanged. uPAR was present on the cell surface and molecularly intact, but radioligand binding analysis with uPA and anti-uPAR antibodies revealed that it was in a previously undetected cryptic form unable to bind uPA. This was not due to direct interactions between uPAR and CD82, as they neither co-localized on the cell surface nor could be co-immunoprecipitated. However, expression of CD82 led to a redistribution of uPAR to focal adhesions, where it was shown by double immunofluorescence labeling to co-localize with the integrin alpha(5)beta(1), which was also redistributed in the presence of CD82. Co-immunoprecipitation experiments showed that, in the presence of CD82, uPAR preferentially formed stable associations with alpha(5)beta(1), but not with a variety of other integrins, including alpha(3)beta(1). These data suggest that CD82 inhibits the proteolytic function of uPAR indirectly, directing uPAR and alpha(5)beta(1) to focal adhesions and promoting their association with a resultant loss of uPA binding. This represents a novel mechanism whereby tetraspanins, integrins, and uPAR, systems involved in cell adhesion and migration, cooperate to regulate pericellular proteolytic activity and may suggest a mechanism for the tumor-suppressive effects of CD82/KAI1.

Over-expression of components of the urokinase system is well documented in cancer and is thought to enable tumour cells to migrate and invade. Changes in integrin expression are also a common feature of tumours and have been linked to changes in protease activity. It has been shown that the alphavbeta6 integrin is neo-expressed in a number of epithelial carcinomas and in wound healing situations. We therefore investigated whether alphavbeta6 is able to modulate a key regulator of proteolysis, the urokinase receptor. We report that epithelial cells expressing full-length alphavbeta6 exhibit decreased urokinase receptor expression and function. Furthermore, this novel modulation requires the C-terminal 11 amino acids of the cytoplasmic tail of the beta6 integrin subunit. Cells expressing alphavbeta3, however, did not affect urokinase receptor expression. De novo expression of beta6 by melanoma cells and beta3 by epithelial cells did not influence urokinase receptor expression or function, suggesting that modulation of urokinase system is both integrin subunit and cell-specific.

Maspin is a member of the serpin family of protease inhibitors and is a tumor suppressor gene acting at the level of tumor invasion and metastasis. This in vivo activity correlates with the ability of maspin to inhibit cell migration in vitro. This behavior suggests that maspin inhibits matrix-degrading proteases, such as those of the plasminogen activation system, in a similar manner to the serpin PAI-1. However, there is controversy concerning the protease inhibitory activity of maspin. It is devoid of activity against a wide range of proteases, in common with other non-inhibitory serpins, but has recently been reported to inhibit plasminogen activators associated with cells and other biological surfaces (Sheng, S. J., Truong, B., Fredrickson, D., Wu, R. L., Pardee, A. B., and Sager, R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 499-504; McGowen, R., Biliran, H., Jr., Sager, R., and Sheng, S. (2000) Cancer Res. 60, 4771-4778). We have compared the effects of maspin with those of PAI-1 in a range of situations in which plasminogen activation is potentiated, reflecting the biological context of this proteolytic system: urokinase-type plasminogen activator bound to its receptor on the surface of tumor cells, tissue-type plasminogen activator specifically bound to vascular smooth muscle cells, fibrin, and the prion protein. Maspin was found to have no inhibitory effect in any of these situations, in contrast to the efficient inhibition observed with PAI-1, but nevertheless maspin inhibited the migration of both tumor and vascular smooth muscle cells. We conclude that maspin is a non-inhibitory serpin and that protease inhibition does not account for its activity as a tumor suppressor.

Prion diseases are associated with the conversion of the normal prion protein, PrP(C), to the infectious disease form PrP(Sc). Discrimination between these isoforms would significantly enhance diagnosis of these diseases, and it has recently been reported that PrP(Sc) is specifically recognized by the serine protease zymogen plasminogen (Fischer et al. (2000) Nature 408, 479). Here we have tested the hypothesis that PrP is a regulator of the plasminogen activation system. The effect of recombinant PrP, either containing copper (holo-PrP) or devoid of it (apo-PrP), on plasminogen activation by both uPA and tPA was determined. PrP had no effect on plasminogen activation by uPA. By contrast, the activity of tPA was stimulated by up to 280-fold. This was observed only with the apo-PrP isoforms. The copper-binding octapeptide repeat region of PrP was involved in the effects, as a mutant lacking this region failed to stimulate plasminogen activation, although a synthetic peptide corresponding to this region was unable to stimulate tPA activity. Competition experiments demonstrated that, in addition to plasminogen binding, the stimulation required a high-affinity interaction between tPA and PrP (K(d) < 2.5 nM). Kinetic analysis revealed a template mechanism for the stimulation, suggesting independent binding sites for tPA and plasminogen. Lack of copper-binding may be an early event in the conversion of PrP(C) to PrP(Sc), and our data therefore suggest that tPA-catalyzed plasminogen activation may provide the basis for a sensitive detection system for the early stages of prion diseases and also play a role in the pathogenesis of these diseases.

A variety of proteases have the potential to degrade the extracellular matrix (ECM), thereby influencing the behaviour of cells by removing physical barriers to cell migration, altering cell-ECM interactions or releasing ECM-associated growth factors. The plasminogen activation system of serine proteases is particularly implicated in this pericellular proteolysis and is involved in pathologies ranging from cancer invasion and metastasis to fibroproliferative vascular disorders and neurodegeneration. A central mechanism for regulating plasmin generation is through the binding of the two plasminogen activators to specific cellular receptors: urokinase-type plasminogen activator to the glycolipid-anchored membrane protein uPAR, and tissue plasminogen activator to a type-II transmembrane protein recently identified on vascular smooth muscle cells. These binary complexes interact with membrane-associated plasminogen to form higher order activation complexes that greatly reduce the Km for plasminogen activation and, in some cases, protect the proteases from their cognate serpin inhibitors. Various other proteins that are involved in cell adhesion and migration also interact with these complexes, modulating the activity of this efficient and spatially restricted proteolytic system. Recent observations demonstrate that certain forms of the prion protein can stimulate tissue plasminogen activator-catalysed plasminogen activation, which raises the possibility that these proteases may also have a role in the pathogenesis of the transmissible spongiform encephalopathies.

The generation of the broad specificity serine protease plasmin in the pericellular environment is regulated by binding of the urokinase-type plasminogen activator (uPA) to its specific glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor, uPAR. This interaction potentiates the reciprocal activation of the cell-associated zymogens pro-uPA and plasminogen. To further study the role of uPAR in this mechanism, we have expressed two directly membrane-anchored chimeric forms of uPA, one anchored by a C-terminal GPI-moiety (GPI-uPA), the other with a C-terminal transmembrane peptide (TM-uPA). These were expressed in the monocyte-like cell lines U937 and THP-1, which are excellent models for kinetic and mechanistic studies of cell-surface plasminogen activation. In both cell-lines, GPI-uPA activated cell-associated plasminogen with characteristics both qualitatively and quantitatively indistinguishable from those of uPAR-bound uPA. By contrast, TM-uPA activated cell-associated plasminogen less efficiently. This was due to effects on the Km for plasminogen activation (which was increased up to five-fold) and the efficiency of pro-uPA activation (which was decreased approximately four-fold). These observations suggest that uPAR serves two essential roles in mediating efficient cell-surface plasminogen activation. In addition to confining uPA to the cell-surface, the GPI-anchor plays an important role by increasing accessibility to substrate plasminogen and, thus, enhancing catalysis. However, the data also demonstrate that, in the presence of an alternative mechanism for uPA localization, uPAR is dispensable and, therefore, unlikely to participate in any additional interactions that may be necessary for the efficiency of this proteolytic system. In these experiments zymogen pro-uPA was unexpectedly found to be constitutively activated when expressed in THP-1 cells, suggesting the presence of an alternative plasmin-independent proteolytic activation mechanism in these cells. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator (tPA) specifically, saturably, and with relatively high affinity (K(d) 25 nM), and this binding potentiates the activation of cell-associated plasminogen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322). We have observed that this binding can be efficiently competed by DFP-inactivated tPA and S478A-tPA but not by tPA inactivated with H-D-Phe-Pro-Arg-chloromethyl ketone (PPACK). VSMC-bound tPA also exhibited a markedly reduced inhibition by PPACK, displaying biphasic kinetics with second-order rate constants of 7. 5 x 10(3) M(-1) s(-1) and 0.48 x 10(3) M(-1) s(-1), compared with 7. 2 x 10(3) M(-1) s(-1) in the solution phase. By contrast, tPA binding to fibrin was competed equally well by all forms of tPA, and its inhibition was unaltered. These effects were shown to extend to the physiological tPA inhibitor, plasminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 complex did not compete tPA binding to VSMC, and the inhibition of bound tPA was reduced by 30-fold. The behavior of the various forms of tPA bound to VSMC correlated with conformational changes in tPA detected by CD spectroscopy. These data suggest that tPA binds to its specific high affinity site on VSMC by a novel mechanism involving the serine protease domain of tPA and distinct from its binding to fibrin. Furthermore, reciprocally linked conformational changes in tPA appear to have functionally significant effects on both the interaction of tPA with its VSMC binding site and the susceptibility of bound tPA to inhibition.

Plasminogen activators play a role in the response of the vessel wall to injury, presumably by mediating the degradation of extracellular matrix (ECM) by vascular smooth muscle cells (VSMCs) that is necessary for their migration and proliferation. We have therefore investigated the ability of VSMCs to assemble specific cell surface plasminogen-activating systems. Urokinase-type plasminogen activator (uPA) bound to a single class of site on VSMCs (kd, 2 nmol/L), binding of pro-uPA resulted in a large potentiation of plasmin generation and both were competed by antibodies to the uPA receptor (uPAR). Tissue-type plasminogen activator (tPA) also bound to VSMCs as determined by functional assay, with the binding isotherms showing two classes of binding site with apparent kds of 25 and 300 nmol/L. tPA binding to the higher affinity site caused a greater than 90-fold enhancement of the activation of cell bound plasminogen, whereas the lower affinity binding, mediated primarily by the ECM, had little effect on tPA activity. The high-affinity binding of tPA to VSMCs resulted in an eightfold greater potential for plasmin generation than the binding of uPA, with this difference increasing to 15-fold after thrombin stimulation of the cells due to a 1.8-fold increase in tPA binding. These data show a novel specific tPA receptor on VSMCs that may be important for the regulation of plasminogen activation in various vascular pathologies.

SummaryMatriptase is a type-II transmembrane serine protease involved in epithelial homeostasis in both health and disease, and is implicated in the development and progression of a variety of cancers. Matriptase mediates its biological effects both via as yet undefined substrates and pathways, and also by proteolytic cleavage of a variety of well-defined protein substrates, several of which it shares with the closely-related protease hepsin. Development of targeted therapeutic strategies will require discrimination between these proteases. Here we have investigated cyclic microproteins of the squash Momordica cochinchinensis trypsin-inhibitor family (generated by total chemical synthesis) and found MCoTI-II to be a high-affinity (Ki 9 nM) and highly selective (> 1,000-fold) inhibitor of matriptase. MCoTI-II efficiently inhibited the proteolytic activation of pro-hepatocyte growth factor (HGF) by matriptase but not by hepsin, in both purified and cell-based systems, and inhibited HGF-dependent cell scattering. MCoTI-II also selectively inhibited the invasion of matriptase-expressing prostate cancer cells. Using a model of epithelial cell tight junction assembly, we also found that MCoTI-II could effectively inhibit the re-establishment of tight junctions and epithelial barrier function in MDCK-I cells after disruption, consistent with the role of matriptase in regulating epithelial integrity. Surprisingly, MCoTI-II was unable to inhibit matriptase-dependent proteolytic activation of prostasin, a GPI-anchored serine protease also implicated in epithelial homeostasis. These observations suggest that the unusually high selectivity afforded by MCoTI-II and its biological effectiveness might represent a useful starting point for the development of therapeutic inhibitors, and further highlight the role of matriptase in epithelial maintenance.

u-Plasminogen activator (uPA) is one of two highly specific mammalian proteases responsible for the activation of plasminogen by specific cleavage of an Arg-Val bond to form the broad substrate specificity protease plasmin. This activity gives it a central role both in the haemostatic process (via the dissolution of fibrin = fibrinolysis) and in the generation of pericellular proteolytic activity, which is implicated in many pathologies involving tissue remodeling/degradation …

Plasminogen activation catalyzed by the urokinase-type plasminogen activator (uPA) constitutes a reciprocal zymogen activation system, as plasmin can efficiently activate pro-uPA, the single-chain zymogenic form of the protease. We have previously shown that the overall efficiency of this plasminogen activation system is greatly enhanced by its assembly on the cell surface, involving binding of pro-uPA to its cellular binding site uPAR, and the concurrent cellular binding of plasminogen. We have now studied the effect of a recombinant soluble form of uPAR (residues 1–277) on the proteolytic reactions of this system. In contrast to the increased efficiencies of plasminogen activation and pro-uPA activation observed with cell-surface uPAR, soluble uPAR had an inhibitory effect on both of these individual reactions. Soluble uPAR also caused no increase in the low, but discernible, intrinsic activity of pro-uPA. Consistent with the observations on the isolated reactions, the overall activity of the pro-uPA-mediated plasminogen activation system was significantly inhibited. These observations confirm the previous interpretation of the observations made with cell-surface uPAR that the mechanism of the enhanced plasmin generation is due to the catalytically favorable interaction of uPAR-bound uPA/pro-uPA with cell-bound plasminogen/plasmin, rather than direct effects on the properties of uPA or pro-uPA on binding to uPAR.

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid anchored receptor (uPAR) is essential for the confinement of plasminogen activation to cell surfaces where it is thought to play an important role in cancer cell invasion and metastasis. The receptor binding site of uPA is retained within its isolated growth factor-like module (GFD; residues 4-43). The NH2-terminal domain of uPAR has a primary role in uPA binding, although maintenance of its multidomain structure has been shown to be necessary for the high affinity of this interaction [Ploug, M., Ellis, V., & Dano, K. (1994) Biochemistry 33, 8991-8997]. To identify residues engaged in the uPAR-uPA interaction, we have performed a "protein-protein footprinting" study on preformed uPAR-GFD complexes by chemical modification with tetranitromethane. All six tyrosine residues in uPAR and the single tyrosine residue in GFD (Tyr24) were susceptible to nitration in the native uncomplexed proteins, whereas in the receptor-ligand complexes both Tyr57 of uPAR and Tyr24 of GFD were protected from modification. Modification of uPAR alone led to a parallel reduction in the potential to bind pro-uPA and 8-anilino-1-naphthalenesulfonate, an extrinsic fluorophore reporting on the accessibility of a hydrophobic site involved in uPA binding. These data clearly demonstrate that Tyr57 in the NH2-terminal domain of uPAR and Tyr24 in uPA are intimately engaged in the receptor-ligand interaction, whereas Tyr87 positioned in the linker region between the first two domains of uPAR does not appear to be shielded by the resulting intermolecular interface.

Plasminogen activation is regulated by the interaction between urokinase-type plasminogen activator (uPA) and its specific glycolipid-anchored cell surface receptor (uPAR). uPAR is composed of three homologous domains and is the only multi-domain member of the Ly-6 family of glycolipid-anchored membrane proteins. Recent evidence has highlighted similarities between the individual domains of uPAR and the large family of secreted, single domain snake venom alpha-neurotoxins, suggesting that uPAR may adopt the same gross folding pattern as these structurally well characterized proteins. Structural aspects of the binding between alpha-neurotoxins and the acetylcholine receptor may have a major influence on future studies of the interaction between uPA and uPAR.

Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G α-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on β1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored three-domain membrane protein playing a central role in pericellular plasminogen activation. We have found that urokinase (uPA) can cleave its receptor between domains 1 and 2 generating a cell-associated uPAR variant without ligand-binding properties. In extracts of U937 cells there are two uPAR variants which after complete deglycosylation have apparent molecular masses of 35,000 and 27,000. Analysis with monoclonal antibodies showed that these variants represented the intact uPAR and a two-domain form, uPAR(2+3), lacking ligand-binding domain 1. Trypsin treatment showed that both variants are present on the outside of the cells. Addition to the culture medium of an anticatalytic monoclonal antibody to uPA inhibited the formation of the uPAR(2+3), indicating that uPA is involved in its generation. Purified uPAR can be cleaved directly by uPA as well as by plasmin. The uPA-catalyzed cleavage does not require binding of the protease to the receptor through its epidermal growth factor-like receptor-binding domain, since low molecular weight uPA that lacks this domain also cleaves uPAR. This unusual reaction in which a specific binding protein is proteolytically inactivated by its own ligand may represent a regulatory step in the plasminogen activation cascade.

We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro-u-PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u-PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system.

Brain-derived neurotrophic factor (BDNF) is secreted as either a mature furin-processed form or an unprocessed pro-form. Here, we characterise the extracellular processing of pro-BDNF by the serine protease plasmin. Using recombinant BDNF, maintained in the pro-form by site-directed mutagenesis or inhibition of furin, we demonstrate that plasmin (but not related proteases) is a specific and efficient activator of pro-BDNF. The proteolytic cleavage site is identified as Arg125-Val, within the consensus furin-cleavage motif (RVRR), generating an active form that stimulated neurite outgrowth on TrkB-transfected PC12 cells. Furthermore, we demonstrate that this processing can also occur in the pericellular environment by the action of cell-associated plasminogen activators.

Urokinase-type plasminogen activator (uPA) binds to a specific receptor on various cell types, the bound molecule retaining its enzymatic activity against plasminogen. We have now investigated whether receptor-bound uPA also retains the ability to react with and be inhibited by plasminogen activator inhibitors (PAI-1 and PAI-2). uPA bound to its receptor on human U937 monocyte-like cells was inhibited by PAI-1 (in its active form in the presence of vitronectin fragments) with an association rate constant of 4.5 x 10(6) M-1 s-1, which was 40% lower than that obtained for uPA in solution (7.9 x 10(6) M-1 s-1). The inhibition of uPA by PAI-2 was decreased to a similar extent by receptor binding, falling from 5.3 x 10(5) to 3.3 x 10(5) M-1 s-1. Stimulation of U937 cells with phorbol 12-myristate 13-acetate was accompanied by a further reduction in receptor-bound uPA inhibition by PAI-1 and PAI-2 to 1.7 x 10(6) and 1.1 x 10(5) M-1 s-1, respectively. These constants although lower than those for uPA in solution still represent rather rapid inhibition of the enzyme, and demonstrate that uPA bound to its specific cellular receptor remains available for efficient inhibition by PAI's, which may therefore play a major role in controlling cell-surface plasminogen activation and extracellular proteolytic activity.

Pericellular proteolytic activity affects many aspects of cellular behaviour, via mechanisms involving processing of the extracellular matrix, growth factors and receptors. The serine proteases have exquisitely sensitive regulatory mechanisms in this setting, involving both receptor-bound and transmembrane proteases. Receptor-bound proteases are exemplified by the uPA (urokinase plasminogen activator)/uPAR (uPAR receptor) plasminogen activation system. The mechanisms initiating the activity of this proteolytic system on the cell surface, a critical regulatory point, are poorly understood. We have found that the expression of the TTSP (type II transmembrane serine protease) matriptase is highly regulated in leucocytes, and correlates with the presence of active uPA on their surface. Using siRNA (small interfering RNA), we have demonstrated that matriptase specifically activates uPAR-associated pro-uPA. The uPA/uPAR system has been implicated in the activation of the plasminogen-related growth factor HGF (hepatocyte growth factor). However, we find no evidence for this, but instead that HGF can be activated by both matriptase and the related TTSP hepsin in purified systems. Hepsin is of particular interest, as the proteolytic cleavage sequence of HGF is an ‘ideal substrate’ for hepsin and membrane-associated hepsin activates HGF with high efficiency. Both of these TTSPs can be activated autocatalytically at the cell surface, an unusual mechanism among the serine proteases. Therefore these TTSPs have the capacity to be true upstream initiators of proteolytic activity with subsequent downstream effects on cell behaviour.

We have demonstrated that tissue plasminogen activator (tPA) binds specifically to human vascular smooth muscle cells (VSMC) in a functionally relevant manner, both increasing plasminogen activation and decreasing tPA inhibition (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322; Werner, F., Razzaq, T. M., and Ellis, V. (1999) J. Biol. Chem. 274, 21555-21561). To further understand this system we have now identified and characterized the protein responsible for this binding. Rat VSMC were surface-labeled with 125I, and cell lysates were subjected to an affinity chromatography scheme based on the previously identified tPA binding characteristics. A single radiolabeled protein of 63 kDa bound specifically and was eluted at low pH. This protein was isolated from large scale preparations of VSMC and unambiguously identified as the rat homologue of the human type-II transmembrane protein p63 (CKAP4) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. In confirmation of this, a monoclonal antibody raised against authentic human p63 recognized the isolated protein in Western blotting. Immunofluorescence microscopy demonstrated that p63 was located principally in the endoplasmic reticulum but was also detected in significant quantities on the surface of human VSMC. In support of the hypothesis that p63 is the functional tPA binding site on VSMC, an anti-p63 monoclonal antibody was found to block tPA binding. Furthermore, heterologous expression of an N-terminally truncated mutant of p63, which targets exclusively to the plasma membrane, led to an increase in tPA-catalyzed plasminogen activation. Therefore, p63 on the surface of VSMC may contribute to the functional regulation of the plasminogen activation system in the vessel wall.

It has been emphasized here that plasminogen represents an enormous reservoir of proteolytic potential and that its activation is consequently a highly regulated process. This begins at the level of transcriptional regulation of the two plasmino-gen activators and in some cell types also their regulated secretion. However, the functional regulation of the plasminogen activators is the level at which the com-plexity of this proteolytic system is most apparent. It is now clear that a diversity of mechanisms exist to regulate the activation and activity of the plasminogen activa-tors, and that these include the utilization of these soluble proteins as cell-surface proteases. uPA is clearly the primary plasminogen activator in this context, binding uPAR and forming catalytic complexes that generate plasmin activity with very high efficiency, and furthermore cooperating with protease inhibitors to confine this ac-tivity to the cell surface or pericellular environment. The activity of this proteolytic system, which is thought to be involved in mediating invasive cellular migration, appears to be functionally linked to other systems fundamental to this cellular behavior. A variety of mechanisms have been proposed, but a common feature is direct or indirect interactions with integrins potentially leading to signaling to the cellular cytoskeletal machinery, suggesting a cooperativity between these systems in mediating invasive migration. tPA clearly also has the potential to act as a cell-surface protease, in addition to its fibrin-dependent role in vascular hemostasis. A variety of cellular binding sites for tPA have been identified, some of which appear to regulate plasminogen activation as efficiently as uPAR. In contrast to uPAR, these binding sites may be cell-type specific, although their roles in vivo have yet to be established.

Cell migration over or through the extracellular matrix (ECM) is an integral feature of both physiological and pathological processes. Regulation of the changing cell–ECM interactions involved can be effected by proteolysis and requires strict spatial and temporal targeting of proteinase activity. The versatile use of different proteinase systems, with a variety of localisation mechanisms and cleavage targets, is being revealed by a plethora of studies using in vitro models. This mini review reflects the status of our knowledge of strategies for the localisation of proteolytic activity effected during cell migration.

Peptides corresponding to residues from Loops I and III of platelet-derived growth factor–BB (PDGF-BB) were examined for their potential to act as PDGF antagonists. We have identified two peptides which directly stimulated DNA synthesis in human dermal fibroblasts and a cyclic peptide which inhibited PDGF-induced DNA synthesis. The inhibitory action of cyclic PDGF-BB73-81, on DNA synthesis was shown to be restricted to cells which express PDGF receptors. Also cyclic PDGF-BB73-81 specifically competed for 125I-labelled PDGF-BB but not for 125I-labelled EGF binding to their respective cellular receptors. The cyclic peptide therefore provides a minimum structure to investigate PDGF/receptor interactions and our findings confirm the importance of the loop configuration of PDGF-BB73-81 in the native molecule. The cyclic peptide may constitute a basis for developing more potent inhibitors of PDGF action.

The receptor for urokinase-type plasminogen activator (uPAR) is an integral membrane protein that specifically binds urokinase-type plasminogen activator (uPA) and plays a crucial role in cell surface plasmin generation. We have previously found that transforming growth factor-beta, type 1 (TGF-beta 1), increases uPAR gene transcription in the human lung carcinoma cell line A549 and now report that also epidermal growth factor (EGF) and the tumour promoter phorbol 12-myristate 13-acetate (PMA) cause increased uPAR transcription and that PMA and TGF-beta 1 in addition increase the stability of uPAR mRNA, while EGF has no effect on this parameter. All three compounds also increase the uPAR protein level, as measured by cell-binding experiments with radiolabelled ligand. The increase in uPAR protein level was however considerably lower with all three compounds than the increase in mRNA level, suggesting that they also exert a translational or post-translational control. Accompanying the increase in the number of uPAR molecules there was a proportional decrease in their ligand-binding affinity, the mechanism of which is unknown. Platelet-derived growth factor, basic fibroblast growth factor and cyclic AMP analogues did not induce any change in the uPAR mRNA level in A549 cells. Previous studies have shown that expression of uPA and its type-1 inhibitor is regulated by a variety of cytokines in a cell-specific manner. The present study indicates that cytokines in addition influence cell surface plasminogen activation by regulating uPAR expression.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

We have observed that a murine IgG1 monoclonal antibody directed against human urokinase-type plasminogen activator (uPA) greatly potentiates pro-uPA-mediated plasminogen activation. This effect was dependent on the interaction between the immunoglobulin and the kringle domain of pro-uPA and could be competed efficiently by kringle-containing proteolytic fragments of uPA. In addition, the potentiation could also be competed by the lysine analog 6-aminohexanoic acid, an antagonist of plasminogen binding. This unexpected plasminogen binding dependence was found to be due to a carboxyl-terminal lysine residue on the immunoglobulin gamma chain, which by analogy with other proteins represents a potential binding site for plasminogen. Removal of this residue with carboxypeptidase B resulted in a complete abolition of the potentiation. It appears therefore that the potentiatory effect involves a novel mechanism with the antibody acting to provide a specific template for the assembly of a ternary complex involving pro-uPA/uPA and plasminogen, enabling them to interact in a catalytically favorable manner. This interpretation was confirmed by studying the kinetics of plasminogen activation by the complex between active, two-chain uPA and the antibody, which resulted in an overall 50-fold increase in reaction efficiency (kcat/Km), primarily due to a reduction in Km from 20 to 0.1 microM. Pro-uPA activation by plasmin was also accelerated, although to a lesser extent. The potentiation due to complex formation also provides a mechanism for the initiation of this system, dependent only on the low intrinsic proteolytic activity of the zymogen forms. The effects observed here, mediated by ternary complex formation, simulate the effects we have previously observed on assembly of the uPA receptor-mediated cellular plasminogen activation system and may therefore represent a mechanistic model for both its activity and initiation.

Publisher Summary This chapter discusses the methods that are developed for the study of urokinase-type plasminogen activator (uPa) and plasmin activity in relation to urokinase-type plasminogen activator (uPAR). These methods may also have a more general applicability in the study of other cell surface-associated protease systems, particularly, those that may have an intimate involvement with the plasminogen activation system, for example, the matrix metalloproteases. Numerous studies have delineated a system whereby the generation of cell surface proteolytic activity can be mediated by uPAR, the cellular receptor for the urokinase-type plasminogen activator (uPA). Because of the saturability of the inhibitory effect of isolated uPAR it is possible, under the experimental conditions to derive an approximation to the dissociation constant for the uPA-uPAR interaction in solution. The requirement for quantitative assays to investigate the functional effects of cell surface uPAR on the activity of the uPA-catalyzed plasminogen activation system presents a number of difficulties, particularly with respect to sensitivity and specific quantification.

The rate of inactivation of human coagulation factor Xa by the plasma proteinase inhibitors antithrombin III and alpha 1-antitrypsin has been studied in the presence of the accessory components which constitute the prothrombinase complex. The rate of inactivation of factor Xa by antithrombin III was found to be decreased in the presence of phospholipid vesicles with high affinity for factor Xa. The second-order rate constant for the reaction fell from 6.21 X 10(4) to 3.40 X 10(4) M-1 min-1 in the presence of 20 microM phospholipid. Purified factor Va had no effect on the rate of inactivation of factor Xa in the absence of phospholipid. In the presence of phospholipid, factor Va increased the protective effect displayed by phospholipid, further reducing the rate constant to 2.20 X 10(4) M-1 min-1. The rate of inactivation of factor Xa by alpha 1-antitrypsin was unaffected under these conditions. Platelet-bound prothrombinase complex was formed by incubation of factor Xa with washed human platelets activated by a mixture of collagen and thrombin. The prothrombinase activity was inhibited by antithrombin III was a second-order rate constant of 0.85 X 10(4) M-1 min-1. This rate was obtained in both the presence and absence of exogenous factor Va. Platelet factor 3 vesicles, isolated from platelet aggregation supernatants, also formed prothrombinase complex in the presence of factor Va, and this was inhibited by antithrombin III at the same rate as the platelet-bound complex. There was no protection of the platelet-bound prothrombinase complex from inhibition by alpha 1-antitrypsin.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.

The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by uPA specifically bound to its receptor on the human monocytoid cell-line U937 and demonstrate this process to have properties differing widely from those observed for uPA in solution. The solution-phase reaction was characterized by a Km of 25 microM and for the cell-associated reaction this fell 40-fold to 0.67 microM, below the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding antagonist. In contrast to intact cells, purified uPA receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937 cells) was observed to partially inhibit uPA-catalyzed Plg activation, although activity against low molecular weight substrates was retained. Therefore, the cellular binding of Plg appears to be of critical importance for the efficient activation of Plg by receptor-bound uPA. Plasmin generated in the cell-surface Plg activation system described here was also observed to be protected from its principal physiological inhibitor alpha-2-antiplasmin. Together, these data demonstrate that the cell surface constitutes the preferential site for Plg activation when uPA is bound to its specific cellular receptor, which therefore has the necessary characteristics to play an efficient role in the generation of pericellular proteolytic activity.