The blockage of the high-affinity lysine binding sites of plasminogen by EACA significantly inhibits prourokinase-induced plasminogen activation

Prourokinase-induced plasminogen activation is complex and involves three distinct reactions: (1) plasminogen activation by the intrinsic activity of prourokinase; (2) prourokinase activation by plasmin; (3) plasminogen activation by urokinase. To further understand some of the mechanisms involved, the effects of epsilon-aminocaproic acid (EACA), a lysine analogue, on these reactions were studied. At a low range of concentrations (10-50 microM), EACA significantly inhibited prourokinase-induced (Glu-/Lys-) plasminogen activation, prourokinase activation by Lys-plasmin, and (Glu-/Lys-) plasminogen activation by urokinase. However, no inhibition of plasminogen activation by Ala158-prourokinase (a plasmin-resistant mutant) occurred. Therefore, the overall inhibition of EACA on prourokinase-induced plasminogen activation was mainly due to inhibition of reactions 2 and 3, by blocking the high-affinity lysine binding interaction between plasmin and prourokinase, as well as between plasminogen and urokinase. These findings were consistent with kinetic studies which suggested that binding of kringle 1-4 of plasmin to the N-terminal region of prourokinase significantly promotes prourokinase activation, and that binding of kringle 1-4 of plasminogen to the C-terminal lysine158 of urokinase significantly promotes plasminogen activation. In conclusion, EACA was found to inhibit, rather than promote, prourokinase-induced plasminogen activation due to its blocking of the high-affinity lysine binding sites on plasmin(ogen).     

Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments

Human tissue-type plasminogen activator (t-PA) consists of five domains designated (starting from the N-terminus) finger, growth factor, kringle 1, kringle 2, and protease. The binding of t-PA to lysine-Sepharose and aminohexyl-Sepharose was found to require kringle 2. The affinity for binding the lysine derivatives 6-aminohexanoic acid and N-acetyllysine methyl ester was about equal, suggesting that t-PA does not prefer C-terminal lysine residues for binding. Intact t-PA and a variant consisting only of kringle 2 and protease domains were found to bind to fibrin fragment FCB-2, the very fragment that also binds plasminogen and acts as a stimulator of t-PA-catalyzed plasminogen activation. In both cases, binding could completely be inhibited by 6-aminohexanoic acid, pointing to the involvement of a lysine binding site in this interaction. Furthermore, the second site in t-PA involved in interaction with fibrin, presumably the finger, appears to interact with a part of fibrin, different from FCB-2.     

Identification of glycated and acetylated lysine residues in human α2-antiplasmin

BackgroundThe post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues.AimWe sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin.MethodsWe analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of β-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM β-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer.ResultsEleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with β-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with β-d-glucose or ASA. Incubation with concomitant ASA and β-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM β-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM β-d-glucose was associated the absence of acetylation at that site.ConclusionHuman α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.     

Serpin-serine protease binding kinetics: alpha 2-antiplasmin as a model inhibitor.

We have examined in detail the kinetics of binding of the serpin alpha 2-antiplasmin to the serine proteases alpha-chymotrypsin and plasmin. These represent model systems for serpin binding. We find, in contrast to earlier published results with alpha 2-antiplasmin and plasmin, that binding is reversible, and slow binding kinetics can be observed, under appropriate conditions. Binding follows a two-step process with both enzymes, with the formation of an initial loose complex which then proceeds to a tightly bound complex. In the absence of lysine and analogues, equilibrium between alpha 2-antiplasmin and plasmin is achieved rapidly, with an overall inhibition constant (Ki') of 0.3 pM. In the presence of tranexamic acid or 6-aminohexanoic acid, lysine analogues that mimic the effects of fibrin, plasmin binding kinetics are changed such that equilibrium is reached slowly following a lag phase after mixing of enzyme and inhibitor. The Ki' is also affected, rising to 2 pM in the presence of 6-aminohexanoic acid concentrations above 15 mM. Thus extrapolation to the in vivo situation indicates that complex formation in the presence of fibrin will be delayed, allowing a burst of enzyme activity following plasmin generation, but a tight, pseudoirreversible complex will result eventually. Chymotrypsin is more weakly inhibited by alpha 2-antiplasmin, exhibiting an overall Ki' of 0.1 nM, after two-stage complex formation. The inhibition constant for the initial loose complex (Ki) is very similar for both enzymes. The difference in binding strength between the two enzymes is accounted for by the dissociation rate constant of the second step of complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding site of α2-plasmin inhibitor to plasminogen

Peptide T-11, a carboxyl terminal tryptic fragment of α₂-plasmin inhibitor, inhibits the reversible first step of the reaction between plasmin and α₂-plasmin inhibitor. To elucidate which amino-acid residues played a important role in the inhibitory activity of peptide T-11, we prepared the various synthetic derivatives of peptide T-11 and determined the peptide concentration that inhibited the apparent rate constant of the reaction between plasmin and α₂-plasmin inhibitor by 50% (IC₅₀). Peptide III, which lacked the residues Gly-1 to Pro-7 of peptide I (peptide T-11), had a strong inhibitory activity, like peptide I (IC₅₀: peptide 1, 7 μM; peptide III, 13 μM). The peptides that lacked the Leu-9 and Lys-10 or Lys-26 of peptide III showed much weaker activity, and the loss of amidation of the C-terminal lysine of peptide III also markedly reduced the inhibitory activity, Peptide III competitivef inhibited the binding of [¹⁴C]tranexamic acid to kringle 1 + 2 + 3 (K1–3) and kringle 4 (K4) in a binding assay performed by the gel-diffusion method. The respectively dissociation constants (K_d) of peptide III for K1–3 and K4 were 0.85 μM and 35.2 μM. These data suggest that the amino residue of Lys-10 and the carboxylic acid of Lys-26 in peptide T-11 play crucial roles in the ionic binding of α₂-plasmin inhibitor to the tranexamic acid-binding site (lysine-binding site) of plasminogen. Peptide T-11: H-G-D-K-L-F-G-P-D-L-K-L-V-P-P-M-E-E-D-Y-P-Q-F-G-S-P-K-OH.     

The heparin binding site of human extracellular-superoxide dismutase.

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is major SOD isozyme in extracellular fluids. We revealed the possible structure of the carbohydrate chain of serum EC-SOD with the serial lectin affinity technique. The structure is a biantennary complex type with an internal fucose residue attached to asparagine-linked N-acetyl-D-glucosamine and with terminal sialic acid linked to N-acetyllactosamine. EC-SOD in plasma is heterogeneous with regard to heparin affinity and can be divided into three fractions: A, without affinity; B, with intermediate affinity; and C, with high affinity. It appeared that this heterogeneity is not dependent on the carbohydrate structure upon comparison of EC-SOD A, B, and C. No effect of the glycopeptidase F treatment of EC-SOD C on its heparin affinity supported the results. A previous report showed that both lysine and arginine residues probably at the C-terminal end, contribute to heparin binding. Recombinant EC-SOD C treated with trypsin or endoproteinase Lys C, which lost three lysine residues (Lys-211, Lys-212, and Lys-220) or one lysine residue (Lys-220) at the C-terminal end, had no or weak affinity for the heparin HPLC column, respectively. The proteinase-treated r-EC-SOD C also lost triple arginine residues which are adjacent to double lysine residues. These results suggest that the heparin-binding site may occur on a "cluster" of basic amino acids at the C-terminal end of EC-SOD C. EC-SOD is speculated to be primarily synthesized as type C, and types A and B are probably the result of secondary modifications. It appeared that the proteolytic cleavage of the exteriorized lysine- and arginine-rich C-terminal end in vivo is a more important contributory factor to the formation of EC-SOD B and/or EC-SOD A.     

Effect of streptokinase on the interaction of alpha-2-antiplasmin with different sites of plasmin molecule

Interaction of streptokinase and alpha-2-antiplasmin with plasmin and plasminogen fragments was compared. Binding sites on the enzyme become half-saturated, streptokinase and alpha-2-antiplasmin concentration being 8.5 and 30 nM, respectively. 6-Aminohexanoic acid in concentration of 20 mM reduces the adsorption of streptokinase and and alpha-2-antiplasmin by 20 and 60%, respectively. From all the investigated fragments, streptokinase shows the greatest affinity for mini-plasminogen and alpha-2-antiplasmin for kringles 1-3. Both proteins in the presence of 20 mM 6-aminohexanoic acid do not bind with kringle domains. Arginine dose 0.1 M does not influence streptokinase adsorption on mini-plasminogen and decreases the value of alpha-2-antiplasmin binding with mini-plasminogen by 50%. The data obtained indicate that plasminogen molecule has the sites of the highest affinity for streptokinase on the serine-proteinase domain, however for alpha-2-antiplasmin it is in the kringles 1-3. Streptokinase with equimolar quantity in respect of alpha-2-antiplasmin inhibits the adsorption of alpha-2-antiplasmin on the plasmin by 70% and in the presence of 6-aminohexanoic acid it is inhibited completely. Addition of streptokinase also increases the influence of increasing concentration of the acid. Inhibiting influence of streptokinase decreases, and that of 6-aminohexanoic acid increases, when plasmin is modified with diisopropylfluorophosphate in its active centre. At the same time maximum inhibition of streptokinase adsorption on the plasmin at different concentrations of alpha-2-antiplasmin and 6-aminohexanoic acid accounts for only 20%. We suppose that in the process of complex formation streptokinase competes with alpha-2-antiplasmin for the binding sites on the catalytic domain of the plasmin. Partial or complete blocking of the plasmin active centre contact zone by streptokinase effectively protects it from inhibition by alpha-2-antiplasmin.     

Purification of Human α2-Antiplasmin with Chicken IgY Specific to Its Carboxy-Terminal Peptide

ABSTRACT

α₂-antiplasmin, a plasma glycoprotein of the serpin superfamily, is the primary physiological inhibitor of plasmin, the key enzyme in fibrin degradation. Previous purification methods utilize lengthy multistep protocols with low yields or use monoclonal antibodies that are expensive or difficult to make. With a relatively small investment, a chicken was immunized with keyhole limpet hemocyanin-conjugated to α₂-antiplasmin C-terminal 26 residue synthetic peptide and the peptide-specific antibody (IgY) was isolated from the egg yolks of hens using the peptide affinity column. Based on the interaction between this IgY and α₂-antiplasmin, pure α₂-antiplasmin was isolated from human plasma in two steps: (a) citrated plasma was precipitated with 15% PEG-8000 to remove the bulk of plasma proteins while retaining the majority of α₂-antiplasmin activity; and (b) the α₂-antiplasmin was affinity-purified from the supernatant using the IgY column. Yields were typically 48% and the purity and authenticity of the α₂-antiplasmin were verified by gel electrophoresis, Western Blot analysis, N-terminal sequence, and amino acid analysis.     

Yeast epiarginase regulation,an enzyme-enzyme activity control: identification of residues of ornithine carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities

In the presence of ornithine and arginine, ornithine carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by ornithine for the closure of the catalytic domain.     

Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor.

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.     

Identification of a plasminogen-binding motif in PAM, a bacterial surface protein

Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33,41,52,53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli . The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13–16-amino-acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30-amino-acid synthetic peptide demonstrated that these repeats harbour the major plasminogen-binding site in the ML53 protein, as well as a binding site for the tissue-type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen-binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle-binding determinants.     

Identification of the domains of tissue-type plasminogen activator involved in the augmented binding to fibrin after limited digestion with plasmin

The binding of recombinant tissue-type plasminogen activator (rt-PA) to fibrin increases upon digestion of fibrin with plasmin. Optimal binding is observed following a limited plasmin digestion of fibrin, coinciding with the generation of fibrin fragment X polymers. We studied the involvement of the separate domains of the amino-terminal "heavy" (H) chain of rt-PA in this augmentation of fibrin binding. The fibrin-binding characteristics of a set of rt-PA deletion mutants, lacking either one or more of the structural domains of the H chain, were determined on intact fibrin matrices and on fibrin matrices that were subjected to limited digestion with plasmin. The augmented fibrin binding of rt-PA is partially abolished when the plasmin-degraded fibrin matrices are subsequently treated with carboxypeptidase B, demonstrating that this increased binding is dependent on the generation of carboxyl-terminal lysine residues in the fibrin matrix. Evidence is provided that this increase of fibrin binding is mediated by the kringle 2 (K2) domain that contains a lysine-binding site. Further increase of the fibrin binding of rt-PA is independent of the presence of carboxyl-terminal lysines. It is shown that the latter increase is not mediated by the K2 domain. Based on our data, we propose that the increase in fibrin binding, unrelated to the presence of carboxyl-terminal lysine residues, is mediated by the finger (F) domain, provided that this domain is correctly exposed in the remainder of the protein.     

Identification of a novel plasmin(ogen)-binding motif in surface displayed alpha-enolase of Streptococcus pneumoniae

The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface-associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal alpha-enolase (Eno) and is subsequently activated to the serine protease plasmin by host-derived tissue plasminogen activator (tPA) or urokinase (uPA). The C-terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)-binding site of Eno by investigating the protein-protein interaction. Plasmin(ogen)-binding activity of C-terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno-plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248-256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)-binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing alpha-enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen-binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen-binding motif in the pathogenesis of pneumococcal diseases.     

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-A resolution.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.     

Kinetics of the reactions between streptokinase, plasmin and alpha 2-antiplasmin.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 S 10(7) M-1 s-1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 X 10(-11) M. This plasmin-streptokinase complex is 10(5) times less reactive towards alpha 2-antiplasmin than plasmin, the inhibition rate constant being 1.4 X 10(2) M-1 s-1. The loss of reactivity of the streptokinase-plasmin complex towards alpha 2-antiplasmin is independent of the lysine binding sites in plasmin since low-Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6-aminohexanoic acid, are both equally unreactive towards alpha 2-antiplasmin on reaction with streptokinase. The plasmin-streptokinase complex binds to Sepharose-lysine and Sepharose-fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human alpha 2-antiplasmin (k1 = 1.6 X 10(6) M-1 s-1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 microM streptokinase).     

Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and 1730 microM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region (EDP I, Glu-plasminogen, Lys-plasminogen, and the plasmin heavy chain) and did not react with those lacking an EDP I region [miniplasminogen, the plasmin light chain or EDP II (kringle 4)] or with tissue plasminogen activator or prothrombin, which also contain kringles. By immunoblotting analyses, a chymotryptic degradation product of Mr 20,000 was derived from EDP I that retained reactivity with the antibody. The high-affinity lysine binding site was equally available to the antibody probe in Glu- and Lys-plasminogen and also appeared to be unoccupied in the plasmin-alpha 2-antiplasmin complex. alpha 2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative Dissection of the Binding Contributions of Ligand Lysines of the Receptor-associated Protein (RAP) to the Low Density Lipoprotein Receptor-related Protein (LRP1)

Although lysines are known to be critical for ligand binding to LDL receptor family receptors, relatively small reductions in affinity have been found when such lysines have been mutated. To resolve this paradox, we have examined the specific binding contributions of four lysines, Lys-253, Lys-256, Lys-270, and Lys-289, in the third domain (D3) of receptor-associated protein (RAP), by eliminating all other lysine residues. Using D3 variants containing lysine subsets, we examined binding to the high affinity fragment CR56 from LRP1. With this simplification, we found that elimination of the lysine pairs Lys-253/Lys-256 and Lys-270/Lys-289 resulted in increases in K_d of 1240- and 100,000-fold, respectively. Each pair contributed additively to overall affinity, with 61% from Lys-270/Lys-289 and 39% from Lys-253/Lys-256. Furthermore, the Lys-270/Lys-289 pair alone could bind different single CR domains with similar affinity. Within the pairs, binding contributions of Lys-270 ≫ Lys-256 > Lys-253 ∼ Lys-289 were deduced. Importantly, however, Lys-289 could significantly compensate for the loss of Lys-270, thus explaining how previous studies have underestimated the importance of Lys-270. Calorimetry showed that favorable enthalpy, from Lys-256 and Lys-270, overwhelmingly drives binding, offset by unfavorable entropy. Our findings support a mode of ligand binding in which a proximal pair of lysines engages the negatively charged pocket of a CR domain, with two such pairs of interactions (requiring two CR domains), appropriately separated, being alone sufficient to provide the low nanomolar affinity found for most protein ligands of LDL receptor family members.     

Limited proteolysis of tumor cells increases their plasmin-binding ability

Mild proteolytic treatment of SW1116 tumor cells with trypsin or plasmin increases their plasmin-binding ability considerably by increasing the number of binding sites without altering their affinity. This mechanism may be operative for increasing the concentration of active plasmin at the surface of tumor cells. C-terminal lysine residues are involved in plasmin binding to cells, since treatment of cells with carboxypeptidase B decreases this binding by 50%.     

Features of the interaction between alpha2-antiplasmin and plasminogen/plasmin

The kinetic of plasmin, Va1442-plasmin, Lys530-plasmin inhibition reaction by alpha 2-antiplasmin as well as interaction of the inhibitor with different derivatives of the plasminogen and its fragments were studied. It was shown that plasmin, mini- and micro-plasmin activity decreased by 97, 88 and 85%, respectively, for equimolar ratio 1:1 of the inhibitor. The value of the inhibition reached its maximum in 1-2, 5-10 and 10-15 min, respectively. The constants of the complex formation rate were 1.4 x 10(6); 1.7 x 10(5) and 6.2 x 10(4) M-1s-1 for the plasmin, mini- and micro-plasmin with alpha 2-antiplasmin, respectively. Both 10(-2) M 6-aminohexanoic acid and 10(-1) M arginine reduced the complex formation rate between plasmin, mini-plasmin and alpha 2-antiplasmin to the value of the rate reaction between micro-plasmin and inhibitor. alpha 2-Antiplasmin bound with all investigated derivatives and fragments of plasminogen. The amount of inhibitor decreased in the series: plasmin, kringle 1-3, kringle 4, mini-plasminogen, micro-plasminogen. The kringle 1-4 and kringle 5 were determined to control the rate of reaction between enzyme and inhibitor, being not necessary for the inhibition. The comparison of the inhibitor interaction with DPP-plasmin, mini-plasminogen and micro-plasminogen displayed the possibility of the additional region existence in catalytic domain. This region participated in the complex with alpha 2-antiplasmin formation. It is supposed that the multisite interaction between plasmin and alpha 2-antiplasmin provides for the specificity and efficiency the inhibitor action.