Exploring the potential of a structural alphabet-based tool for mining multiple target conformations and target flexibility insight.

Protein flexibility is often implied in binding with different partners and is essential for protein function. The growing number of macromolecular structures in the Protein Data Bank entries and their redundancy has become a major source of structural knowledge of the protein universe. The analysis of structural variability through available redundant structures of a target, called multiple target conformations (MTC), obtained using experimental or modeling methods and under different biological conditions or different sources is one way to explore protein flexibility. This analysis is essential to improve the understanding of various mechanisms associated with protein target function and flexibility. In this study, we explored structural variability of three biological targets by analyzing different MTC sets associated with these targets. To facilitate the study of these MTC sets, we have developed an efficient tool, SA-conf, dedicated to capturing and linking the amino acid and local structure variability and analyzing the target structural variability space. The advantage of SA-conf is that it could be applied to divers sets composed of MTCs available in the PDB obtained using NMR and crystallography or homology models. This tool could also be applied to analyze MTC sets obtained by dynamics approaches. Our results showed that SA-conf tool is effective to quantify the structural variability of a MTC set and to localize the structural variable positions and regions of the target. By selecting adapted MTC subsets and comparing their variability detected by SA-conf, we highlighted different sources of target flexibility such as induced by binding partner, by mutation and intrinsic flexibility. Our results support the interest to mine available structures associated with a target using to offer valuable insight into target flexibility and interaction mechanisms. The SA-conf executable script, with a set of pre-compiled binaries are available at http://www.mti.univ-paris-diderot.fr/recherche/plateformes/logiciels.

Soluble urokinase plasminogen activator receptor and incidence of venous thromboembolism.

Raised plasma levels of the soluble urokinase plasminogen activator receptor (suPAR) have been associated with increased incidence of cardiovascular diseases. Whether suPAR is associated with venous thromboembolism (VTE) is largely unknown. The purpose of the present study was to investigate the relationship between suPAR and incidence of VTE in a cohort study. suPAR was measured in 5,203 subjects (aged 46-68 years, 58 % women) from the general population, who participated in the Malmö Diet and Cancer (MDC) study between 1991 and 1994. Incident cases of VTE were identified from the Swedish patient register during a mean follow-up of 15.7 years. Of 5,203 subjects with measurements of suPAR, 239 had VTE during follow-up (127 venous thrombosis, 86 lung embolism, 26 both). Incidence of VTE was significantly higher in subjects with suPAR levels in the top quartile. Adjusted for age and sex, the HR (4th vs 1st quartile) was 1.74 (95 %CI: 1.2-2.6, p for trend=0.003). After adjustments for risk factors, the HR was 1.66 (95 %CI: 1.1-2.5, p for trend=0.016). High level of suPAR was a risk indicator for incidence of VTE in this population-based cohort study. The causal relationships between suPAR and VTE remain to be explored.

Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a).

Apo(a), the distinguishing protein component of lipoprotein(a) [Lp(a)], exhibits sequence similarity to plasminogen and can inhibit binding of plasminogen to cell surfaces. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. The ability of apo(a) to inhibit pericellular plasminogen activation on vascular cells was therefore evaluated. Two isoforms of apo(a), 12K and 17K, were found to significantly decrease tissue-type plasminogen activator-mediated plasminogen activation on human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes and macrophages. Lp(a) purified from human plasma decreased plasminogen activation on THP-1 monocytes and HUVECs but not on THP-1 macrophages. Removal of kringle V or the strong lysine binding site in kringle IV10 completely abolished the inhibitory effect of apo(a). Treatment with carboxypeptidase B to assess the roles of carboxyl-terminal lysines in cellular receptors leads in most cases to decreases in plasminogen activation as well as plasminogen and apo(a) binding; however, inhibition of plasminogen activation by apo(a) was unaffected. Our findings directly demonstrate that apo(a) inhibits pericellular plasminogen activation in all three cell types, although binding of apo(a) to cell-surface receptors containing carboxyl-terminal lysines does not appear to play a major role in the inhibition mechanism.

[Structure and functions of plasminogen/plasmin system].

The main physiological function of plasmin is a blood clot fibrinolysis and restore normal blood flow. To date, however, it became apparent that in addition to thrombolysis plasminogen/plasmin system plays an important physiological and pathological role in the degradation of extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation and tumor cells migration. This review focuses on the structural features of plasminogen, the regulation of its activation by physiological plasminogen activators, inhibitors of plasmin and plasminogen activators, the role of the plasminogen binding to fibrin, cellular receptors and extracellular ligands in performing various functions by formed plasmin.

Inhibitors of urokinase type plasminogen activator and cytostatic activity from crude plants extracts.

In view of the clear evidence that urokinase type plasminogen activator (uPA) plays an important role in the processes of tumor cell metastasis, aortic aneurysm, and multiple sclerosis, it has become a target of choice for pharmacological intervention. The goal of this study was thus to determine the presence of inhibitors of uPA in plants known traditionally for their anti-tumor properties. Crude methanol extracts were prepared from the leaves of plants (14) collected from the subtropical dry forest (Guanica, Puerto Rico), and tested for the presence of inhibitors of uPA using the fibrin plate assay. The extracts that tested positive (6) were then partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol, in a sequential manner. The resulting fractions were then tested again using the fibrin plate assay. Extracts from leaves of Croton lucidus (C. lucidus) showed the presence of a strong uPA inhibitory activity. Serial dilutions of these C. lucidus partitions were performed to determine the uPA inhibition IC50 values. The chloroform extract showed the lowest IC50 value (3.52 µg/mL) and hence contained the most potent uPA inhibitor. Further investigations revealed that the crude methanol extract and its chloroform and n-butanol partitions did not significantly inhibit closely related proteases such as the tissue type plasminogen activator (tPA) and plasmin, indicating their selectivity for uPA, and hence superior potential for medicinal use with fewer side effects. In a further evaluation of their therapeutic potential for prevention of cancer metastasis, the C. lucidus extracts displayed cytostatic activity against human pancreatic carcinoma (PaCa-2) cells, as determined through an MTS assay. The cytostatic activities recorded for each of the partitions correlated with their relative uPA inhibitory activities. There are no existing reports of uPA inhibitors being present in any of the plants reported in this study.

Three decades of research on plasminogen activator inhibitor-1: a multifaceted serpin.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator and therefore plays an important role in the plasminogen-plasmin system. PAI-1 is involved in a variety of cardiovascular diseases (mainly through inhibition of t-PA) as well as in cell migration and tumor development (mainly through inhibition of u-PA and interaction with vitronectin). PAI-1 is a unique member of the serpin superfamily, exhibiting particular unique conformational and functional properties. Because of its involvement in various biologic and pathophysiologic processes, PAI-1 has been the subject of many studies, including extensive structural investigations, in vitro cell biologic studies, in vivo animal studies, and epidemiologic studies. The review provides an overview on the current knowledge on PAI-1.

Structural basis for activation of an integral membrane protease by lipopolysaccharide.

Omptins constitute a unique family of outer membrane proteases that are widespread in Enterobacteriaceae. The plasminogen activator (Pla) of Yersinia pestis is an omptin family member that is very important for development of both bubonic and pneumonic plague. The physiological function of Pla is to cleave (activate) human plasminogen to form the plasma protease plasmin. Uniquely, lipopolysaccharide (LPS) is essential for the catalytic activity of all omptins, including Pla. Why omptins require LPS for enzymatic activity is unknown. Here, we report the co-crystal structure of LPS-free Pla in complex with the activation loop peptide of human plasminogen, its natural substrate. The structure shows that in the absence of LPS, the peptide substrate binds deep within the active site groove and displaces the nucleophilic water molecule, providing an explanation for the dependence of omptins on LPS for enzymatic activity.

Regulation of cell migration and invasion by specific modules of uPA: mechanistic insights and specific inhibitors.

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

Structural and functional comparison of proteolytic enzymes from plant latex and snake venoms.

This work describes classification, functions, location, inhibition, activation, and therapeutic applications of proteases from snake venoms and vegetables. Snake venoms and vegetables can present toxins that unchain necrosis or proteolysis due to the direct cytotoxic action of venom proteases. These proteases are potential tools in the development of drugs for the prevention and treatment of several illnesses. We report herein mainly fibrinogenolytic metallo proteases and serine proteases ("thrombin-like"). These enzymes are extensively used in the treatment and prevention of thrombotic disorders, since they serve as defibrinogenating agents. The therapeutic uses of fibrin(ogen)olytic metallo proteases hold promise for clinical application due to potential in reversing the effects of thrombosis; this has been shown to be an alternative approach to the prevention and treatment of cardiovascular disorders, which are among the most prominent causes of mortality around the world. Plant proteases can be utilized for many cellular and molecular activities, in antibacterial and anticancer therapies, and in the treatment of snakebites, inhibiting snake venom activities such as blood-clotting, defibrinogenation, and fibrin(ogen)olytic and hemorrhagic actions. These toxins also display potential for clinical use in the treatment of hemostatic disorders.

Urokinase-a very popular cardiovascular agent.

Urokinase (UK) [EC 3.4.99.26] is a serine protease that activates plasminogen to plasmin, which in turn degrades fibrin clots. Hence, UK finds its value as an important anti-thromboembolic drug. Plasmin has diverse physiological roles apart from its fibrinolytic role in the regulation of blood clotting. It has been implicated in complement activation, cell migration, wound healing, and generation of localized extracellular proteolysis during tissue remodelling, pro-hormone conversion, carcinogenesis and neoplasia. Among the plasminogen activators, UK provides a superior alternative for the simple reasons of its being more potent as compared to tissue-plasminogen activator and non-antigenic by virtue of its human origin unlike streptokinase. Based on these observations, UK is a very popular cardiovascular agent. Hence, UK, as one of the most potent plasminogen activators is attracting a great deal of attention. We will summarize recent patents related to the occurrence, mechanism of action, structure and function, physico-chemical properties, in vitro production, cloning and expression, purification and applications of UK.

Substrate specificity and screening of the integral membrane protease Pla.

This paper reports a study to find small peptide substrates for the important virulence factor of Yersinia pestis, plasminogen activator, Pla. The method used to find small substrates for this protease is reported along with studies examining the ability of these peptides to inhibit activity of the enzyme. Through the use of parallel synthesis and positional scanning, small tripeptides were identified that are viable substrates for the protease.

Design of a novel plasminogen activator based on the structure of hirudin.

Using a phage library, seven peptide sequences with high affinity to human microplasminogen were obtained. Caseinolytic assay indicated that only the synthesized peptide P07 had slight fibrinolytic activity. To enhance its plasminogen activation ability, peptide P07 was fused into loop 32-35 of hirudin. In vitro assay demonstrated that this hirudin-like fusion protein can activate human plasminogen and retain the function of thrombin inhibition. Fusing the sequence ''SPDASRL'' into hirudin generated a plasminogen activation activity 100 times higher than peptide P07 in chromogenic and radial caseinolytic assay. This significant functional improvement might originate from a more specific active structure due to the hirudin scaffold.

Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro.

During ovarian follicle growth, there is expansion of the basal lamina and changes in the follicular extracellular matrix (ECM) that are mediated in part by proteolytic enzyme cascades regulated by tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). One PA inhibitor, serine protease inhibitor-E2 (SERPINE2) is expressed in granulosa but not theca cells, and expression changes with follicle development. In this study, we hypothesized that PA and SERPINE2 expression/secretion by granulosa cells are regulated by FSH and growth factors. SERPINE2 mRNA and protein levels, tPA gene expression and uPA secretion were stimulated by FSH. Insulin-like growth factor-I stimulated SERPINE2 secretion and uPA activity, and decreased secreted tPA activity and gene expression. Bone morphogenetic protein-7 increased SERPINE2 secretion and expression and tPA secretion. In contrast, fibroblast growth factor-2 inhibited tPA secretion and SERPINE2 secretion and expression. Epidermal growth factor inhibited SERPINE2 secretion and expression, but increased secreted tPA activity. Estradiol and SERPINE2 secretion were highly positively correlated, but estradiol did not alter SERPINE2 expression. These data demonstrate that SERPINE2 expression and protein secretion are regulated by FSH and growth factors in non-luteinizing bovine granulosa cells. As estradiol is a known marker of follicle health, and SERPINE2 is an anti-apoptotic factor, we propose that SERPINE2 is involved in the regulation of atresia in bovine follicles.

Production of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1) in tobacco is hampered by proteolysis.

The high fibrin specificity of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1 or desmoteplase (INN)) makes it a promising candidate for the treatment of acute ischemic stroke. In the current study we explored the use of transgenic tobacco plants and BY-2 suspension cells as alternative production platforms for this drug. Four different N-terminal signal peptides, from plants and animals, were used to translocate the recombinant DSPAalpha1 protein to the endomembrane system. Intact recombinant DSPAalpha1 was produced in transgenic plants and BY-2 cells, although a certain degree of degradation was observed in immunoblotted extracts. The choice of signal peptide had no major influence on the degradation pattern or recombinant protein accumulation, which reached a maximum level of 38 microg/g leaf material. N-terminal sequencing of purified, His6-tagged DSPAalpha1 revealed only minor changes in the position of signal peptide cleavage compared to the same protein expressed in Chinese hamster ovary cells. However, correctly processed recombinant DSPAalpha1 was also detected. The enzymatic activity of the recombinant protein was confirmed using an in vitro assay with unpurified and purified samples, demonstrating that plants are suitable for the production of functional DSPAalpha1. In contrast to whole plant cell extracts, no recombinant DSPAalpha1 was detected in the culture supernatant of transgenic BY-2 cells. Further analysis showed that recombinant DSPAalpha1 is subject to proteolysis and that endogenous secreted BY-2 proteases are responsible for DSPAalpha1 degradation in the culture medium. The addition of a highly concentrated protease inhibitor mixture or 5 mM EDTA reduced DSPAalpha1 proteolysis, improving the accumulation of intact product in the culture medium. Strategies to improve the plant cell suspension system for the production of secreted recombinant proteins are discussed.

Regulation of urokinase receptor proteolytic function by the tetraspanin CD82.

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.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251Õ. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251Õ. StreptaseÕ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The UnitinaseÕ and SolustrepÕ formulations were the weakest, showing about 50 percent activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251Õ activity per vial, StreptaseÕ (75.7 ± 5.0 units) and StreptonaseÕ (94.7 ± 4.6 units) had the highest activity, while UnitinaseÕ (31.0 ± 2.4 units) and StrekÕ (32.9 ± 3.3 units) had the weakest activity. SolustrepÕ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

Monolithic peptidyl sorbents for comparison of affinity properties of plasminogen activators.

Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein's denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA-EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.

Localization of plasminogen activator activity within normal and injured lungs by in situ zymography.

During inflammatory lung injury, the fibrinolytic activity that is normally present within bronchoalveolar lavage (BAL) fluid (BALF) is often suppressed due to increased levels of inhibitors, including plasminogen activator inhibitor (PAI)-1. Despite this suppression, BALF frequently contains fibrin degradation products, indicating persistence of fibrinolytic activity within the lung. To address this discrepancy and determine the sites where plasminogen activation is occurring, we developed an in situ zymographic technique for frozen sections of lung tissue that localizes plasminogen activator activity at the cellular level. After validating the method using enzyme inhibitors and mice with genetic manipulations of their plasminogen system genes, we applied the technique to lungs of normal and bleomycin-exposed mice. In normal mice, plasminogen activator activity was localized to bronchial epithelial cells, cells of the alveolar walls, and alveolar macrophages. After bleomycin exposure, in situ zymography showed that, despite loss of fibrinolytic activity within BALF, abundant enzymatic activity was associated with aggregates of inflammatory cells. PAI-1-deficient mice that are protected from bleomycin-induced fibrosis had preserved plasminogen activator activity in BALF and increased tissue activity, as determined by in situ zymography. We conclude that analysis of BALF does not adequately reflect the fibrinolytic activity that persists within microenvironments of the lung during inflammation.

Nonlysine-analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin-like activity.

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 microg.mL(-1). These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.

Domain-dependent action of urokinase on smooth muscle cell responses.

BACKGROUND: Single-chain urokinase-type plasminogen activator (sc-uPA) is one of the key serine proteases involved in modulating cellular and extracellular matrix responses during tissue remodeling. Sc-uPA is composed of three domains: aminoterminal fragment (ATF), kringle domain, and carboxyterminal fragment (CTF). sc-uPA is readily cleaved into these three domain fragments in vitro, each of which is biologically active; however, their roles in the microenvironment of the vessel wall are poorly understood. PURPOSE: The purpose of this study was to determine the role of each domain of sc-uPA on vascular smooth muscle cell (SMC) proliferation and migration. METHODS: SMCs were cultured in vitro. Assays of DNA synthesis, cell proliferation, and migration were performed in response to sc-uPA, ATF, kringle, and CTF in the presence and absence of the plasmin inhibitors epsilon-aminocaproic acid (EACA) and aprotinin, the Galphai inhibitor pertussis toxin, and the mitogen-activated protein kinase 1 (the upstream regulator of the extracellular-signal regulated kinase [ERK]) inhibitor PD98059. RESULTS: sc-uPA produced dose-dependent increases in DNA synthesis and cell proliferation. These responses were dependent on the CTF domain and were sensitive to plasmin inhibitors, pertussis toxin, and PD98059. Sc-uPA also induced SMC migration, which could be elicited by both ATF and kringle. Migration to sc-uPA, ATF, and kringle was both pertussis toxin and PD98059 sensitive, but importantly was plasmin-independent. CONCLUSION: sc-uPA induces SMC proliferation and migration, which are domain-dependent and mediated in part by Galphai-linked, ERK-dependent processes, while only the mitogenic response is protease dependent. These findings suggest that migration is linked to a G-protein coupled nonprotease receptor, while proliferation is associated with a G-protein coupled protease receptor.