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.     

Conversion of Glu-plasminogen to Lys-plasminogen is necessary for optimal stimulation of plasminogen activation on the endothelial cell surface

When Glu-plasminogen is bound to cells, plasmin (Pm) formation by plasminogen (Pg) activators is markedly enhanced compared with the reaction in solution. It is not known whether the direct activation of Glu-Pg by Pg activators is promoted on the cell surface or whether plasminolytic conversion of Glu-Pg to the more readily activated Lys-Pg is necessary for enhanced Pm formation on the cell surface. To distinguish between these potential mechanisms, we tested whether Pm formation on the cell surface could be stimulated in the absence of conversion of Glu-Pg to Lys-Pg. Rates of activation of Glu-Pg, Lys-Pg, and a mutant Glu-Pg, [D646E]Glu-Pg, by either tissue Pg activator (t-PA) or urokinase (u-PA) were compared when these Pg forms were either bound to human umbilical vein endothelial cells (HUVEC) or in solution. ([D646E]Glu-Pg can be cleaved at the Arg(561)-Val(562) bond by Pg activators but does not possess Pm activity subsequent to this cleavage because of the mutation of Asp(646) of the serine protease catalytic triad.) Glu-Pg activation by t-PA was enhanced on HUVEC compared with the solution phase by 13-fold. In contrast, much less enhancement of Pg activation was observed with [D646E]Glu-Pg ( approximately 2-fold). Although the extent of activation of Lys-Pg on cells was similar to that of Glu-Pg, the cells afforded minimal enhancement of Lys-Pg activation compared with the solution phase (1.3-fold). Similar results were obtained when u-PA was used as activator. When Glu-Pg was bound to the cell in the presence of either t-PA or u-PA, conversion to Lys-Pg was observed, but conversion of ([D646E]Glu-Pg to ([D646E]Lys-Pg was not detected, consistent with the conversion of Glu-Pg to Lys-Pg being necessary for optimal enhancement of Pg activation on cell surfaces. Furthermore, we found that conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was markedly enhanced ( approximately 20-fold) on the HUVEC surface, suggesting that the stimulation of the conversion of Glu-Pg to Lys-Pg is a key mechanism by which cells enhance Pg activation.     

Binding and activation of plasminogen on immobilized immunoglobulin G. Identification of the plasmin-derived Fab as the plasminogen-binding fragment

We have found that tissue plasminogen activator catalyzes the binding of plasminogen (Pg) to immunoglobulin G (IgG) immobilized on a surface. This enhancement is due to the formation of plasmin, since plasmin treatment of immobilized IgG produced a 20-fold increase in Pg binding. Pg binding is lysine site dependent and reversible. The augmentation of Pg binding by plasmin is specific as other proteases produced significantly less or no effect. Immobilized plasmin-treated IgG also specifically binds Pg in plasma. IgG-immobilized Pg is activated by tissue plasminogen activator, and a significant portion of the plasmin formed remains bound to the IgG. The Pg reactive species in a plasmin-treated IgG digest was identified as the Fab fragment by chromatography utilizing the immobilized high affinity lysine-binding site of plasminogen. Specificity of the interaction was further demonstrated by immunoblot-ligand analysis which demonstrated that the plasmin-derived Fab fragment bound Pg whereas papain-derived Fab or plasmin-derived Fc fragments did not. These data suggest that Pg binds to the new COOH-terminal lysine residue of the plasmin-derived Fab. Pg also binds to an immobilized immune complex following plasmin treatment. These findings indicate that surface-bound IgG localizes plasminogen thus extending the spectrum of activity of the plasmin system to immunologic reactions.     

Role of lysine binding sites in activation of plasminogen by streptokinase

The function of lysine-binding sites in kringle domains K1-4 and K5 of plasminogen (Pg) during its activation by streptokinase (SK) was studied. Activation rates of Glu- and Lys-Pg exceed activation rate of mini- and micro-Pg 26 and 40 times, respectively. 6-Animohexanoic acid (6-AHA) in concentrations from 10(-5) to 10(-2) M inhibits activation of Glu-, Lys- and mini-Pg and does not impact the activation of micro-Pg. Complete inhibition of Lys-Pg activation occurs with presence of 10(-3) M 6-AHA while 90% inhibition of mini-Pg activation and 70% inhibition of Glu-Pg activation occur with 10(-2) M 6-AHA. Isolated kringles K1-3 and K4 of Pg inhibit activation of Glu-Pg by SK and concentrations [I]50 are 4.0 and 8.1 x 10(-6) M, respectively. Catalytic activity of Glu-Pg-SK, Lys-Pg-SK and Pm-SK complexes with respect to S 2251 is not inhibited by 6-AHA in concentrations from 10(-5) to 10(-2) M. Activation of substrate Pg by Pm-SK complex is also inhibited by 6-AHA in concentrations from 10(-5) to 10(-2) M; however, this effect of inhibition is significantly weaker than that with activation by SK. Cleavage of C-terminal Lys or chemical modification of NH2-groups of amino acid residues in SK molecule also results in the decrease of the Glu-Pg activation rate. Lysin-binding sites in K1-4 and K5 of Pg molecule are important at different steps of Pg activation process which includes formation of equimolar complex; structural reorganizations resulted in formation of active center in Pg; and binding of substrate Pg with Pg-SK complex. Lysin-binding sites in K1-4 of Pg are necessary for maintenance of high rate of Pg activation by SK.     

Kinetics of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine residues of fibrin degradation products and removal of plasminogen-binding sites

Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the k(cat) and K(m) of Glu(1)-plasminogen (Glu-Pg)-binding site removal are 2.34 s(-1) and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 μm(-1) s(-1). The corresponding values of Lys(77)/Lys(78)-plasminogen (Lys-Pg)-binding site removal are 0.89 s(-1) and 96 nm implying a catalytic efficiency of 9.23 μm(-1) s(-1). These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 μm(-1) s(-1). Interestingly, this value increases to 3.85 μm(-1) s(-1) in the presence of Glu-Pg. These changes are due to a decrease in K(m). This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.     

The role of carbohydrate side chains of plasminogen in its activation by staphylokinase

Kinetic parameters (k(Pg) and K(Pg)) were determined for activation of Glu-plasminogen (Glu-Pg) and Lys-plasminogen (Lys-Pg) type I (with N-linked carbohydrate chain at Asn-289) and type II (with unsubstituted Asn-289) by plasmin-staphylokinase (Pm-STA) complex. The K(Pg) values for Glu-Pg I and Lys-Pg I (17.1 and 11.2 microM, respectively) were higher than those for Glu-Pg II and Lys-Pg II (14.9 and 5.4 microM, respectively), while only minor differences in the k(Pg) values were observed between plasminogens type I and type II. Soluble fibrin significantly increased the k(Pg)/K(Pg) values for activation of all four plasminogens due to a decrease in the K(Pg) values but did not alter the k(Pg) values. However, the activation of plasminogens type I was stimulated by fibrin lesser degree than that of plasminogens type II. These findings indicate that N-glycosylation of kringle 3 of plasminogen decreases the stability of Pm-STA-Pg ternary enzyme-substrate complex in solution as well as interferes with its formation and rearrangement on the fibrin surface.     

Mechanism of inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5--a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis--on kinetic parameters (k(Pg) and K(Pg)) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect the k(Pg) value, but increases the value K(Pg) urokinase plasminogen activation. A decrease in the k(Pg) value and an increase in the K(Pg) value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA by mixed type. Such an influence ofangiostatin on the kinetic constants ofthe urokinase plasminogen activation suggests that angiostatin dose dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex that leads to a decrease in k(Pg) and an increase in K(Pg) of plasminogen activation. Inhibition constants by angioststin (Ki) of plasminogen-activator activities of uPA and tPA determined by Dixon method were found to be 0.59 +/- 0.04 and 0.12 +/- 0.05 microM, respectively.     

Features of plasminogen activation in a complex with antiplasminogen monoclonal antibody IV-1C

Antiplasminogen monoclonal antibody IV-1c (IV-1c) with antigenic determinant in V709-G718 site of plasminogen (Pg) protease domain (Druzhina N.N. et al. 1996.) can induce catalytic activity in Pg moiety of the complex. Catalytic activity appeared in Pg-IV-1c complex after approximately 2 h lag-period. Rate of Lys-Pg activation was higher then that of Glu-Pg. Amidolytic activity of Pg-SK equimolar complex was completely inhibited by IV-1c at 2:1 = Pg:IV-1c molar ratio. At constant Glu-Pg concentration increasing of the IV-1c concentration to equimolar of Pg accelerated Pg activation. Subsequent increase of IV-1c concentration inhibited the Pg activation sharply. Increasing of Glu-Pg concentration at constant IV-1c one did not inhibit Glu-Pg activation in Pg-IV-1c complex. The rate dependence of Pg activation from Glu-Pg-IV-1c complex concentration curve had bell-shaped form with maximum at 500 nM. Electrophoretic analysis of components of Glu-Pg-IV-1c complex showed that Lys-Pg and Lys-Pm were not observed at 100 nM complex concentration for 6 h period of reaction. At 680 nM concentration Glu-Pg-IV-1c complex these forms appeared in initial moments of reaction activation after lag-period. Kinetic scheme and peculiarities of Pg activation reaction in Pg-IV-1c complex are discussed.     

Mechanism of action of θ-amino acids on plasminogen activation and fibrinolysis induced by staphylokinase

Stimulation of Lys-plasminogen (Lys-Pg) and Glu-plasminogen (Glu-Pg) activation under the action of staphylokinase and Glu-Pg activation under the action of preformed plasmin-staphylokinase activator complex (Pm-STA) by low concentrations and inhibition by high concentrations of omega-amino acids (>90-140 mM) were found. Maximal stimulation of the activation was observed at concentrations of L-lysine, 6-aminohexanoic acid (6-AHA), and trans-(4-aminomethyl)cyclohexanecarboxylic acid 8.0, 2.0, and 0.8 mM, respectively. In contrast, the Lys-Pg activation rate by Pm-STA complex sharply decreased when concentrations of omega-amino acids exceeded the above-mentioned values. It was found that formation of Pm-STA complex from a mixture of equimolar concentrations of staphylokinase and Glu-Pg or Lys-Pg is stimulated by low concentrations (maximal at 10 mM) of 6-AHA. Negligible increase in the specific activities of plasmin and Pm-STA complex was detected at higher concentrations of 6-AHA (to maximal at 70 and 50 mM, respectively). Inhibitory effects of omega-amino acids on the rate of fibrinolysis induced by staphylokinase, Pm-STA complex, and plasmin were compared. It was found that inhibition of staphylokinase-induced fibrinolysis by omega-amino acids includes blocking of the reactions of Pm-STA complex formation, plasminogen activation by this complex, and lysis of fibrin by forming plasmin as a result of displacement of plasminogen and plasmin from the fibrin surface. Thus, the slow stage of Pm-STA complex formation plays an important role in the mechanism of action of omega-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to alpha-->beta change of Glu-Pg conformation, stimulation of Pm-STA complex formation leads to increase in Glu-Pg activation rate in the presence of low concentrations of omega-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by omega-amino acids is responsible for appearance of long lag phases on curves of fibrinolysis induced by staphylokinase.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: A key event in melanoma cell invasiveness in vitro

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on Me Wo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Catalytic properties of Glu-plasminogen in a complex with monoclonal antibody IV-1c

Earlier it was shown that anti-plasminogen monoclonal antibody IV-1c was able to induce a catalytic activity in plasminogen. IV-1c activates plasminogen by binding to plasminogen protease domain with antigen binding site and to lysine-binding sites by C-terminal lysines of gamma-chains. The effect of plasminogen and IV-1c concentration on rate of catalytic activity induce in Pg-IV-1c complex has been investigated. It was found that IV-1c inhibited an activation reaction at concentrations higher of equimolar to Glu-Pg. Glu-Pg did not inhibit reaction of activation in higher to IV-1c concentrations. Role of IV-1c gamma-chain C-terminal lysine concentration in Pg activation is discussed.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Thrombin-activable fibrinolysis inhibitor attenuates (DD)E-mediated stimulation of plasminogen activation by reducing the affinity of (DD)E for tissue plasminogen activator. A potential mechanism for enhancing the fibrin specificity of tissue plasminogen activator

A complex of d-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k(cat) and increasing K(m) for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and d-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.     

alpha Domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator

The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.     

Identification and characterization of novel tPA- and plasminogen-binding sites within fibrin(ogen) alpha C-domains

Molecular defects in the alphaC-domains of some abnormal fibrinogens have been associated with impaired fibrin-mediated activation of plasminogen (Pg) by its activator tPA, suggesting the involvement of these domains in fibrinolysis. To test this suggestion, we expressed in E. coli the alphaC-fragment (residues Aalpha221-610) corresponding to the entire alphaC-domain as well as its NH(2)- and COOH-terminal halves (residues Aalpha221-391 and Aalpha392-610) and tested their effects on activation of Pg and their interaction with Pg and tPA. When the activation was monitored by cleavage of a chromogenic substrate with newly formed plasmin, the reaction was much more efficient in the presence of the alphaC-fragment. This stimulation was abolished upon digestion of the alphaC-fragment with plasmin. In surface plasmon resonance experiments, both tPA and Pg bound to the immobilized alphaC-fragment with K(d)s of 33 and 32 nM, respectively. Similar results were obtained by ELISA. This binding occurred via independent sites since saturating amounts of Pg did not prevent binding of tPA and vice versa. Both sites were localized in the COOH-terminal half of the alphaC-domain since the Aalpha392-610 fragment bound both tPA and Pg and was an effective stimulator whereas Aalpha221-391 was inactive. These results indicate that the fibrinogen alphaC-domains contain novel high-affinity tPA- and Pg-binding sites that play an important role in the regulation of fibrinolysis.     

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.     

Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride

The urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) are very similar serine proteases with the same physiological function, the activation of plasminogen. An increased amount or activity of uPA but not tPA has been detected in human cancers. The PAs are weak proteolytic enzymes, but they activate plasminogen to plasmin, a strong proteolytic enzyme largely responsible for the malignant properties of cancers. It has been shown recently that the administration of uPA inhibitors can reduce tumor size. Inhibitors of uPA could therefore be used as anti-cancer and anti-angiogenesis agents. It has been found that amiloride competitively inhibits the catalytic activity of uPA but not tPA. Modification of this chemical could therefore produce a new class of uPA specific inhibitors and a new class of anti-cancer agents. The X-ray structure of the uPA complex with amiloride is not known. There are structural differences in the specificity pocket of uPA and tPA. However, the potential energy of binding amiloride is lower outside this cavity in the case of tPA. A region responsible for binding amiloride to tPA has been proposed as the loop B93-B101, reached in negatively charged amino acids present in tPA but not uPA.     

Epsilon amino caproic acid inhibits streptokinase-plasminogen activator complex formation and substrate binding through kringle-dependent mechanisms

Lysine side chains induce conformational changes in plasminogen (Pg) that regulate the process of fibrinolysis or blood clot dissolution. A lysine side-chain mimic, epsilon amino caproic acid (EACA), enhances the activation of Pg by urinary-type and tissue-type Pg activators but inhibits Pg activation induced by streptokinase (SK). Our studies of the mechanism of this inhibition revealed that EACA (IC(50) 10 microM) also potently blocked amidolytic activity by SK and Pg at doses nearly 10000-fold lower than that required to inhibit the amidolytic activity of plasmin. Different Pg fragments were used to assess the role of the kringles in mediating the inhibitory effects of EACA: mini-Pg which lacks kringles 1-4 of Glu-Pg and micro-Pg which lacks all kringles and contains only the catalytic domain. SK bound with similar affinities to Glu-Pg (K(A) = 2.3 x 10(9) M(-1)) and to mini-Pg (K(A) = 3.8 x 10(9) M(-)(1)) but with significantly lower affinity to micro-Pg (K(A) = 6 x 10(7) M(-)(1)). EACA potently inhibited the binding of Glu-Pg to SK (K(i) = 5.7 microM), but was less potent (K(i) = 81.1 microM) for inhibiting the binding of mini-Pg to SK and had no significant inhibitory effects on the binding of micro-Pg and SK. In assays simulating substrate binding, EACA also potently inhibited the binding of Glu-Pg to the SK-Glu-Pg activator complex, but had negligible effects on micro-Pg binding. Taken together, these studies indicate that EACA inhibits Pg activation by blocking activator complex formation and substrate binding, through a kringle-dependent mechanism. Thus, in addition to interactions between SK and the protease domain, interactions between SK and the kringle domain(s) play a key role in Pg activation.     

Mechanism of the inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5, a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis, on kinetic parameters (k _Pg and K _Pg) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect on the k _Pg value, but increases the value of K _Pg plasminogen activation by urokinase. A decrease in the k _Pg value and an increase in the K _Pg value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is a competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA with a mixed type. Such an influence of angiostatin on the kinetic constants of the plasminogen activation by urokinase suggests that angiostatin dose-dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In the case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with the formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex, which leads to a decrease in k _Pg and an increase in K _Pg of the plasminogen activation. Inhibition constants by angiostatin (K _i) of plasminogen-activator activities of uPA and tPA determined by the Dixon method were found to be 0.59 ± 0.04 and 0.12 ± 0.05 μM, respectively.     

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

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