Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin.

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.     

Structural/functional characterization of the alpha 2-plasmin inhibitor C-terminal peptide

The alpha(2)-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin. It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment. The latter, peptide Asn(398)-Lys(452), and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively). CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure. Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K(a) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 3.2 mM(-)(1)), all of which are known to exhibit lysine-binding capability. The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N(alpha)-acetyllysine, a mimic of a C-terminal Lys residue. The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles. (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS. We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys(452) most definitely enhances the binding. This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain.     

Conformation of Lys-plasminogen and the kringle 1-3 fragment of plasminogen analyzed by small-angle neutron scattering

Native human Glu-plasminogen (Glu1-Asn791) was previously shown to have a radius of gyration of 39 A and a shape best described by a prolate ellipsoid [Mangel, W. F., Lin, B., & Ramakrishnan, V. (1990) Science 248, 69-73]. Upon occupation of a weak lysine-binding site, the shape reversibly changes to that best described by a Debye random coil with a radius of gyration of 56 A. Conversion from the closed to the open form is not accompanied by any change in secondary structure, hence the closed conformation is formed by interaction between domains, the five kringles and the protease domain, and this is abolished upon conversion to the open form. Here we analyzed by small-angle neutron scattering the conformations of human Lys-plasminogen (Lys78-Asn791) and the fragment K1-3 that contains the first three kringles of plasminogen (Tyr80-Val338 or Tyr80-Val354). The shape of Lys-plasminogen was best described by a Debye random coil with a radius of gyration of 51 A, and occupation of its lysine-binding sites by 6-aminohexanoic acid did not dramatically alter its conformation. Thus Lys-plasminogen was in the open form, similar to that of Glu-plasminogen with its lysine-binding sites occupied. The fragment K1-3 in the absence or presence of 6-aminohexanoic acid had a shape best described equally either by an elongated prolate ellipsoid or by a Debye random coil, with a radius of gyration of 29 A. Our model for the two forms of plasminogen is that, in the closed form, domain interaction generates a compact, almost globular, structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two classes of lysine-binding sites of plasminogen molecule

Affinity of plasminogen fragments K1, K2-3, K4 and K5 for 6-aminophenyl-Sepharose was investigated to characterize the lysine-binding sites of the protein. K1 and K5 fragments were bound to the affinity column, whereas kringle 2-3 and kringle 4 were not. The results obtained and data known from literature have indicate that two types of lysine-binding sites are present in the plasminogen molecule. Both positively and negatively charged groups of the ligand are necessary for binding with the first-type sites (K4 and K2-3). The interaction between ligands and the second-type sites localized in kringles 1 and 5 is provided by their positively charged group only.     

Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein

The X-ray crystal structure of a complex of a modified recombinant kringle-2 domain of human plasminogen, K2Pg[C4G/E56D/L72Y] (mK2Pg), containing an upregulated lysine-binding site, bound to a functional 30 residue internal peptide (VEK-30) from an M-type protein of a group A Streptococcus surface protein, has been determined by molecular replacement methods using K4Pg as a model, and refined at 2.7 A resolution to a R-factor of 19.5 %. The X-ray crystal structure shows that VEK-30 exists as a nearly end-to-end alpha-helix in the complex with mK2Pg. The final structure also revealed that Arg17 and His18 of VEK-30 served as cationic loci for Asp54 and Asp56 of the consensus lysine-binding site of mK2Pg, while Glu20 of VEK-30 coordinates with Arg69 of the cationic binding site of mK2Pg. The hydrophobic ligand-binding pocket in mK2Pg, consisting primarily of Trp60 and Trp70, situated between the positive and negative centers of the lysine-binding site, is utilized in a novel manner in stabilizing the interaction with VEK-30 by forming a cation-pi-electron-mediated association with the positive side-chain of Arg17 of this peptide. Additional lysine-binding sites, as well as exosite electrostatic and hydrogen bonding interactions involving Glu9 and Lys14 of VEK-30, were observed in the structural model. The importance of these interactions were tested in solution by investigating the binding constants of synthetic variants of VEK-30 to mK2Pg, and it was found that, Lys14, Arg17, His18, and Glu20 of VEK-30 were the most critical amino acid binding determinants. With regard to the solution studies, circular dichroism analysis of the titration of VEK-30 with mK2Pg demonstrated that the peptidic alpha-helical structure increased substantially when bound to the kringle module, in agreement with the X-ray results.This investigation is the first to delineate structurally the mode of interaction of the lysine-binding site of a kringle with an internal pseudo-lysine residue of a peptide or protein that functionally interacts with a kringle module, and serves as a paradigm for this important class of interactions.     

Antiangiogenic kringles derived from human plasminogen and apolipoprotein(a) inhibit fibrinolysis through a mechanism that requires a functional lysine-binding site

Many proteins in the fibrinolysis pathway contain antiangiogenic kringle domains. Owing to the high degree of homology between kringle domains, there has been a safety concern that antiangiogenic kringles could interact with common kringle proteins during fibrinolysis leading to adverse effects in vivo. To address this issue, we investigated the effects of several antiangiogenic kringle proteins including angiostatin, apolipoprotein(a) kringles IV(9)-IV(10)-V (LK68), apolipoprotein(a) kringle V (rhLK8) and a derivative of rhLK8 mutated to produce a functional lysine-binding site (Lys-rhLK8) on the entire fibrinolytic process in vitro and analyzed the role of lysine binding. Angiostatin, LK68 and Lys-rhLK8 increased clot lysis time in a dose-dependent manner, inhibited tissue-type plasminogen activator-mediated plasminogen activation on a thrombin-modified fibrinogen (TMF) surface, showed binding to TMF and significantly decreased the amount of plasminogen bound to TMF. The inhibition of fibrinolysis by these proteins appears to be dependent on their functional lysine-binding sites. However, rhLK8 had no effect on these processes owing to an inability to bind lysine. Collectively, these results indicate that antiangiogenic kringles without lysine binding sites might be safer with respect to physiological fibrinolysis than lysine-binding antiangiogenic kringles. However, the clinical significance of these findings will require further validation in vivo.     

Direct identification of lysine-33 as the principal cationic center of the omega-amino acid binding site of the recombinant kringle 2 domain of tissue-type plasminogen activator.

We have generated site-specific mutants of the kringle 2 domain of tissue-type plasminogen activator [( K2tPA]) in order to identify directly the cationic center of the protein that is responsible for its interaction with the carboxyl group of important omega-amino acid effector molecules, such as epsilon-amino caproic acid (EACA). Molecular modeling of [K2tPA], docked with EACA, based on crystal structures of the kringle 2 region of prothrombin and the kringle 4 domain of human plasminogen, clearly shows that Lys33 is the only positively charged amino acid in [K2tPA] that is sufficiently proximal to the carboxyl group of the ligand to stabilize this interaction. In order to examine directly the importance of this particular amino acid residue in this interaction, we have constructed, expressed, and purified three recombinant (r) mutants of [K2tPA], viz., Lys33Thr, Lys33Leu, and Lys33Arg, and found that only the last variant retained significant ability to interact with EACA and several of its structural analogues at neutral pH. In addition, another mutated r-[K2tPA], i.e., Lys33His, interacts very weakly with omega-amino acids at neutral pH and much more strongly at lower pH values where His33 would be expected to undergo protonation. This demonstrates that any positively charged amino acid at position 33 satisfies the requirement for mediation of significant bindings to this class of molecules. Since, in other kringles, positively charged residues at amino acid sequence positions homologous to Lys68, Arg70, and Arg71 of [K2tPA] have been found to participate in kringle interactions with EACA-like compounds, we have also examined the binding of EACA, and some of its analogues, to three additional r-[K2tPA] variants, i.e., Lys68Ala, Arg70Ala, and Arg71Ala. In each case, binding of these omega-amino acids to the variant kringles was observed, with only the Lys68Ala variant showing a slightly diminished capacity for this interaction. These investigations provide clear and direct evidence that Lys33 is the principal cationic site in wild-type r-[K2tPA] that directly interacts with the carboxyl group of omega-amino acid effector molecules.     

A high affinity interaction of plasminogen with fibrin is not essential for efficient activation by tissue-type plasminogen activator

Fibrin (Fn) enhances plasminogen (Pg) activation by tissue-type plasminogen activator (tPA) by serving as a template onto which Pg and tPA assemble. To explore the contribution of the Pg/Fn interaction to Fn cofactor activity, Pg variants were generated and their affinities for Fn were determined using surface plasmon resonance (SPR). Glu-Pg, Lys-Pg (des(1-77)), and Mini-Pg (lacking kringles 1-4) bound Fn with K(d) values of 3.1, 0.21, and 24.5 μm, respectively, whereas Micro-Pg (lacking all kringles) did not bind. The kinetics of activation of the Pg variants by tPA were then examined in the absence or presence of Fn. Whereas Fn had no effect on Micro-Pg activation, the catalytic efficiencies of Glu-Pg, Lys-Pg, and Mini-Pg activation in the presence of Fn were 300- to 600-fold higher than in its absence. The retention of Fn cofactor activity with Mini-Pg, which has low affinity for Fn, suggests that Mini-Pg binds the tPA-Fn complex more tightly than tPA alone. To explore this possibility, SPR was used to examine the interaction of Mini-Pg with Fn in the absence or presence of tPA. There was 50% more Mini-Pg binding to Fn in the presence of tPA than in its absence, suggesting that formation of the tPA-Fn complex exposes a cryptic site that binds Mini-Pg. Thus, our data (a) indicate that high affinity binding of Pg to Fn is not essential for Fn cofactor activity, and (b) suggest that kringle 5 localizes and stabilizes Pg within the tPA-Fn complex and contributes to its efficient activation.     

Characterization of the high-affinity interaction between human plasminogen and pro-urokinase

Activation of human Glu-plasminogen, Lys-plasminogen and low-Mr plasminogen (lacking lysine-binding sites) by pro-urokinase (pro-UK), obtained from a human lung adenocarcinoma cell line (Calu-3, ATCC), obeys Michaelis-Menten kinetics. Activation occurs with a comparable affinity (Km 0.40-0.77 microM), while the catalytic rate constant (kcat) is comparable for Glu-plasminogen (0.0022s-1) and low-Mr plasminogen (0.0034 s-1), but is somewhat higher for Lys-plasminogen (0.0106 s-1). The rate of activation of plasminogen by pro-UK is not significantly influenced by the presence of 6-aminohexanoic acid, purified fragments LBS I or LBS II or histidine-rich glycoprotein, indicating that the high affinity of pro-UK for plasminogen is not mediated via the high-affinity lysine-binding site of plasminogen located in kringles 1-3 (LBS I) nor via the low-affinity lysine-binding site comprised within kringle 4 (LBS II). The site(s) in plasminogen involved in the high-affinity interaction with pro-UK thus appear to be located within the low-Mr plasminogen moiety.     

A 1H-NMR study of isolated domains from human plasminogen. Structural homology between kringles 1 and 4

Kringles 1 and 4 from human plasminogen are polypeptide domains of Mr approximately equal to 10000 each of which can be isolated by proteolysis of the zymogen. They have been studied by 1H-NMR spectroscopy at 300 MHz and 600 MHz. The spectra, characteristic of globular structures, show striking analogies that point to a close conformational relatedness among the two kringles, consistent with their high degree of amino acid conservancy and homology. The interaction of both kringles with p-benzylaminesulfonic acid (BASA), an antifibrinolytic drug that binds to a lysine-binding site, results in better resolved, narrower lines for both spectra. Aromatic and methyl-region spectra of BASA complexes of kringles 1 and 4 were compared and the latter was studied by two-dimensional NMR spectroscopy. Analysis of the CH3 multiplets in terms of their resonance patterns, and the amino acid compositions and sequences of the two kringles, leads to the identification of most signals and to some assignments. In particular, a doublet at -1 ppm, exhibited by both kringles and also found in reported proton spectra of homologous bovine prothrombin fragments, has been assigned to Leu46, a residue that is conserved in all of the kringles studied to date by 1H-NMR. Since this resonance is somewhat more sensitive to BASA than other methyl signals, it is likely that Leu46 is proximal to the lysine-binding site. Nuclear Overhauser experiments reveal that Leu46 is surrounded by a cluster of closely interacting hydrophobic and aromatic side chains. Kringle 4 was also compared with a derivative chemically modified at Trp72 with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide. As judged from the proton spectra, the modified kringle 4 retains globularity and is perturbed mainly in the aromatic region, in analogy to that which is observed for the unmodified kringle upon BASA binding. Furthermore, although previous studies have indicated no retention of the modified kringle by lysine-Sepharose, the NMR studies point to a definite interaction between BASA and the kringle derivative. The spectroscopic data also suggest that the His31 imidazole is not significantly affected by the ligand and that the lysine-binding site is structured mostly by hydrophobic side chains, including Trp72 in the case of kringle 4, and probably Tyr72 in kringle 1.     

On the biological significance of the specific interaction between fibrin,plasminogen and antiplasmin

The influence of antiplasmin on the interaction between fibrin and plasminogen was studied in plasma and in a purified system. The amount of plasminogen bound to fibrin was quantitated using trace amounts of ¹²⁵I-labeled Glu-plasminogen (plasminogen with NH₂-terminal glutamic acid) or ¹²⁵I-labeled Lys-plasminogen (NH₂-terminal lysine).When whole plasma was clotted, 5.2% of Glu-plasminogen was associated with the fibrin clot. In plasma clotted in the presence of 20 mM 6-amino-hexanoic acid only 1.4% of the plasminogen was bound to fibrin, indicating that about 4% of the plasma plasminogen specifically binds to fibrin. With Lys-plasminogen these values were approximately twice as high.When antiplasmin-depleted plasma was used, only slightly higher amounts of both types of plasminogen were associated with the fibrin. The adsorbed plasminogen was not significantly eluted with plasma or with purified antiplasmin at physiological concentrations.These findings indicate that antiplasmin does not play a significant role in the inhibition of the binding of plasminogen to fibrin or the dissociation of the plasminogen · fibrin complex.These observations in conjunction with previous findings on the kinetics of the plasmin-antiplasmin reaction suggest that the lysine-binding site of plasminogen, which is responsible both for its interaction with fibrin and its interaction with antiplasmin, plays an important role in the very fast neutralization of plasmin formed in circulating blood and serves to attach plasminogen to fibrin and thereby sequestrate plasmin formed in loco from circulating antiplasmin.     

Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation

Binding of plasminogen to fibrin and cell surfaces is essential for fibrinolysis and pericellular proteolysis. We used surface plasmon resonance and enzyme kinetic analyses to study the effect of two mAbs (A10.2, CPL15) on plasminogen binding and activation at fibrin surfaces. A10.2 is directed against the lysine-binding site (LBS) of kringle 4, whereas CPL15 recognises a region in kringle 1 outside the LBS. In the presence of CPL15 and A10.2 mAbs, binding of plasminogen (K(d)=1.16+/-0.22 micromol/l) to fibrin was characterised by a mAb concentration-dependent bell-shaped isotherm. A progressive increase in the concentration of mAbs at the surface was also detected, and reached a plateau corresponding to the maximum of plasminogen bound. These data indicated that at low mAb concentration, bivalent plasminogen-mAb-plasminogen ternary complexes are formed, whereas at high mAb concentration, a progressive shift to monovalent plasminogen-mAb binary complexes is observed. Plasmin formation in the presence of mAbs followed a similar bell-shaped profile. Monovalent Fab fragments of mAb A10.2 showed no effect on the binding of plasminogen, confirming the notion that a bivalent mAb interaction is essential to increase plasminogen binding and activation at the surface of fibrin.     

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.     

On the specific interaction between the lysine-binding sites in plasmin and complementary sites in alpha2-antiplasmin and in fibrinogen.

Plasminogen and plasminogen derivatives which contain lysine-binding sites were found to decrease the reaction rate between plasmin and alpha2-antiplasmin by competing with plasmin for the complementary site(s) in alpha2-antiplasmin. The dissocwation constant Kd for the interaction between intact plasminogen (Glu-plasminogen) and alpha2-antiplasmin is 4.0 microM but those for Lys-plasminogen or TLCK-plasmin are about 10-fold lower indicating a stronger interaction. The lysine-binding site(s) which is situated in triple-loops 1--3 in the plasmin A-chain is mainly responsible for the interaction with alpha2-antiplasmin. The interaction between Glu-plasminogen and alpha2-antiplasmin furthermore enhances the activation of Glu-plasminogen by urokinase to a comparable extent as 6-aminohexanoic acid, suggesting that similar conformational changes occur in the proenzyme after complex formation. Fibrinogen, fibrinogen digested with plasmin, purified fragment E and purified fragment D interfere with the reaction between plasmin and alpha2-antiplasmin by competing with alpha2-antiplasmin for the lysine-binding site(s) in the plasmin A-chain. The Kd obtained for these interactions varied between 0.2 microM and 1.4 microM; fragment E being the most effective. Thus the fibrinogen molecule contains several complementary sites to the lysine-binding sites located both in its NH2-terminal and COOH-terminal regions; these sites are to a large extent.     

Apolipoprotein(a): structure-function relationship at the lysine-binding site and plasminogen activator cleavage site

Apolipoprotein(a) [apo(a)] is the distinctive glycoprotein of lipoprotein Lp(a), which is disulfide linked to the apo B100 of a low density lipoprotein particle. Apo(a) possesses a high degree of sequence homology with plasminogen, the precursor of plasmin, a fibrinolytic and pericellular proteolytic enzyme. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogen-like kringle 4 and single copies of kringle 5, and the protease region including the backbone positions for the catalytic triad (Ser, His, Asp). A lysine-binding site that is similar to that of plasminogen kringle 4 is present in apo(a) kringle IV type 10. These kringle motifs share some amino acid residues (Asp55, Asp57, Phe64, Tyr62, Trp72, Arg71) that are key components of their lysine-binding site. The spatial conformation and the function of this site in plasminogen kringle 4 and in apo(a) kringle IV-10 seem to be identical as indicated by (i) the ability of apo(a) to compete with plasminogen for binding to fibrin, and (ii) the neutralisation of the lysine-binding function of these kringles by a monoclonal antibody that recognises key components of the lysine-binding site. In contrast, the lysine-binding site of plasminogen kringle 1 contains a Tyr residue at positions 64 and 72 and is not recognised by this antibody. Plasminogen bound to fibrin is specifically recognised and cleaved by the tissue-type plasminogen activator at Arg561-Val562, and is thereby transformed into plasmin. A Ser-Ile substitution at the activation cleavage site is present in apo(a). Reinstallation of the Arg-Val peptide bond does not ensure cleavage of apo(a) by plasminogen activators. These data suggest that the stringent specificity of tissue-type plasminogen activator for plasminogen requires molecular interactions with structures located remotely from the activation disulfide loop. These structures ensure second site interactions that are most probably absent in apo(a).     

Specific interaction of angiostatin with integrin alpha(v)beta(3) in endothelial cells

Angiostatin, the N-terminal four kringles (K1-4) of plasminogen, blocks tumor-mediated angiogenesis and has great therapeutic potential. However, angiostatin's mechanism of anti-angiogenic action is unclear. We found that bovine arterial endothelial (BAE) cells adhere to angiostatin in an integrin-dependent manner and that integrins alpha(v)beta(3), alpha(9)beta(1), and to a lesser extent alpha(4)beta(1), specifically bind to angiostatin. alpha(v)beta(3) is a predominant receptor for angiostatin on BAE cells, since a function-blocking antibody to alpha(v)beta(3) effectively blocks adhesion of BAE cells to angiostatin, but an antibody to alpha(9)beta(1) does not. epsilon-Aminocaproic acid, a Lys analogue, effectively blocks angiostatin binding to BAE cells, indicating that an unoccupied Lys-binding site of the kringles may be required for integrin binding. It is known that other plasminogen fragments containing three or five kringles (K1-3 or K1-5) have an anti-angiogenic effect, but plasminogen itself does not. We found that K1-3 and K1-5 bind to alpha(v)beta(3), but plasminogen does not. These results suggest that the anti-angiogenic action of angiostatin may be mediated via interaction with alpha(v)beta(3). Angiostatin binding to alpha(v)beta(3) does not strongly induce stress-fiber formation, suggesting that angiostatin may prevent angiogenesis by perturbing the alpha(v)beta(3)-mediated signal transduction that may be necessary for angiogenesis.     

X-ray crystallographic structure of the angiogenesis inhibitor, angiostatin, bound to a peptide from the group A streptococcal surface protein PAM

The crystal structure of the human Pg-derived angiogenesis inhibitor, angiostatin, complexed to VEK-30, a peptide from the group A streptococcal surface protein, PAM, was determined and refined to 2.3 A resolution. This is the first structure of angiostatin bound to a ligand and provides a model of the interaction between Pg and streptococcal-derived pathogenic proteins. VEK-30 contains a "through-space isostere" for C-terminal lysine, wherein Arg and Glu side chains, separated by one helical turn, bind within the bipolar angiostatin kringle 2 (K2) domain lysine-binding site. VEK-30 also makes several contacts with K2 residues that exist outside of the canonical LBS and are not conserved among the other Pg kringles, thus providing a molecular basis for the selectivity of VEK-30 for K2. The structure also shows that Pg kringle domains undergo significant structural rearrangement relative to one another and reveals dimerization between two molecules of angiostatin and VEK-30 related by crystallographic symmetry. This dimerization, which exists only in the crystal structure, is consistent with the parallel coiled-coil full-length PAM dimer expected from sequence similarities and homology modeling.     

Structural/functional properties of the Glu1-HSer57 N-terminal fragment of human plasminogen: conformational characterization and interaction with kringle domains.

The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of approximately 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 1H-NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rK1, Ka approximately 4.6 mM(-1); rK2, Ka approximately 3.3 mM(-1); K4, Ka approximately 6.2 mM-'; K5, K, 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Nalpha-acetyl-L-lysine methyl ester (Ka < 0.6 mM(-l), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3. which is devoid of a LBS. CB-NTP and L-NTP 1H-NMR spectra were assigned and interproton distances estimated from 1H-1H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys5O within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectra- and exhibit fast amide 1H-2H exchange in 2H2O, suggestive of high flexibility.     

Functional hierarchy of plasminogen kringles 1 and 4 in fibrinolysis and plasmin-induced cell detachment and apoptosis

Plasmin(ogen) kringles 1 and 4 are involved in anchorage of plasmin(ogen) to fibrin and cells, an essential step in fibrinolysis and pericellular proteolysis. Their contribution to these processes was investigated by selective neutralization of their lysine-binding function. Blocking the kringle 1 lysine-binding site with monoclonal antibody 34D3 fully abolished binding and activation of Glu-plasminogen and prevented both fibrinolysis and plasmin-induced cell detachment-induced apoptosis. In contrast, blocking the kringle 4 lysine-binding site with monoclonal antibody A10.2 did not impair its activation although it partially inhibited plasmin(ogen) binding, fibrinolysis and cell detachment. This remarkable, biologically relevant, distinctive response was not observed for plasmin or Lys-plasminogen; each antibody inhibited their binding and activation of Lys-plasminogen to a limited extent, and full inhibition of fibrinolysis required simultaneous neutralization of both kringles. Thus, in Lys-plasminogen and plasmin, kringles 1 and 4 act as independent and complementary domains, both able to support binding and activation. We conclude that Glu-/Lys-plasminogen and plasmin conformations are associated with transitions in the lysine-binding function of kringles 1 and 4 that modulate fibrinolysis and pericellular proteolysis and may be of biological relevance during athero-thrombosis and inflammatory states. These findings constitute the first biological link between plasmin(ogen) transitions and functions.     

The effect of kringles K1-3, K4 and K5 on lysis of fibrin clots caused by the activation of Glu- and Lys-plasminogen by a tissue activator

Kringles K1-3, K4 and K5 are studied for their effect on tissue plasminogen activator-induced fibrin clot lysis in the presence of Glu- and Lys-plasminogen. It is established that kringles K4 and K5 inhibit fibrinolysis of Glu-plasminogen, and K1-3--that of Lys-plasminogen. The role of plasminogen molecule kringles in the plasminogen interaction with fibrin polymer is discussed.