Pg Binding Research Articles

Human salivary protein-derived peptides specific-salivary SIgA antibodies enhanced by nasal double DNA adjuvant in mice play an essential role in preventing Porphyromonas gingivalis colonization: an in-vitro study

BackgroundWe previously showed that fimbriae-bore from Poryphyromonas gingivalis (Pg), one of the putative periodontopathogenic bacteria specifically bound to a peptide domain (stat23, prp21) shared on statherin or acidic proline-rich protein 1 (PRP1) molecule of human salivary proteins (HSPs). Here, we investigated whether the nasal administration of DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 as double DNA adjuvant (dDA) with stat23 and prpr21 induces antigen (Ag)-specific salivary secretory IgA (SIgA) antibodies (Abs) in mice. Further, we examined that stat23- and prpr21-specific salivary SIgA Abs induced by dDA have an impact on Pg-binding to human whole saliva-coated hydroxyapatite beads (wsHAPs).Material and methodsC57BL/6N mice were nasally immunized with dDA plus sta23 or/and prp21 peptide as Ag four times at weekly intervals. Saliva was collected one week after the final immunization and was subjected to Ag-specific ELISA. To examine the functional applicability of Ag-specific SIgA Abs, SIgA-enriched saliva samples were subjected to Pg binding inhibition assay to wsHAPs.ResultsSignificantly elevated levels of salivary SIgA Ab to stat23 or prp21 were seen in mice given nasal stat23 or prp21 with dDA compared to those in mice given Ag alone. Of interest, mice nasally given the mixture of stat23 and prp21 as double Ags plus dDA, resulted in both stat23- and prp21-specific salivary SIgA Ab responses, which are mediated through significantly increased numbers of CD11c+ dendritic cell populations and markedly elevated Th1 and Th2 cytokines production by CD4+ T cells in the mucosal inductive and effector tissues. The SIgA Ab-enriched saliva showed significantly reduced numbers of live Pg cells binding to wsHAPs as compared with those in mice given double Ags without dDA or naïve mice. Additionally, saliva from IgA-deficient mice given nasal double Ags plus dDA indicated no decrease of live Pg binding to wsHAPs.ConclusionThese findings show that HSP-derived peptides-specific salivary SIgA Abs induced by nasal administration of stat23 and prp21 peptides plus dDA, play an essential role in preventing Pg attachment and colonization on the surface of teeth, suggesting a potency that the SIgA may interrupt and mask fimbriae-binding domains in HSPs on the teeth.

Integration of VEK-30 peptide enhances fibrinolytic properties of staphylokinase.

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α-2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK-30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C-terminal end of SAK with a RGD (Arg-Gly-Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK-30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat /Km , of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2- antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin-specific manner and its ability to clear platelet-rich plasma clot faster than SAK.

Preparation of n‐3 Polyunsaturated Phosphatidylglycerol from Salmon Roe Lipids by Phospholipase D and In Vitro Digestion

AbstractPhosphatidylglycerol (PG) is a highly functional phospholipid (PL), which has many physiological functions. However, naturally occurring PG binding n‐3 polyunsaturated fatty acid (n‐3 PUFA) is low in content, resulting in a scarcity of industrial bio‐resources of n‐3 PUFA enriched PG. The current study investigates the preparation of salmon roe PG (SRPG) from three types of salmon roe lipids and glycerol via phospholipase D (PLD)‐mediated transphosphatidylation. The yields of SRPG obtained from salmon roe total lipid (SRTL) and salmon roe PL (SRPL) are higher than those obtained from purified salmon roe phosphatidylcholine (SRPC) in aqueous system. Following a 24 h reaction with 0.75 U PLD, SRTL, and SRPL yield up to 96.4 mol% and 96.7 mol% SRPG, respectively. In addition, more fatty acids are released from synthesized SRPG via hydrolysis by pancreatic enzymes than from SRPC and soybean PC in in vitro digestion model. Fatty acids at the sn‐2 position of SRPG are completely liberated by 0.04 U of phospholipase A2 (PLA2) during a 6 h reaction, whereas fatty acids of SRPC are partially unhydrolyzed even after a 24 h reaction. Our results suggest that SRPG converted from salmon lipids by PLD is a functional PL with high bioavailability of n‐3 PUFAs.Practical Applications: Phosphatidylglycerol rich in n‐3 PUFAs is prepared from salmon roe lipids (SRPG) catalyzed by PLD. The SRPG yields reach 96.4 mol% and 96.7 mol% of phosphatidylcholine contained in SRTL and SRPL, respectively, in aqueous reaction system. Fatty acids rich in n‐3 PUFAs at sn‐2 position of prepared SRPG are rapidly liberated by PLA2 in an in vitro digestion model.

Computational simulations and experimental validation of structure- physicochemical properties of pristine and functionalized graphene: Implications for adverse effects on p53 mediated DNA damage response

Recent reports indicated DNA damaging potential of few-layer graphene in human cell systems. Here, we used computational technique to understand the interaction of both pristine (pG) or carboxyl functionalized graphene (fG) of different sizes (1, 6, and 10nm) with an important DNA repair protein p53. The molecular docking study revealed strong interaction between pG and DNA binding domains (DBD) of p53 with binding free energies (BE) varying from −12.0 (1nm) to −34 (6nm)kcal/mol, while fG showed relatively less interaction with BE varying from −6.7 (1nm) to −11.1 (6nm)kcal/mol. Most importantly, pG or fG bound p53-DBDs could not bind to DNA. Further, microarray analysis of human primary endothelial cells revealed graphene intervention on DNA damage and its structure-properties effect using comet assay studies. Thus, computational and experimental results revealed the structure-physicochemical property dependent adverse effects of graphene in DNA repair protein p53.

Bio-informatics analysis of renal carcinoma gene matrix metalloproteinase-7

Renal cancer is one of the common malignant tumors of the urinary system, seriously threatening human being's health. The current discoveries, however, are far enough for efficient and secure treatment of renal cancer. The aim was to explore the mechanism of matrix metalloproteinase-7 (MMP-7) protein in renal carcinoma cell metastasis by bioinformatics analysis. Bioinformatics methods were used to analyze the composition of amino acids, as well as transmembrane structure, coiled coils, subcellular localization, signal peptide, functions and structures at all levels. It showed that the gene MMP-7 totally had 1131 bp. A peptide chain containing 267 amino acids was encoded in the coding region. Based on random coil, α helix, and further super-helix, it had formed a stable neutral hydrophilic protein. The subcellular location analysis indicated that the protein was located outside the cell. The mature peptide started from the 18th amino acid, and its front-end was the sequence of the signal peptide, belonging to the secreted protein. Analysis of the functional domain showed that this protein had two functional domains, the PG binding domain, and the zinc finger binding domain. Moreover, the protein, which was cross-linked with it, was also one related to cancer cell proliferation and metastasis. To sum up, MMP-7 is a stable neutral hydrophilic secreted protein, and it may play a vital role in the invasion and metastasis of cancer cells.

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin

Fibrin (Fn) clots formed from γ'-fibrinogen (γ'-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γA-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ'-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ'-Fn, and/or altered cross-linking. Clots formed from γ'-Fg lysed more slowly than those formed from γA-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ'-Fn with an association rate constant 22% lower than that to γA-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ'-Fn than with γA-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ'-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ'-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ'-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis.

Binding of Tissue-type Plasminogen Activator to the Glucose-regulated Protein 78 (GRP78) Modulates Plasminogen Activation and Promotes Human Neuroblastoma Cell Proliferation in Vitro

The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in K(m) and an increase in the V(max) for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence (98)LIGRTWNDPSVQQDIKFL(115). We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth.

Covalent Stabilization Of Coagulation Factor Xa Enhances Its Fibrinolytic Function In Vitro and In Vivo: A Novel Cofactor-Based Thrombolytic Agent

The key “clot-busting” enzyme, tissue plasminogen activator (tPA), functions to activate plasminogen (Pg) to plasmin, which directly cleaves and solubilizes the clot. tPA requires a cofactor for physiologically significant plasmin generation. Fibrin is thought to be the only essential tPA cofactor and consequently localizes fibrinolysis. Limited proteolysis of fibrin enhances this tPA cofactor function by exposing C-terminal lysines that are integral to new Pg and tPA binding sites. Our recent work has suggested that the initiating steps of fibrinolysis are accelerated not just by fibrin, but also by localized auxiliary cofactors. These proteins can be cleaved by plasmin more efficiently than fibrin to reveal C-terminal lysine. Our focus has been clotting factor Xa (FXa), whose sequential plasmin-mediated derivatives FXaβ and Xa33/13 acquire C-terminal lysines that facilitate Pg binding and tPA-cofactor function. We now report that in plasma Xa33/13 is rapidly degraded, and hypothesize that preventing formation of Xa33/13 will prolong the fibrinolytic cofactor activity of FXaβ in plasma. To impair the production of Xa33/13 and stabilize the FXaβ activity, an ∼25 angstrom tetraethylene glycol (TEG) steric group was linked to the active site. A C-terminal lysine was tethered to the TEG to further enhance the activity (Xa-K). Treatment with purified plasmin showed that Xa-K was converted to the β-form without further proteolysis to the Xa33/13 analogue. Using conventional turbidity assays, the time for thrombin (3 nM)-induced plasma clots to reach 50% fibrinolysis (25 pM tPA) was shortened ∼8-fold by 1 nM Xa-K, whereas unmodified FXa at 200-fold higher concentration was insignificant. Identically modified trypsin-K had no effect on fibrinolysis. Using Doppler ultrasound to follow blood flow in surgically exposed mouse carotid arteries, the effect of Xa-K on shortening the time to reperfusion was evaluated after complete occlusion was induced by ferric chloride. The tPA variant Tenecteplase (TNKase) injected into the tail vein (17 µg/g) completely restored blood flow by ∼30 min, which was reduced to ∼14 min in the presence of Xa-K (0.5 µg/g, n=10). At a sub-therapeutic dose (9 µg/g, n=4) TNKase did not restore blood flow by the experimental end-point (60 minutes). However, complete reperfusion was observed by ∼20 min when combined with Xa-K (0.5 µg/g, n=8), showing that adjunctive Xa-K can reduce the required amount of TNKase. In the absence of TNKase, a dose-dependent effect on reperfusion was observed when Xa-K was injected alone (0.5 and 1.1 µg/g n=5), indicating an effect on endogenous tPA. Western blots of plasma from mice achieving complete reperfusion showed animals administered Xa-K alone, but not TNKase alone, had no systemic plasminogen or fibrinogen fragmentation. Furthermore, XaK appears to have significantly attenuated the systemic effect of TNKase during adjunctive treatment, possibly due to localization by Xa-K to the site of the clot. These results support a role for auxiliary cofactors in fibrinolysis and suggest that Xa-K may have novel thrombolytic applications. Disclosures:Pryzdial:Canadian Blood Services: Employment, Inventor, Inventor Patents & Royalties.

Full Time Course Kinetics of the Streptokinase-Plasminogen Activation Pathway

Our previously hypothesized mechanism for the pathway of plasminogen (Pg) activation by streptokinase (SK) was tested by the use of full time course kinetics. Three discontinuous chromogenic substrate initial rate assays were developed with different quenching conditions that enabled quantitation of the time courses of Pg depletion, plasmin (Pm) formation, transient formation of the conformationally activated SK·Pg* catalytic complex intermediate, formation of the SK·Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. Analysis of full time courses of Pg activation by five concentrations of SK along with activity-based titrations of SK·Pg* and SK·Pm formation yielded rate and dissociation constants within 2-fold of those determined previously by continuous measurement of parabolic chromogenic substrate hydrolysis and fluorescence-based equilibrium binding. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK·Pg* complex catalyzes an initial cycle of Pg proteolytic conversion to Pm that acts as a trigger. Higher affinity binding of the formed Pm to SK outcompetes Pg binding, terminating the trigger cycle and initiating the bullet catalytic cycle by the SK·Pm complex that converts the residual Pg into Pm. The new assays can be adapted to quantitate SK-Pg activation in the context of SK- or Pg-directed inhibitors, effectors, and SK allelic variants. To support this, we show for the first time with an assay specific for SK·Pg* that fibrinogen forms a ternary SK·Pg*·fibrinogen complex, which assembles with 200-fold enhanced SK·Pg* affinity, signaled by a perturbation of the SK·Pg* active site.

A phase plane graph based model of the ovulatory cycle lacking the "positive feedback" phenomenon

When hormones during the ovulatory cycle are shown in phase plane graphs, reported FSH and estrogen values form a specific pattern that resembles the leaning “&" symbol, while LH and progesterone (Pg) values form a "boomerang" shape. Graphs in this paper were made using data reported by Stricker et al. [Clin Chem Lab Med 2006;44:883–887]. These patterns were used to construct a simplistic model of the ovulatory cycle without the conventional "positive feedback" phenomenon. The model is based on few well-established relations:hypothalamic GnRH secretion is increased under estrogen exposure during two weeks that start before the ovulatory surge and lasts till lutheolysis.the pituitary GnRH receptors are so prone to downregulation through ligand binding that this must be important for their function.in several estrogen target tissue progesterone receptor (PgR) expression depends on previous estrogen binding to functional estrogen receptors (ER), while Pg binding to the expressed PgRs reduces both ER and PgR expression.Some key features of the presented model are here listed:High GnRH secretion induced by the recovered estrogen exposure starts in the late follicular phase and lasts till lutheolysis. The LH and FSH surges start due to combination of accumulated pituitary GnRH receptors and increased GnRH secretion. The surges quickly end due to partial downregulation of the pituitary GnRH receptors (64% reduction of the follicular phase pituitary GnRH receptors is needed to explain the reported LH drop after the surge). A strong increase in the lutheal Pg blood level, despite modest decline in LH levels, is explained as delayed expression of pituitary PgRs. Postponed pituitary PgRs expression enforces a negative feedback loop between Pg levels and LH secretions not before the mid lutheal phase.Lutheolysis is explained as a consequence of Pg binding to hypothalamic and pituitary PgRs that reduces local ER expression. When hypothalamic sensitivity to estrogen is diminished due to lack of local ERs, hypothalamus switches back to the low GnRH secretion rate, leading to low secretion of gonadotropins and to lutheolysis. During low GnRH secretion rates, previously downregulated pituitary GnRH receptors recover to normal levels and thus allow the next cycle.Possible implications of the presented model on several topics related to reproductive physiology are shortly discussed with some evolutionary aspects including the emergence of menopause.

Abstract 4610: Nuclear interaction between estrogen and progesterone receptors in experimental breast cancer is blocked by antiestrogens but not by RU 486

Abstract The expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) in breast cancer is used as a prognostic factor and identifies a group of tumors which may respond to an endocrine therapy aimed to block estrogen synthesis or ER activation. There is however, compelling evidence that indicates that PR is also involved in breast cancer growth. We have developed an experimental model of breast cancer in BALB/c female mice in which mammary carcinomas are induced by the administration of medroxyprogesterone acetate (MPA). The hormone-dependent (HD) tumor, C4-HD, grows in vivo only in the presence of MPA, and expresses ERα and both PR isoforms (PRA and PRB). PR are essential for tumor growth in this model, since antisense oligonucleotides of PR and different antiprogestins inhibit their growth. Interestingly, 17β-estradiol (E2) and the pure antiestrogen ICI 182780 (ICI) also induce tumor regression. It is controversial how progesterone (Pg) induces non genomic effects. Although it has been demonstrated that Pg binding to cytosolic PRB activates c-Src through an interaction with ERα, others have reported that this interaction is not necessary for c-Src activation in human T47D breast cancer cells. In our Laboratory, we have shown a nuclear physical association between ERα and PR in C4-HD tumors, and we postulate that this interaction is necessary to induce progestin stimulated cell proliferation. The aim of this study was to further investigate this nuclear interaction in C4-HD and in T47D epithelial cells. We found, by confocal immunofluorescence, that MPA (10nM) treatment induced an early increase in the nuclear co-localization between ERα and PRA in both tumor models (p<0.001). These results were corroborated by co-immunoprecipitation assays showing that both PR isoforms interact with ERα in the cell nuclei. We also found nuclear interaction between both pSer118 ERα and pSer294 PR after 15 minutes of MPA incubation in C4-HD cells (p<0.001), indicating that this complex could be transcriptionally active. E2 (1nM) and ICI (1μM) treatment inhibited this interaction whereas the antiprogestin RU 486 (RU, 10nM), which also inhibits cell proliferation as E2 and ICI, behaved as a PR agonist supporting previous data of our lab that indicates that RU activates MAPK as MPA, and that the inhibitory effects induced by RU are due to genomic events. Our data support the hypothesis that ERα is involved in MPA-induced cell proliferation; we postulate however that the nuclear interaction between ERα and PR participates in the progestin induced stimulatory effects in these breast cancer models. In addition, the results shown herein reinforce our previous findings in which we showed early agonistic effects of RU mediating non genomic events, despite inducing tumor regression. These results highlight the importance of targeting both ERα and PR in breast cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4610.

Solution structure of the complex of VEK-30 and plasminogen kringle 2

The solution structure of the complex containing the isolated kringle 2 domain of human plasminogen (K2(Pg)) and VEK-30, a 30-amino acid residue internal peptide from a streptococcal M-like plasminogen (Pg) binding protein (PAM), has been determined by multinuclear high-resolution NMR. Complete backbone and side-chain assignments were obtained from triple-resonance experiments, after which structure calculations were performed and ultimately refined by restrained molecular simulation in water. We find that, in contrast with the dimer of complexes observed in the asymmetric unit of the crystal, global correlation times and buoyant molecular weight determinations of the complex and its individual components showed the monomeric nature of all species in solution. The NMR-derived structure of K2(Pg) in complex with VEK-30 presents a folding pattern typical of other kringle domains, while bound VEK-30 forms an end-to-end alpha-helix (residues 6-27) in the complex. Most of the VEK-30/K2(Pg) interactions in solution occur between a single face of the alpha-helix of VEK-30 and the lysine binding site (LBS) of K2(Pg). The canonical LBS of K2(Pg), consisting of Asp54, Asp56, Trp60, Arg69, and Trp70 (kringle numbering), interacts with an internal pseudo-lysine of VEK-30, comprising side-chains of Arg17, His18, and Glu20. Site-specific mutagenesis analysis confirmed that the electrostatic field formed by the N-terminal anionic residues of the VEK-30 alpha-helix, viz., Asp7, and the non-conserved cationic residues of K2(Pg), viz., Lys43 and Arg55, play additional important roles in the docking of VEK-30 to K2(Pg). Structural analysis and kringle sequence alignments revealed several important features related to exosite binding that provide a structural rationale for the high specificity and affinity of VEK-30 for K2(Pg).

Homology modeling of Mycoplasma pneumoniae enolase and its molecular interaction with human plasminogen.

Alpha (alpha)-enolase (e), a glycolytic enzyme, has an alternative role as a surface receptor of several bacteria mediating plasminogen (pg) binding. It is also recognized as a virulence factor of some pathogenic bacteria facilitating plasminogen activation and host cell invasion. A mycoplasmal alpha-enolase is also a plasminogen binding protein. Molecular interactions of enolase from Mycoplasma pneumoniae with host plasminogen would be useful for exploring the pathogen-host interaction. In an attempt to identify plasminogen binding sites of M. pneumoniae enolase, homology modeling and docking studies were conducted to obtain modeled structures of the M. pneumoniae enolase-plasminogen complex. The refined model was validated further by standard methods. Molecular docking revealed hydrogen bonding of eLys70-pgTyr50, eAsn165-pgThr66, eAla168-pgGlu21, eAsp17-pgLys70, and eAsn213-pgPro68/pgAsn69. Substantial decreases in accessible surface area (ASA) were observed and in concurrence with hydrogen bond pattern. These findings provide a detailed prediction of key residues that interact at the protein-protein interface. Our theoretical prediction is consistent with known biochemical data. The predicted interaction complex can be of great assistance in understanding structural insights, which is necessary to pathogen and host-component interaction. The ability of M. pneumoniae enolase to bind plasminogen may be indicative of an important role in invasion of this pathogen to host.

Rapid-reaction Kinetic Characterization of the Pathway of Streptokinase-Plasmin Catalytic Complex Formation

Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys(414) binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK.Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys(414) deletion mutant (SKDeltaK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKDeltaK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKDeltaK414 was approximately 10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK.Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys(414) binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes.

Non‐enzymatic glycation of type I collagen diminishes collagen–proteoglycan binding and weakens cell adhesion

Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans(1) (PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content-effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, and had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.

An assay for measuring functional activated thrombin-activatable fibrinolysis inhibitor in plasma

Thrombin-activatable fibrinolysis inhibitor (TAFI), also called procarboxypeptidase U (proCPU), is a plasma zymogen that can be activated by thrombin, the thrombin–thrombomodulin complex, or plasmin. The activated form of TAFI (TAFIa, CPU) removes C-terminal lysine residues of plasmin-modified fibrin (FN′) that mediates a positive feedback mechanism in plasminogen (Pg) activation, thereby attenuating fibrinolysis. The plasma concentration of TAFI is approximately 75 nM. Because the half-maximal effect of TAFIa occurs at 1 nM, only approximately 1.3% of TAFI needs to be activated to exert an effect on clot lysis. The assay is performed by mixing soluble FN′ covalently attached to a quencher and fluorescein-labeled Pg. The sample containing TAFIa is then added, and the rate of fluorescence increase due to removal of C-terminal lysine from FN′ and loss of Pg binding is measured with a fluorescence plate reader. The assay was shown to be sensitive for TAFIa at a concentration as low as 12 pM. The intraassay variability and interassay variability of the assay were 6.3 and 8.3%, respectively. This assay was not confounded by the naturally occurring TAFI Thr325Leu polymorphism that affects the thermal stability of TAFIa or endogenous plasminogen in plasma.

Binding of the COOH-terminal Lysine Residue of Streptokinase to Plasmin(ogen) Kringles Enhances Formation of the Streptokinase·Plasmin(ogen) Catalytic Complexes

Streptokinase (SK) activates human fibrinolysis by inducing non-proteolytic activation of the serine proteinase zymogen, plasminogen (Pg), in the SK.Pg* catalytic complex. SK.Pg* proteolytically activates Pg to plasmin (Pm). SK-induced Pg activation is enhanced by lysine-binding site (LBS) interactions with kringles on Pg and Pm, as evidenced by inhibition of the reactions by the lysine analogue, 6-aminohexanoic acid. Equilibrium binding analysis and [Lys]Pg activation kinetics with wild-type SK, carboxypeptidase B-treated SK, and a COOH-terminal Lys414 deletion mutant (SKDeltaK414) demonstrated a critical role for Lys414 in the enhancement of [Lys]Pg and [Lys]Pm binding and conformational [Lys]Pg activation. The LBS-independent affinity of SK for [Glu]Pg was unaffected by deletion of Lys414. By contrast, removal of SK Lys414 caused 19- and 14-fold decreases in SK affinity for [Lys]Pg and [Lys]Pm binding in the catalytic mode, respectively. In kinetic studies of the coupled conformational and proteolytic activation of [Lys]Pg, SKDeltaK414 exhibited a corresponding 17-fold affinity decrease for formation of the SKDeltaK414.[Lys]Pg* complex. SKDeltaK414 binding to [Lys]Pg and [Lys]Pm and conformational [Lys]Pg activation were LBS-independent, whereas [Lys]Pg substrate binding and proteolytic [Lys]Pm generation remained LBS-dependent. We conclude that binding of SK Lys414 to [Lys]Pg and [Lys]Pm kringles enhances SK.[Lys]Pg* and SK.[Lys]Pm catalytic complex formation. This interaction is distinct structurally and functionally from LBS-dependent Pg substrate recognition by these complexes.

Role of Plasminogen-Fibrin Interaction in Plasminogen Activation by t-PA.

Plasminogen (Pg) is the zymogen of the fibrinolytic enzyme plasmin (Pn). Fibrin (Fn) promotes the activation of Pg by tissue-type plasminogen activator (t-PA) by serving as a template that brings t-PA and Pg into close proximity. In addition, proteolysis of Fn by Pn generates carboxyl-terminal lysine residues that provide nascent high affinity binding sites for Glu-Pg, thereby promoting Pg activation. Pg activation is also enhanced when Glu-Pg is converted to Lys-Pg, a derivative with higher Fn affinity. Therefore, kinetic and functional data suggest that Pg binding to Fn is a key aspect of efficient Pg activation. To explore this concept, we performed binding and kinetic analyses with Mini-Pg, an elastase-derived fragment of Glu-Pg with reduced Fn affinity. Binding of Glu-Pg, Lys-Pg and Mini-Pg to immobilized fibrinogen (Fg) and fibrin monomer (Fm) was monitored by surface plasmon resonance. The affinity of Glu-Pg for Fm is 4-fold higher than that for Fg with Kd values of 3.1 and 12.5 μM, respectively, whereas Lys-Pg binds with high affinity to both Fg and Fm (Kd values of 0.25 and 0.21 μM, respectively). In contrast, Mini-Pg demonstrates weak binding to Fg and Fm with Kd values of 10.5 and 24.5 μM, respectively. To complement the binding experiments, kinetic studies of Pg activation by t-PA were performed in the absence or presence of native Fn clots by monitoring Pn formation using a Pn-directed chromogenic substrate. As expected, the catalytic efficiency of Lys-Pg or Mini-Pg activation by t-PA in the absence of cofactor was higher than that of Glu-Pg (kcat/KM values of 1.3, 0.325, and 0.026 μM−1 min−1, respectively). The catalytic efficiency of Glu-Pg activation by t-PA is 500-fold higher in the presence of Fn than it is in its absence. The stimulatory effect of fibrin was maintained with Lys and Mini-Pg with over 100-fold enhancement in catalytic efficiency of activation. The fibrin-dependent increase in catalytic efficiency was expressed predominantly through a decrease in KM, with values of >20 and 0.2 μM for Glu-Pg activation in the absence and presence of fibrin, respectively. Lys and Mini-Pg also expressed similar enhancements in catalytic efficiency through a decrease in KM. Thus, despite a 100-fold range in their affinities for Fn, the activation of Mini-Pg, Glu-Pg and Lys-Pg by t-PA are all enhanced by at least 2 orders of magnitude in the presence of Fn. These results demonstrate that substrate binding to Fn is not essential for Fn-mediated stimulation of Pg activation by t-PA. To investigate the importance of the Fn-plasminogen activator interaction, activation studies were carried out using urokinase-type plasminogen activator (u-PA), an activator without Fn affinity. In contrast to the results with t-PA, Fn did not enhance u-PA-mediated activation of Glu, Lys or Mini-Pg. The lack of fibrin stimulation of Pg activation by u-PA suggests that the Pg-Fn interaction is not essential to Pg activation. Therefore, the cofactor role of Fn is expressed predominantly through interaction and stimulation of t-PA. These findings support the hypothesis that Fn binding to t-PA may expose cryptic binding sites in the activator that stabilize the formation of the enzyme-substrate complex.

Regulation of Tissue Factor Signaling by Tissue Factor Pathway Inhibitor.

Tissue factor initiates the coagulation cascade as well as cellular signaling through the activation of cell surface protease activated receptors (PARs), relevant to inflammation, tumor metastasis and angiogenesis. Tissue factor pathway inhibitor-1 (TFPI-1) is the major regulator of TF initiated coagulation, but recombinant TFPI-1 (rTFPI-1) has shown limited efficacy on sepsis-associated inflammatory responses and mortality. In TNF-stimulated human umbilical vein endothelial cells (HUVECs), we demonstrate with specific blocking antibodies that endogenous TFPI effectively controls TF procoagulant activity as well as TF-dependent signaling through PAR1 and PAR2. The effect of rTFPI-1 on coagulation and signaling was tested in TF-transfected CHO-cells or in HUVECs transduced with TF to overcome the inhibitory threshold of endogenous TFPI-1. Under conditions that specifically measure signaling of the ternary TF-VIIa-Xa complex, 2.5–5 nM rTFPI-1 inhibited TF-dependent Xa generation by >80% but the signaling of the ternary complex through PAR1 at these concentrations of rTFPI-1 was only marginally inhibited. Moreover, studies with proteoglycan-deficient CHO cells provided genetic evidence that PG binding facilitates the inhibitory activities of rTFPI-1 on coagulation and signaling. In HUVECs that co-expressed PAR2 with TF, three different signaling responses of the ternary TF-VIIa-Xa complex were inhibited by ~ 50% at 1.2–2.5 nM rTFPI-1, but the inhibitory potency of rTFPI-1 to block Xa generation was consistently higher relative to its effect on signaling. These data demonstrate that coagulation initiation phase signaling can occur in the absence of the activation of coagulation. The results also establish that endogenously expressed TFPI-1, known to be entirely GPI-anchored in endothelial cells, is a potent, negative regulator of ternary complex-mediated PAR signaling. The efficacy of rTPFI-1 appears to be influenced by the presence of PAR2, further emphasizing the close reciprocal signaling linkage of PAR2 and TF demonstrated in angiogenesis and cellular signaling targeting the TF cytoplasmic domain by phosphorylation.

Streptokinase Triggers Conformational Activation of Plasminogen through Specific Interactions of the Amino-terminal Sequence and Stabilizes the Active Zymogen Conformation

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of Ile(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking Ile(1) (SK(2--414)). SK(2--414) retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--15), whereas Pg(562--569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.