Analogue 6-aminohexanoic Acid Research Articles

Fibrinolysis-resistant carbonylated fibrin detected in thrombi attached to the vascular wall of abdominal aortic aneurysms

In this study, we investigated how carbonylation of fibrinogen by acrolein modified its indispensable function to enhance fibrinolysis after being converted to fibrin and contributed to generating a fibrinolysis-resistant fibrin clot. Acrolein-treated fibrinogen was subjected to tissue plasminogen activator-induced fibrinolysis assay and the effect of lysine residue carbonylation in fibrinogen on fibrinolysis was analyzed. The acrolein-treated fibrinogen-derived fibrin clot appeared more resistant to fibrinolysis and the N-acetyl 3-formyl-3,4-dehydropiperidino (FDP)-Lysine levels in the lysed solution were positively correlated with the duration of clot lysis. The lysine analog 6-amino hexanoic acid (6AHA), which mimics the C-terminal lysine of fibrin, was carbonylated and its enhancing effect on Glu1-plasminogen activation was evaluated. After incubation with acrolein, 6AHA was converted to N-acetyl FDP-6AHA, losing its ability to enhance Glu1-plasminogen activation. These results suggest that fibrinogen carbonylation by acrolein to generate N-acetyl FDP-Lysine resulted in the generation of fibrinolysis-resistant fibrin by attenuating the C-terminal lysine-dependent activation of the Glu1-plasminogen. In abdominal aortic aneurysms, fibrin(ogen) containing the acrolein adduct N-acetyl FDP-Lysine was detected in the vascular wall-attached thrombi. These results suggest that this mechanism is likely involved in the modification of fibrinolysis-resistant thrombi and to their persistence for a long period.

Small size apolipoprotein(a) isoforms enhance inflammatory and proteolytic potential of collagen-primed monocytes

BackgroundAtherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease.Design and methodsThe aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen.ResultsApo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites.ConclusionsThese results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.

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.

Plasminogen activator inhibitor-1 impairs plasminogen activationmediated vascular smooth muscle cell apoptosis

The role of plasminogen activator inhibitor-1 (PAI-1) in vascular smooth muscle cell (VSMC) apoptosis mediated by plasminogen activation was studied with the use of aorticVSMC derived from mice with deficiency of PAI-1 (PAI-1 (-/-) ), tissue-type (t-PA (-/-) ) or urokinase-type (u-PA (-/-) ) plasminogen activator or from wildtype (WT) mice with corresponding genetic background. Plasminogen incubated with confluent VSMC was activated in a concentration-dependent and saturable manner for all four cell types, with maximal activation rates that were comparable for WT, u-PA (-/-) and t-PA (-/-) cells, but about two-fold higher for PAI-1 (-/-) cells. Plasminogen activation was impaired by addition of the lysine analogue 6-aminohexanoic acid, and by addition of t-PA and u-PA neutralizing antibodies, suggesting that it depends on binding to cell surface COOH-terminal lysine residues, and on plasminogen activator activity. Morphological alterations consistent with apoptosis were observed much earlier in PAI-1 (-/-) than in WT VSMC. Without addition of plasminogen, the apoptotic index was similar for all four cell types, whereas after incubation with physiological plasminogen concentrations, it was greater in PAI-1 (-/-) VSMC, as compared to WT, t-PA (-/-) or u-PA (-/-) VSMC. Furthermore, the apoptotic rate paralleled the release of plasmin. Thus, plasmin-mediated apoptosis of VSMC occurs via plasminogen activation by either t-PA or u-PA and is impaired by PAI-1.

Nonlysine-analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin-like activity.

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 microg.mL(-1). These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.

Soluble Tissue Factor Interferes with Angiostatin-mediated Inhibition of Endothelial Cell Proliferation by Lysine-specific Interaction with Plasminogen Kringle Domains

Experimental and clinical data suggest that tissue factor (TF), the major initiator of blood coagulation cascade, as well as proteases and components of the fibrinolytic system are involved in tumor growth at least in some solid tumors via effects on angiogenesis. Whereas the pro- and anti-angiogenic effects of the plasminogen/plasmin system and plasminogen kringle domains, respectively, are well characterized, the pathways responsible for the pro-angiogenic properties of TF remain poorly understood. To learn more about the biological significance of the recently described binding of plasminogen to the extracellular domain of TF, we examined the effects of soluble TF (sTF) on angiostatin-inhibited proliferation of endothelial cells. In solid phase binding assays, we found that sTF binds specifically to plasminogen, to the plasminogen kringle domains K1-3, K1-5, K4, as well as to mini-plasminogen. Inhibition of binding of plasminogen and its kringle domains to sTF by the lysine analog 6-aminohexanoic acid (AHA) suggests that lysine-binding sites are involved in plasminogen interaction with TF. Moreover, in the presence of sTF, the inhibitory effect of K1-5 on bFGF-mediated HUVEC proliferation was dose-dependently and saturably abolished. This suggests that TF can interfere with the antagonistic effect of K1-5 on endothelial cell proliferation. In contrast, sTF by itself had no effect on the endothelial cell proliferation. Whereas the interference of TF with K1-5-mediated effect was prevented by AHA, this lysine analog did not abolish the proliferation inhibition of K1-5. In conclusion, the binding of sTF to the plasminogen fragment K1-5 seems to antagonize the anti-angiogenic effects of this plasminogen fragment.

Inhibition of neutrophil activation by fibrinogen.

Physiological levels of human fibrinogen markedly inhibited the chemotactic activity of human neutrophils triggered by zymosan-activated serum (ZAS), C5a, or IL-8 in a Boyden chamber assay. Fibrinogen also slightly inhibited the N-formyl-methionyl leucyl-phenylalanine (FMLP)-induced migration of human neutrophils. Albumin was devoid of the inhibitory activities displayed by fibrinogen in this system. The inhibition of chemotaxis by fibrinogen was dose-dependent and saturable. Fibrinogen placed in the upper compartment of the Boyden chamber produced a larger inhibition than that obtained with fibrinogen placed in the lower compartment. Lysine as well as the lysine analog 6-aminohexanoic acid (AHA) decreased the inhibitory capacity of fibrinogen. In contrast, both arginine and glutamine failed to suppress the fibrinogen-mediated inhibition of neutrophil chemotaxis. AHA counteracts the inhibition of ZAS-induced chemotaxis by anti-CD18 monoclonal antibody, suggesting that lysine binding sites are required for integrin function in chemotaxis. Fibrinogen also inhibited, in a dose-dependent manner, the oxygen consumption of neutrophils activated by opsonized zymosan. Taken together, the present results indicate that fibrinogen modulates neutrophil functions and suggest that in addition to its role in blood coagulation, circulating fibrinogen may be involved in regulation of the inflammatory response.

Study of tissue‐type plasminogen activator binding sites on fibrin using distinct fragments of fibrinogen

It is well established that tissue-type plasminogen activator (t-PA) binds to the D region of fibrin(ogen) and that two distinct CNBr fragments of fibrinogen (FCB), FCB-2 and FCB-5, comprising parts of this region, stimulate plasminogen activation by t-PA. In the present work, ligand-binding studies were performed to characterize the interactions between t-PA and the corresponding fibrin regions using a well defined model of a fibrin surface and both FCB-2 and FCB-5 in liquid and solid phase. Binding isotherms showed a characteristic Langmuir adsorption saturation profile. The dissociation constants determined for the binding of t-PA to immobilized FCB-2 (Kd = 0.70 +/- 0.10 nM) and FCB-5 (Kd = 0.47 +/- 0.08 nM) were of the same order of magnitude as the Kd for fibrin binding (Kd = 1 +/- 0.2 nM). The specificity of the binding was demonstrated by the ability of soluble FCB-2 and FCB-5 to inhibit t-PA binding to solid-phase fibrin (Ki = 3.3 microM and 6.4 microM, respectively). The binding of t-PA to fibrin and to immobilized FCB-2 was partially inhibited by the lysine analogue 6-aminohexanoic acid (Ki = 123 +/- 47 microM and 364 microM, respectively) but was not modified by carboxypeptidase B, thus indicating involvement of internal lysine residues. Removal of lysine residues by treatment with, successively, plasmin and carboxypeptidase B, produced only a partial inhibition of t-PA binding, thus confirming the existence of both a lysine-dependent and a lysine-independent mechanism of binding of t-PA to both fibrin and FCB-2. In contrast, the binding of t-PA to FCB-5 was not significantly affected by 6-aminohexanoic acid. Altogether, these data indicate that the mechanism of binding of t-PA to fibrin involves mainly a lysine-independent interaction with the D region which is contributed by sequences present in FCB-5 and FCB-2; contribution to binding by a lysine-dependent interaction was detected only in FCB-2 and is probably of minor relevance as suggested by the limited effect of 6-aminohexanoic acid.

Potentiation of plasminogen activation by an anti-urokinase monoclonal antibody due to ternary complex formation. A mechanistic model for receptor-mediated plasminogen activation.

We have observed that a murine IgG1 monoclonal antibody directed against human urokinase-type plasminogen activator (uPA) greatly potentiates pro-uPA-mediated plasminogen activation. This effect was dependent on the interaction between the immunoglobulin and the kringle domain of pro-uPA and could be competed efficiently by kringle-containing proteolytic fragments of uPA. In addition, the potentiation could also be competed by the lysine analog 6-aminohexanoic acid, an antagonist of plasminogen binding. This unexpected plasminogen binding dependence was found to be due to a carboxyl-terminal lysine residue on the immunoglobulin gamma chain, which by analogy with other proteins represents a potential binding site for plasminogen. Removal of this residue with carboxypeptidase B resulted in a complete abolition of the potentiation. It appears therefore that the potentiatory effect involves a novel mechanism with the antibody acting to provide a specific template for the assembly of a ternary complex involving pro-uPA/uPA and plasminogen, enabling them to interact in a catalytically favorable manner. This interpretation was confirmed by studying the kinetics of plasminogen activation by the complex between active, two-chain uPA and the antibody, which resulted in an overall 50-fold increase in reaction efficiency (kcat/Km), primarily due to a reduction in Km from 20 to 0.1 microM. Pro-uPA activation by plasmin was also accelerated, although to a lesser extent. The potentiation due to complex formation also provides a mechanism for the initiation of this system, dependent only on the low intrinsic proteolytic activity of the zymogen forms. The effects observed here, mediated by ternary complex formation, simulate the effects we have previously observed on assembly of the uPA receptor-mediated cellular plasminogen activation system and may therefore represent a mechanistic model for both its activity and initiation.

One-step purification of tissue-type plasminogen activator using affinity chromatography with a special monoclonal antibody under mild conditions

We have previously isolated a monoclonal antibody, designated as 1-3-1, specific for tissue-type plasminogen activator (t-PA). We have shown that t-PA dissociates from 1-3-1 in the presence of the lysine analogue 6-aminohexanoic acid (6-AHA). Here we describe a method for the one-step immunoaffinity purification of t-PA from conditioned melanoma cell medium, using 1-3-1 immobilised on Sepharose under mild elution conditions, favourable for t-PA. The yield of t-PA (antigen or total protein) from a 1-3-1-Sepharose column, when eluted using a buffer supplemented with 0.2 M 6-AHA at neutral pH, was as effective as other buffers that involve a strong pH-change, i.e., pH 2-3. However, the enzymatic activity of the t-PA purified with 6-AHA was 25 to 30% higher, as compared with t-PA eluted using a pH change. This resulted in a markedly higher specific activity of t-PA purified with 0.2 M 6-AHA, as compared with t-PA purified using a strong pH-change. The purity of t-PA, purified using the present method, was very high, as determined by gel electrophoresis. An additional advantage of the present procedure is that the mild elution conditions prolong the column life.

Stimulation of plasmin activity by oleic acid.

The amidolytic activity of plasmin with the chromogenic substrate H-D-valyl-L-leucyl-L-lysine p-nitroanilide (S-2251) is stimulated by oleic acid in a dose-dependent and saturable fashion. The activity of plasmin on S-2251 in the presence of oleic acid followed a sigmoidal kinetic pattern, with an almost 4-fold stimulation of activity at 60 microM-oleic acid. Half-maximal stimulation occurred at an oleic acid level of 19.5 microM. The amino acid analogue 6-aminohexanoic acid (AHA), which is known to bind to lysine-binding sites in plasmin, suppressed the stimulatory effect of oleic acid in a concentration-dependent manner; at 0.3 mM-AHA, about 70% of the oleic acid-dependent enhancement of plasmin activity was abolished. The l/v versus 1/[S] plot for plasmin changed in the presence of oleic acid from a linear to a non-linear curve, suggesting positive co-operativity. 14C-labelled oleic acid bound to plasmin, and the bound ligand was displaced by an excess of unlabelled oleic acid. Oleic acid also produced a marked (40-fold) stimulation of the plasminogen-dependent cleavage of S-2251 by urokinase. A half-maximal effect on plasminogen activation was obtained at 40 microM-oleic acid. The present findings suggest that the ability of oleic acid to stimulate plasmin activity and to enhance the conversion of plasminogen to plasmin depends on the interaction of oleic acid with specific lysine-binding sites in plasmin.

Characterization of the binding of plasminogen to fibrin surfaces: The role of carboxy-terminal lysines

In the present study we have quantitatively characterized the interaction of purified human Glu- and Lys-plasminogen with intact and degraded fibrin by ligand-binding experiments using a radioisotopic dilution method and antibodies against human plasminogen. A fibrinogen monolayer was covalently linked to a solid support with polyglutaraldehyde and was treated with thrombin or with thrombin and then plasmin to respectively obtain intact and degraded fibrin surfaces. Under these conditions, a well-defined surface of fibrin is obtained (410 +/- 4 fmol/cm2) and, except for a 39-kDa fragment, most of the fibrin degradation products remain bound to the support. New binding sites for plasminogen were detected on the degraded surface of fibrin. These sites were identified as carboxy-terminal lysine residues both by inhibition of the binding by the lysine analogue 6-aminohexanoic acid and by carboxy-terminal end-group digestion with carboxypeptidase B. The binding curves exhibited a characteristic Langmuir adsorption isotherm saturation profile. The data were therefore analyzed accordingly, assuming a single-site binding model to simplify the analysis. Equilibrium dissociation constants (Kd) and the maximum number of binding sites (Bmax) were derived from linearized expression of the Langmuir isotherm equation. The Kd for the binding of Glu-plasminogen to intact fibrin was 0.99 +/- 0.17 microM and for degraded fibrin was 0.66 +/- 0.22 microM. The Kd for the binding of Lys-plasminogen to intact fibrin was 0.41 +/- 0.22 microM and for degraded fibrin was 0.51 +/- 0.12 microM.(ABSTRACT TRUNCATED AT 250 WORDS)