Lysine-binding Site Of Kringle Research Articles

Screening, characterization and specific binding mechanism of aptamers against human plasminogen Kringle 5

Plasminogen Kringle 5 is one of the most potent cytokines identified to inhibit the proliferation and migration of vascular endothelial cells. Herein, six aptamer candidates that specifically bind to Kringle 5 were generated by the systematic evolution of ligands by exponential enrichment (SELEX). After 10 rounds of screening against Kringle 5, a highly enriched ssDNA pool was sequenced and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. The preferred aptamer KG-4, which demonstrated a low dissociation constant (Kd) of ∼ 432 nM and excellent selectivity for Kringle 5. A conserved “motif” of eight bases located at the stem-loop intersection, common to the aptamer, was further confirmed as the recognition element for binding with Kringle 5. The bulge formed by the motif and depression on the lysine binding site of Kringle 5 were both located at the binding interface, and the “induced fit” between their structures played a central role in the recognition process. Kringle 5 interacts KG-4 primarily through enthalpy-driven van der Waals forces and hydrogen bond. The key nucleotides A34 and C35 at motif on KG-4 and the positively charged amino acids in the loop 1 and loop 4 regions on Kringle 5 play a major role in the interaction. Furthermore, KG-4 dose-dependently reduced the proliferation inhibition of vascular endothelial cells by Kringle 5 and had a blocking effect on the function of Kringle 5 in inhibiting migration and promoting apoptosis of vascular endothelial cells in vitro. This study put a new light on protein-aptamer binding mechanism and may provide insight into the treatment of ischemic diseases by target depletion of Kringle 5.

Inhibition of pericellular plasminogen activation by apolipoprotein(a): Roles of urokinase plasminogen activator receptor and integrins αMβ2 and αVβ3

Background and aimsLipoprotein(a) (Lp(a)) is a causal risk factor for cardiovascular disorders including coronary heart disease and calcific aortic valve stenosis. Apolipoprotein(a) (apo(a)), the unique glycoprotein component of Lp(a), contains sequences homologous to plasminogen. Plasminogen activation is markedly accelerated in the presence of cell surface receptors and can be inhibited in this context by apo(a). MethodsWe evaluated the role of potential receptors in regulating plasminogen activation and the ability of apo(a) to mediate inhibition of plasminogen activation on vascular and monocytic/macrophage cells through knockdown (siRNA or blocking antibodies) or overexpression of various candidate receptors. Binding assays were conducted to determine apo(a) and plasminogen receptor interactions. ResultsThe urokinase-type plasminogen activator receptor (uPAR) modulates plasminogen activation as well as plasminogen and apo(a) binding on human umbilical vein endothelial cells (HUVECs), human acute monocytic leukemia (THP-1) cells, and THP-1 macrophages as determined through uPAR knockdown and overexpression. Apo(a) variants lacking either the kringle V or the strong lysine binding site in kringle IV type 10 are not able to bind to uPAR to the same extent as wild-type apo(a). Plasminogen activation is also modulated, albeit to a lower extent, through the Mac-1 (αMβ2) integrin on HUVECs and THP-1 monocytes. Integrin αVβ3 can regulate plasminogen activation on THP-1 monocytes and to a lesser extent on HUVECs. ConclusionsThese results indicate cell type-specific roles for uPAR, αMβ2, and αVβ3 in promoting plasminogen activation and mediate the inhibitory effects of apo(a) in this process.

Apolipoprotein(a) Inhibits In Vitro Tube Formation in Endothelial Cells: Identification of Roles for Kringle V and the Plasminogen Activation System

Elevated plasma concentrations of lipoprotein(a) are associated with increased risk for atherothrombotic diseases. Apolipoprotein(a), the unique glycoprotein component of lipoprotein(a), is characterized by the presence of multiple kringle domains, and shares a high degree of sequence homology with the serine protease zymogen plasminogen. It has been shown that angiostatin, a proteolytic fragment of plasminogen containing kringles 1–4, can effectively inhibit angiogenesis. Moreover, proteolytic fragments of plasminogen containing kringle 5 are even more potent inhibitors of angiogenesis than angiostatin. Despite its strong similarity with plasminogen, the role of apolipoprotein(a) in angiogenesis remains controversial, with both pro- and anti-angiogenic effects reported. In the current study, we evaluated the ability of apolipoprotein(a) to inhibit VEGF- and angiopoietin-induced tube formation in human umbilical cord endothelial cells. A 17 kringle-containing form of recombinant apo(a) (17K), corresponding to a well-characterized, physiologically-relevant form of the molecule, effectively inhibited tube formation induced by either VEGF or angiopoietin-1. Using additional recombinant apolipoprotein(a) (r-apo(a)) variants, we demonstrated that this effect was dependent on the presence of an intact lysine-binding site in kringle V domain of apo(a), but not on the presence of the functional lysine-binding site in apo(a) kringle IV type 10; sequences within in the amino-terminal half of the molecule were also not required for the inhibitory effects of apo(a). We also showed that the apo(a)-mediated inhibition tube formation could be reversed, in part by the addition of plasmin or urokinase plasminogen activator, or by removal of plasminogen from the system. Further, we demonstrated that apo(a) treated with glycosidases to remove sialic acid was significantly less effective in inhibiting tube formation. This is the first report of a functional role for the glycosylation of apo(a) although the mechanisms underlying this observation remain to be determined in the context of angiogenesis.

Apolipoprotein(a)-Dependent Inhibition of Pericellular Plasminogen Activation Is Mediated by Specific Cellular Receptors

Abstract 2236Lipoprotein(a) [Lp(a)] has been identified as an independent risk factor for cardiovascular diseases such as coronary heart disease. Lp(a) levels vary over 1000-fold within the human population and Lp(a) possesses both proatherogenic and prothrombotic properties due to the LDL-like moiety and apolipoprotein(a) [apo(a)] components, respectively. Apo(a) is highly homologous to plasminogen and thus can potentially interfere with plasminogen activation. Plasmin generated in the context of fibrin mediates the breakdown of blood clots, which are the causative factors in heart attacks and strokes. 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. Previous studies have suggested that apo(a) may inhibit pericellular plasminogen activation on the basis of observations that apo(a) decreases plasminogen binding to cells. We have undertaken analysis of the mechanism by which apo(a) may interfere with pericellular plasminogen activation to allow for a more definitive description of the role of Lp(a) within the vasculature.Plasminogen activation was found to be markedly inhibited by the recombinant apo(a) variant 17K, in a dose dependent manner, on human umbilical vein endothelial cells (HUVECs), human monocytic leukemia cells (THP-1), THP-1 macrophages, and smooth muscle cells. The strong lysine binding site in kringle IV type 10, as well as kringle V appear to be required for this effect since apo(a) variants lacking these elements (17KΔAsp and 17KΔV, respectively) failed to inhibit activation. However, the role of lysine-dependent binding of apo(a) itself to the cells is not clear. Carboxypeptidase treatment of cells did not decrease apo(a) binding, and apo(a) does not compete directly for plasminogen binding to the cells. Rather, apo(a) and plasminogen may bind to the cells as a complex.We next attempted to identify the cell-surface receptor(s) that mediate plasminogen activation on the cell surface as well as its inhibition by apo(a). Urokinase-type plasminogen activator receptor (uPAR) has been previously shown to bind to urokinase-type plasminogen activator (uPA), vitronectin, and β3 integrins. uPAR is involved in the remodeling of the extracellular matrix (ECM) through regulation of plasminogen activation. We found evidence that uPAR is a potential receptor for both plasminogen and apo(a). Knockdown of uPAR in HUVECs results in decreased binding of plasminogen, 17K and, to a lesser extent, 17KΔAsp and 17KΔV. Similar experiments in SMCs revealed no changes in binding. A decrease in tPA-mediated plasminogen activation following uPAR knockdown occurred in HUVECs, and addition of 17K did not result in any further decrease. Overexpression of uPAR in THP-1 macrophages leads to greater than a two fold increase in 17K and plasminogen binding. Plasminogen activation increases over two-fold as a result of overexpression of uPAR, while 17K blunts the effect of uPAR overexpression. These results indicate that uPAR plays a crucial role in both plasminogen and apo(a) binding to the cell surface of specific cells and inhibition by apo(a) of plasminogen activation.Macrophage-1-antigen (Mac-1) receptor consists of CD11b (αM) and CD18 (β2) integrin and has been previously shown to recognize uPA and control migration and adhesion. Furthermore, αVβ3 has been previously shown to bind to vitronectin and the uPA-uPAR complex which promotes cell adhesion through binding of both vitronectin and αVβ3 integrins. We found that blocking the αM, β2, or αVβ3 receptors with monoclonal antibodies in THP-1 cells leads to a decrease in plasminogen activation, as well as a blunting of the inhibitory effects of apo(a) on plasminogen activation. These results indicate a role for Mac-1 and αVβ3 in apo(a) binding and inhibition of plasminogen activation.In conclusion, we have demonstrated, for the first time, the role of specific receptors in binding of apo(a) to vascular cell surfaces and in mediating the inhibitory effect of apo(a) on pericellular plasminogen activation. Disclosures:No relevant conflicts of interest to declare.

Characteristics of plasminogen binding to Trypanosoma cruzi epimastigotes

The binding constants of the interaction between plasminogen and Trypanosoma cruzi epimastigotes were determined. An indirect method in which the bound plasminogen is detached from the cell by ɛ-aminocaproic acid and a direct method through biotinylated plasminogen were used. The analyses revealed a dissociation constant ( K d) from 0.4 to 1.2 μM, these values being compatible with recognition in vivo. Moreover, epimastigotes from the gut of Rhodnius prolixus were able to bind plasminogen from the blood meal. Fragments derived from elastase digestion of plasminogen were tested for their ability to bind T. cruzi cells. The fragment with highest ability to interact with the parasite was miniplasminogen that bound in a concentration-dependent and saturable manner with a K d similar to that for plasminogen. This binding was inhibited by ɛ-aminocaproic acid indicating that the lysine-binding site of kringle 5 may be responsible for the interaction of plasminogen with T. cruzi.

Prion protein stimulates tissue‐type plasminogen activator‐mediated plasmin generation via a lysine‐binding site on kringle 2

Recombinant human prion-protein (PrP23-231) stimulates plasminogen activation by tissue-type plasminogen activator (t-PA). The stimulatory activity is conserved in the N-terminal fragment (PrP23-110). It has further been shown by others that PrP(c) binds to kringle-domains of plasminogen. We compared the stimulatory activity of recombinant PrP23-231 and PrP23-110 on plasminogen activation catalyzed by t-PA, urokinase (u-PA), streptokinase and Desmodus salivary plasminogen activator (DSPAalpha1). As these plasminogen activators are distinct, with respect to their kringle domains we studied their binding to immobilized PrP23-110. Plasminogen activation was measured in a chromogenic assay in vitro and binding studies were carried out using surface plasmon resonance technology. We found that recombinant full-length prion protein, PrP23-231, and PrP23-110 specifically stimulate t-PA mediated plasminogen activation. Two hundred nanomoles per liter of PrP23-110 stimulated 1.8 nmol L(-1) t-PA 48-fold, 180 nmol L(-1) DSPA(alpha1) 2.5-fold, 1.8 nmol L(-1) u-PA 1.1-fold, and 1.8 nmol L(-1) streptokinase 1.8-fold. Our data show no specific binding for streptokinase. In contrast all plasminogen activators carrying a kringle domain bound to PrP23-110. We further studied the effect of lysine on binding to PrP23-110 and on plasminogen activation by DSPA(alpha1) or t-PA. Lysine decreased both the binding of t-PA to PrP23-110 and the stimulation of plasmin generation by t-PA. Both binding and plasminogen activation of DSPA(alpha1) were not influenced by the presence of lysine. All plasminogen activators tested bearing kringle domains bind to PrP23-110. Binding to PrP23-110 is not sufficient for stimulation of plasmin generation. Thus the lysine-binding site of kringle 2 that is unique to t-PA appears to mediate the specific stimulation of plasminogen activation by the cellular prion protein.

Seeking for binding determinants of the prion protein to human plasminogen

Plasminogen (Pg), a pro-protease implicated in neuronal excitotoxicity, has recently been identified as binding to prion protein (PrP) from several species. Although the precise effect of the binding of PrP to plasminogen in the course of prion-caused diseases has not yet been demonstrated, the implications of this important finding for diagnostic applications are straightforward. In this paper we have investigated the possible modes of interaction of PrP with plasminogen, by means of molecular modelling and computational simulation techniques. To this goal, we first exploited the information available for the mK2Pg/VEK-30 complex in order to identify the PrP residues which satisfy the specific electronic and geometric requirements needed to interact with the kringle lysine binding site, we compared the relevant mK2Pg/VEK-30 and mK2Pg/PrP interactions obtained from the simulated protein—protein complexes and we assessed the docking hypothesis utilized for the mK2Pg/PrP complex by simulating the interaction of PrP with the multi-kringle angiostatin, a more realistic model of the physiological target. The results obtained will be instrumental for planning experiments tailored to clarify the role of the plasminogen activator system in prion-related diseases and, eventually, for mimicking dominant binding determinants through structure-based drug design.

Inhibition of Plasminogen Activation by Lipoprotein(a): CRITICAL DOMAINS IN APOLIPOPROTEIN(a) AND MECHANISM OF INHIBITION ON FIBRIN AND DEGRADED FIBRIN SURFACES

Similarity between the apolipoprotein(a) (apo(a)) moiety of lipoprotein(a) (Lp(a)) and plasminogen suggests a potentially important link between atherosclerosis and thrombosis. Lp(a) may interfere with tissue plasminogen activator (tPA)-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoagulable state in vivo. A fluorescence-based system was employed to study the effect of apo(a) on plasminogen activation in the presence of native fibrin and degraded fibrin cofactors and in the absence of positive feedback reactions catalyzed by plasmin. Human Lp(a) and a physiologically relevant, 17-kringle recombinant apo(a) species exhibited strong inhibition with both cofactors. A variant lacking the protease domain also exhibited strong inhibition, indicating that the apo(a)-plasminogen binding interaction mediated by the apo(a) protease domain does not ultimately inhibit plasminogen activation. A variant in which the strong lysine-binding site in kringle IV type 10 had been abolished exhibited substantially reduced inhibition whereas another lacking the kringle V domain showed no inhibition. Amino-terminal truncation mutants of apo(a) also revealed that additional sequences within kringle IV types 1-4 are required for maximal inhibition. To investigate the inhibition mechanism, the concentrations of plasminogen, cofactor, and a 12-kringle recombinant apo(a) species were systematically varied. Kinetics for both cofactors conformed to a single, equilibrium template model in which apo(a) can interact with all three fibrinolytic components and predicts the formation of ternary (cofactor, tPA, and plasminogen) and quaternary (cofactor, tPA, plasminogen, and apo(a)) catalytic complexes. The latter complex exhibits a reduced turnover number, thereby accounting for inhibition of plasminogen activation in the presence of apo(a)/Lp(a).

Evidence that the conformation of unliganded human plasminogen is maintained via an intramolecular interaction between the lysine-binding site of kringle 5 and the N-terminal peptide

Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (alpha) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys50 and Lys62. In contrast, molecules in which NTP residues Lys20 or Lys33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys50 and/or Lys62. These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.

Reaction of human α 2-antiplasmin and plasmin Stopped-flow fluorescence kinetics

The interaction of human plasmin with human α 2-antiplasmin was measured in the presence and absence of lysine-binding ligands using the corresponding active site fluorescence changes. The stopped-flow method allows for direct determination of reliable values of the second order rate constant for the fast association step of plasmin and α 2-antiplasmin in the absence of another interacting compound, e.g. a plasmin substrate. At pH 7.4,25°C, k 1= 2.2 X 10 7 M −1 s −1 was obtained. Substantial reductions in k 1 were seen in the presence of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at concentrations corresponding to lysine-binding site interactions at kringle 4 of plasmin; at saturation the rate constant is reduced 20-fold, whereas the effect of saturation of kringle 1 is only a 2-fold reduction. It is thus found that the interaction of α 2-antiplasmin with the lysine-binding site of kringle 1 is of little importance compared with that of kringle 4 in regulating the inhibition reaction of plasmin with α 2-antiplasmin. Similar results were recently obtained for the bovine plasmin-bovine α 2-antiplasmin reaction (Christensen et al. (1995) Biochem. J. 305, 97–102).

A kringle-specific monoclonal antibody.

Kringle domains are found in several plasma proteins of blood coagulation and fibrinolysis. A murine monoclonal antibody, designated alpha HII-5, was produced against a synthetic peptide representing residues 216-231 of human prothrombin kringle 2. The sequence of the hexadecapeptide (Glu-Asn-Phe-Cys-Arg-Asn-Pro-Asp-Gly-Asp-Glu-Glu-Gly-Val-Gly-Cys) is conserved in several kringle-containing proteins, represents a predicted region of high local hydrophilicity in prothrombin kringle 2, and contains the anionic (Asp-223 and Asp-225) residues that contribute to lysine binding by plasminogen kringle 4. In a solution-phase immunoassay, antibody alpha HII-5 bound prothrombin and the kringle 5 light chain fragment of plasminogen (miniplasminogen), but not plasminogen or plasmin. In contrast, using a solid-phase assay with antigen immobilized onto a surface (polystyrene microtiter plates, glass, or nitrocellulose) antibody alpha HII-5 specifically bound prothrombin, plasminogen, recombinant tissue plasminogen activator (tPA), and the apo(a) subunit of lipoprotein(a). By immunoblotting analysis antibody alpha HII-5 bound determinants on prothrombin fragment 2 and plasminogen kringle 5. These observations suggest that a subset of kringle domains on plasma proteins, including prothrombin kringle 2 and plasminogen kringle 5, contains a homologous antigenic determinant in the region of the kringle lysine-binding site. In contrast to prothrombin kringle 2, the homologous peptide site on plasminogen is not available for antibody binding except when plasminogen is adsorbed to a nonphysiological surface, or when kringles 1-4 are removed.

600 MHz 1H nuclear magnetic resonance studies of the kringle 4 fragment of human plasminogen

Kringle 4, a ~10,000-Da domain in the heavy chain of human plasminogen, has been isolated intact and studied by 1H NMR spectroscopy at 600 MHz. The spectroscopic data indicates that kringle 4 possesses a globular and flexible structure which exhibits relatively fast amide-hydrogen exchange. About 17 NH groups show retarded exchange, with half-lives of ~7 h in 2H 2O at pH ∗ 6.45, 25 °C, which indicates that regions of the kringle are buried and shielded from direct interaction with the solvent. Analysis of the methyl region spectrum accounts for all singlets and doublets in terms of the amino acid composition; resonances from the C- and N-termini residues could be identified from the magnitude of their 3 J couplings and their response to pH titration. It is shown that elastase digestion of plasminogen generates two species of kringle 4, one that terminates with Ala 85 and another that extends to Val 87. The heterogeneity can be resolved by chromatography on CM-Sephadex. The interaction of kringle 4 with BASA ( p-benzylaminesulfonic acid), an antifibrinolytic drug presumed to bind to the plasminogen lysine-binding sites, has been investigated through the effects of added ligand on the kringle spectrum. The kringle lysine-binding site would appear to be integrated by a cluster of interacting His and aromatic residues since many of these resonances follow a definite saturation curve pattern upon BASA titration. In contrast, only minor changes are detected in the aliphatic methyl spectra. The association constant for the BASA-kringle 4 interaction is estimated to be K a ~ 74 m m −1, which should be compared with K a ~ 145 m m −1 previously measured for kringle 1 under identical conditions. It is proposed that residues in the proximity of the Cys 80-Cys 1 disulfide bridge are proximal to, or form part of, the lysine-binding site.