Analysis of plasminogen-binding M proteins of Streptococcus pyogenes

Group A streptococci are common human pathogens that cause a variety of infections. They express M proteins which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express M proteins that bind plasminogen and plasmin with high affinity. The binding is mediated by a 13-amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these M proteins. This sequence binds to kringle 2 in plasminogen, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of plasminogen. It could be demonstrated that plasminogen, absorbed from plasma by growing group A streptococci expressing the plasminogen-binding M proteins, could be activated by exogenous and endogenous streptokinase, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host.     

Isolation of a prokaryotic plasmin receptor. Relationship to a plasminogen activator produced by the same micro-organism.

Plasminogen receptors have been identified on the surface of a number of prokaryotic and eukaryotic cells. A receptor demonstrating high affinity for plasmin with minimal reactivity with the native zymogen Glu-plasminogen has been identified on the surface of certain group A streptococci. In this study the group A streptococcal plasmin receptor has been solubilized and purified to homogeneity. The isolated protein was an Mr approximately 41,000 molecule which retained its ability to bind plasmin following solubilization and affinity purification on a column of enzymatically inactivated human plasmin. The isolated plasmin receptor was compared functionally, antigenically, and physicochemically to the secreted plasminogen activator, streptokinase, produced by the same organism. The Mr approximately 41,000 surface plasmin receptor was shown to be functionally and antigenically distinct from the Mr approximately 48,000 streptokinase molecule produced by the same strain and lacked any plasminogen activator activity. The streptokinase molecule produced by this strain was shown to be closely related to the plasminogen activator protein secreted by other group A and C streptococci. This study represents the first report of the isolation of a plasmin receptor, either prokaryotic or eukaryotic, with functional activity.     

Contribution of plasminogen activation towards the pathogenic potential of oral streptococci

Oral streptococci are a heterogeneous group of human commensals, with a potential to cause serious infections. Activation of plasminogen has been shown to increase the virulence of typical human pathogenic streptococci such as S. pneumoniae. One important factor for plasminogen activation is the streptococcal α-enolase. Here we report that plasminogen activation is also common in oral streptococci species involved in clinical infection and that it depends on the action of human plasminogen activators. The ability to activate plasminogen did not require full conservation of the internal plasminogen binding sequence motif FYDKERKVY of α-enolase that was previously described as crucial for increased plasminogen binding, activation and virulence. Instead, experiments with recombinant α-enolase variants indicate that the naturally occurring variations do not impair plasminogen binding. In spite of these variations in the internal plasminogen binding motif oral streptococci showed similar activation of plasminogen. We conclude that the pathomechanism of plasminogen activation is conserved in oral streptococci that cause infections in human. This may contribute to their opportunistic pathogenic character that is unfurled in certain niches.     

Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

On the basis of 125I-labeled plasminogen activator binding analysis we have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a Kd of 0.8958 x 10(-9) M [corrected]. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.     

The maintenance of high affinity plasminogen binding by group A streptococcal plasminogen-binding M-like protein is mediated by arginine and histidine residues within the a1 and a2 repeat domains

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.     

Complex interactions between bovine plasminogen and streptococcal plasminogen activator PauA

The interactions between bovine plasminogen and the streptococcal plasminogen activator PauA that culminate in the generation of plasmin are not fully understood. Formation of an equimolar activation complex comprising PauA and plasminogen by non-proteolytic means is a prerequisite to the recruitment of substrate plasminogen; however the determinants that facilitate these interactions have yet to be defined. A mutagenesis strategy comprising nested deletions and random point substitutions indicated roles for both amino and carboxyl-terminal regions of PauA and identified further essential residues within the alpha domain of the plasminogen activator. A critical region within the alpha domain was identified using non-overlapping PauA peptides to block the interaction between PauA and bovine plasminogen, preventing formation of the activation complex. Homology modelling of the activation complex based upon the known structures of streptokinase complexed with human plasmin supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.     

A chromogenic assay for the detection of plasmin generated by plasminogen activator immobilized on nitrocellulose using a para-nitroanilide synthetic peptide substrate

A direct solid phase chromogenic assay has been developed for the detection of plasmin (EC 3.4.21.7), generated by the interaction of a nitrocellulose-bound plasminogen activator, using the plasmin specific tripeptide substrate, H-D-valyl-leucyl-lysine - p-nitroaniline. para-Nitroaniline released by the cleavage of the lysine - p-nitroaniline bound by plasmin was derivatized to its diazonium salt and subsequently coupled to N-1-napthylethylenediamine in situ to form a diazoamino of an intense red color at the site of the plasminogen activator. This method was used to assay for the streptococcal plasminogen activator, streptokinase, not only in crude bacterial supernatants, but also to detect streptokinase secreted by individual bacterial colonies. In addition, this solid phase assay was used to identify monoclonal antibodies specific for streptokinase which could inhibit the activation of human plasminogen by streptokinase. This method also permitted simultaneous immunological and biochemical identification of the plasminogen activator, thus permitting unequivocal comparative observations. This assay is quantitative and sensitive to nanogram amounts of activator comparable to those obtained with soluble assays. This method may also be applicable for the detection of other plasminogen activators, such as tissue plasminogen activator, urokinase, and staphylokinase, and also for the detection of immobilized proteases which can cleave other substrates derivatized with p-nitroaniline. The reagents used in this assay are inexpensive and easy to prepare.     

Treatment of mouse L-cells with phorbol myristate acetate induces the secretion of a plasminogen activator inhibitor which binds to human and mouse urokinase and human tissue plasminogen activator

1. Serum-free conditioned medium from L-cells or L-cells treated with the tumor-promotor phorbol myristate acetate (PMA) was analyzed for plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activity. Conditioned medium from control or PMA-treated cells did not contain detectable PA activity when assayed by SDS-PAGE and zymography. 2. Conditioned medium from PMA-treated cells, but not control cells, contained a PAI of Mr = 40,000 da when assayed by reverse zymography. 3. The L-cell PAI formed SDS-stable complexes with purified human (homo sapiens) urokinase and tissue plasminogen activator, as well as, mouse (Mus musculus) urinary PA. 4. These results indicate that biochemical and immunological differences between human and mouse urokinase and human urokinase and human tissue plasminogen activator do not influence the interaction of the L-cell PAI with these enzymes.     

链激酶研究概况及其进展

链激酶(Streptok inase,SK)是世界上最早发现的纤维蛋白酶原激活剂,也是最早作为临床药品治疗血栓性疾病的溶栓酶。它是由A,C,G群链球菌中β-溶血性链球菌分泌的胞外非酶蛋白质,能和纤溶酶原结合,将纤溶酶原激活为纤溶酶,具有溶解血栓的作用。本文详细综述了该酶的性质、在溶栓酶中的地位、研究历史、作用机理等。此外,由于它有半衰期短、不具有纤维蛋白特异性、治疗后出血和血栓易复发的缺点,所以有必要用基因工程的手段进行改造,以达到更好的治疗效果。     

Conservation of critical functional domains in murine plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 is the main physiological regulator of tissue-type plasminogen activator in normal plasma. In addition to its critical function in fibrinolysis, plasminogen activator inhibitor-1 has been implicated in roles in other physiological and pathophysiological processes. To investigate structure-function aspects of mouse plasminogen activator inhibitor-1, the recombinant protein was expressed in Escherichia coli and purified. Five variant recombinant murine proteins (R76E, Q123K, R346A, R101A, and Q123K/R101A) were also generated using site-directed mutagenesis. The variant (R346A) was found to be defective in its inhibitory activity against tissue plasminogen activator relative to its wild-type counterpart. Enzyme-linked immunosorbent assay and surface plasmon resonance experiments demonstrated reduced vitronectin-binding affinity of the (Q123K) variant (K(D) = 1800 nm) relative to the wild-type protein (K(D) = 5.4 nm). Kinetic analyses indicated that the (Q123K) variant had a slower association (k(on) = 2.92 x 10(4) m(-1) s(-1)) to, and a faster dissociation from, vitronectin (k(off) = 5.3 x 10(-2) s(-1)), (wild-type k(on) = 1.03 x 10(6) m(-1) s(-1) and k(off) = 5.27 x 10(-3) s(-1)). The Q123K/R101A variant demonstrated an even lower vitronectin-binding ability. Low density lipoprotein receptor-related protein binding was decreased for the (R76E) variant. It was also demonstrated that the plasminogen activator inhibitor-1/vitronectin complex decreased the interaction of plasminogen activator inhibitor-1 with low density lipoprotein receptor-related protein. These results indicate that the complex interactions traditionally associated with different plasminogen activator inhibitor-1 functions apply to the murine system, thus showing a commonality of subtle functions among different species and evolutionary conservation of this protein. Further, this study provides additional evidence that the human hemostasis system can be studied effectively in the mouse, which is a great asset for investigations with gene-altered mice.     

Stimulation of tPA-dependent provisional extracellular fibrin matrix degradation by human recombinant soluble melanotransferrin

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.     

Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen

A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radioimmunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human an rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply.     

92-kD type IV collagenase mediates invasion of human cytotrophoblasts

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.     

New insights into the tPA-annexin A2 interaction. Is annexin A2 CYS8 the sole requirement for this association?

Annexin A2 has been described as an important receptor for tissue-type plasminogen activator in endothelium and other cell types. Interaction between tissue-type plasminogen activator and its cellular receptor is critical for many of the functions of this protease. The annexin A2 motif that mediates tissue plasminogen activator interaction has been assigned to the hexapeptide LCKLSL in the amino-terminal domain of the protein, and it has been proposed that Cys(8) of this sequence is essential for tPA binding. In an attempt to identify other amino acids critical for tPA-annexin A2 interaction, we have analyzed a set of peptides containing several modifications of the original hexapeptide, including glycine scans, alanine scans, d-amino acid scans, conservative mutations, cysteine blocking, and enantiomer and retroenantiomer sequences. Using a non-radioactive competitive binding assay, we have found that all cysteine-containing peptides, independently of their sequence, compete the interaction between tPA and annexin A2. Cysteine-containing peptides also inhibit tPA binding to the surface of cultured human umbilical vein endothelial cells (HUVEC). Mass spectrometry demonstrates that the peptides bind through a disulfide bond to a cysteine residue of annexin A2, the same mechanism that has been suggested for the inhibition mediated by homocysteine. These data call for a revision of the role of the LCKLSL sequence as the sole annexin A2 structural region required to bind tPA and indicate that further studies are necessary to better define the annexin A2-tPA interaction.     

The production of distinct forms of plasminogen activator by mouse embryonic cells

We have examined cultured parietal endoderm, visceral endoderm, and extraembryonic mesoderm cells from the mouse embryo for production of the protease plasminogen activator. All of these cell types synthesize and secrete the enzyme, but the molecular characteristics of the plasminogen activators differ as defined by the apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gels, the antigenic properties as defined by two antisera to distinct plasminogen activators, and the interaction with an inhibitor present in fetal bovine serum. The parietal endoderm plasminogen activator has a predominant molecular weight of 79,000, is immunoprecipitated and inhibited by an antiserum raised against a human melanoma plasminogen activator, but not by an antiserum against mouse urokinase, and is only partially inhibited by the serum inhibitor. The visceral endoderm and extraembryonic mesoderm plasminogen activators, which are identical by all criteria, have molecular weights of 48,000, are inactivated only by the anti-urokinase antibodies, and are inhibited by an inhibitor in fetal bovine serum. These results establish the presence of at least two different forms of plasminogen activator in the early mouse embryo. The distinctive nature of the enzyme produced by parietal endoderm can be used as a diagnostic marker for this cell type at this stage of development. When F9 teratocarcinoma stem cells are induced to differentiate by retinoic acid and dibutyryl cAMP, they secrete a plasminogen activator of the parietal endoderm type.     

Bacterial plasminogen activators and receptors

Invasive bacterial pathogens intervene at various stages and by various mechanisms with the mammalian plasminogen/plasmin system. A vast number of pathogens express plasmin(ogen) receptors that immobilize plasmin(ogen) on the bacterial surface, an event that enhances activation of plasminogen by mammalian plasminogen activators. Bacteria also influence secretion of plasminogen activators and their inhibitors from mammalian cells. The prokaryotic plasminogen activators streptokinase and staphylokinase form a complex with plasmin(ogen) and thus enhance plasminogen activation. The Pla surface protease of Yersinia pestis resembles mammalian activators in function and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators turn bacteria into proteolytic organisms using a host-derived system. In Gram-negative bacteria, the filamentous surface appendages fimbriae and flagella form a major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules as well as M-protein-related structures have been identified as plasminogen receptors, the former receptor type also occurs on mammalian cells. Plasmin is a broad-spectrum serine protease that degrades fibrin and noncollagenous proteins of extracellular matrices and activates latent procollagenases. Consequently, plasmin generated on or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrices. In a few instances plasminogen activation has been shown to enhance bacterial metastasis in vitro through reconstituted basement membrane or epithelial cell monolayers. In vivo evidence for a role of plasminogen activation in pathogenesis is limited to Y. pestis, Borrelia, and group A streptococci. Bacterial proteases may also directly activate latent procollagenases or inactivate protease inhibitors of human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers.     

Plasminogen activation: biochemistry, physiology, and therapeutics

The mammalian serine protease zymogen, plasminogen, can be converted into the active enzyme plasmin by vertebrate plasminogen activators urokinase (uPA), tissue plasminogen activator (tPA), factor XII-dependent components, or by bacterial streptokinase. The biochemical properties of the major components of the system, plasminogen/plasmin, plasminogen activators, and inhibitors of the plasminogen activators, are reviewed. The plasmin system has been implicated in a variety of physiological and pathological processes such as fibrinolysis, tissue remodeling, cell migration, inflammation, and tumor invasion and metastasis. A defective plasminogen activator/inhibitor system also has been linked to some thromboembolic complications. Recent studies of the mechanism of fibrinolysis in human plasma suggest that tPA may be the primary initiator and that overall fibrinolytic activity is strongly regulated at the tPA level. A simple model for the initiation and regulation of plasma fibrinolysis based on these studies has been formulated. The plasminogen activators have been used for thrombolytic therapy. Three new thrombolytic agents--tPA, pro-uPA, and acylated streptokinase-plasminogen complex--have been found to possess better properties over their predecessors, urokinase and streptokinase. Further improvements of these molecules using genetic and protein engineering tactics are being pursued.     

An Early Look at the Therapeutic Uses of Some New Vasopressin Analogs in Gastroenterology

Natural vasopressins have been used, with varying success, in attempts to stop bleeding from esophageal varices for over two decades. Reasons for lack of success include (a) failure to induce a sufficiently prolonged and constant vasoconstrictor effect at the bleeding site, (b) dangerous side-effects, and (c) release of plasminogen activator induced by the peptides which can lyse any clot as it forms.In the last decade analogs of vasopressin have been developed with a prolonged action, using two separate principles of chemical modification: (1) hormonogens, and (2) blockage of sites of inactivating enzymatic cleavage (in particular “carba” analogs without a disulfide bridge). These two categories of analog are compared here: the carba analogs have the advantages of high potency (higher than the parent hormone) with prolongation, but are also very active on the plasminogen activator release system. The hormonogens combine prolongation with low potency, but have lost not only a releasing action on plasminogen activator, but also, by virtue of altered release kinetics, have effectively lost cardiovascular toxicity.Mechanisms of analog action and receptor interaction are presented, along with initial clinical experiences.     

Activation of peroxisome proliferator-activated receptor-gamma decreases pancreatic cancer cell invasion through modulation of the plasminogen activator system

Cancer cell invasion and metastasis require the concerted action of several proteases that degrade extracellular matrix proteins and basement membranes. Recent reports suggest the plasminogen activator system plays a critical role in pancreatic cancer biology. In the present study, we determined the contribution of the plasminogen activator system to pancreatic cancer cell invasion in vitro. Moreover, the effect of peroxisome proliferator-activated receptor (PPAR)-gamma ligands, which are currently in clinical use as antidiabetic drugs and interestingly seem to display antitumor activities, on pancreatic cancer cell invasion and the plasminogen activator system was assessed. Expression of components of the plasminogen activator system [i.e., urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1, and uPA receptor] was detected in six human pancreatic cancer cell lines. Inhibition of urokinase activity by specific synthetic compounds reduced baseline pancreatic cancer cell invasion. The PPAR-gamma ligands 15-deoxy-Delta12,14-prostaglandin J2 and ciglitazone also attenuated pancreatic cancer cell invasion. This effect was abrogated by dominant-negative PPAR-gamma receptors and pharmacologic PPAR-gamma inhibitors. Moreover, activation of PPAR-gamma by ligands increased plasminogen activator inhibitor-1 and decreased uPA levels in pancreatic cancer cells, and this was accompanied by a reduction in total urokinase activity. The present study shows that the plasminogen activator system plays an integral role in pancreatic cancer cell invasion in vitro. Activation of the nuclear receptor PPAR-gamma by ligands reduced pancreatic cancer cell invasion, which was largely mediated by modulation of the plasminogen activator system. These findings further underscore the potential role of PPAR-gamma ligands as therapeutic agents in pancreatic cancer.