Regulation of plasminogen activation by components of the extracellular matrix

The kinetics of activation of Glu-plasminogen (Glu-Pg) and Lys77-Pg by two-chain recombinant tissue plasminogen activator (t-PA) were determined in the presence of isolated protein components of the extracellular matrix (ECM) and compared to activation in the presence of fibrinogen and fibrinogen fragments and in the absence of added protein. Several ECM protein components were as effective as fibrinogen fragments at stimulating Pg activation. Stimulation of Glu-Pg activation resulted from both a decrease in Km and an increase in Vmax, whereas stimulation of Lys77-Pg was due primarily to increases in Vmax. The most effective stimulators of activation were basement membrane type IV collagen and gelatin which resulted in a 21- and 55-fold increase, respectively, in the kcat/Km of Glu-Pg (relative to a 10-fold increase observed with fibrinogen fragments). Amidolytic activity of t-PA was also enhanced up to 12-fold by ECM proteins. However, plasmin amidolytic activity was unaffected by the presence of added proteins. These data suggest that several ECM-associated proteins can enhanced the activation of Pg in the absence of fibrin.     

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.     

Proteolytic modulation of factor Xa–antithrombin complex enhances fibrinolysis in plasma

Our previous work showed that purified coagulation factor Xa (FXa) acquires fibrinolysis cofactor activity after plasmin-mediated cleavage. The predominant functional species is a non-covalent heterodimer of 33 and 13 kDa, termed Xa33/13, which has predicted newly exposed C-terminal lysines that are important for tissue plasminogen activator (tPA)-mediated plasminogen activation to plasmin. To provide evidence that this mechanism occurs in a physiological context, here we demonstrated the appearance of Xa33 in clotting plasma by western blot analysis. Since the normal fate of FXa is stable association with antithrombin (AT), an AT western blot was conducted, which revealed a band of ~ 13 kDa higher apparent molecular weight than AT that appeared concurrent to Xa33. Sequencing of purified proteins confirmed the generation of Xa13 covalently bound to AT and Xa33 (Xa33/13-AT) by cleavages at Lys–Met339 and Lys–Asp389. Ligand blots demonstrated ¹²⁵I-plasminogen binding to the Xa33 subunit of plasmin-generated Xa33/13-AT. Purified XaAT added to plasma that was induced to clot enhanced the rate of tPA-mediated fibrinolysis by ~ 16-fold. Similarly, purified plasminogen activation by tPA was enhanced by ~ 16-fold by XaAT. Plasmin cleaves XaAT and exposes plasminogen binding sites at least 10-fold faster than FXa. Here we demonstrate a novel function for AT, which accelerates the modulation of FXa into the fibrinolytic form, Xa33/13. The consequent exposure of C-terminal lysine binding sites essential for plasminogen activation enhances fibrinolysis. These results are consistent with a model where auxiliary cofactors link coagulation to fibrinolysis by priming the accelerating role of fibrin.     

Enhanced fibrinolysis by proteolysed coagulation factor Xa

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaα) must be sequentially cleaved by plasmin or autoproteolysis, producing FXaβ and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaα/β or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaα/β to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaα active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaα/β or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaα/β or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaα undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.     

The effect of divalent cations on the conformation and function of human plasminogen

The activation of native human plasminogen (Glu1-Pg) by tissue plasminogen activator, urinary plasminogen activator (u-PA), and streptokinase is inhibited by the divalent cations Ca2+, Mg2+, and Mn2+. This inhibition is accompanied by a conformational change in the molecule as evidenced by a decrease in Stokes' radius and intrinsic fluorescence. Kinetic analysis indicates that Mn2+ acts as an uncompetitive inhibitor of u-PA-catalyzed Glu1-Pg activation. In contrast to the inhibitory effects of divalent cations on Glu1-Pg, Ca2+ and Mg2+ stimulate the activation of proteolytically modified Lys77-Pg. These observations provide further evidence that Glu1-Pg and Lys77-Pg exhibit differential responses to ligands in the microenvironment.     

Substrate specificity of tissue-type and urokinase-type plasminogen activators

Recent studies suggest that plasminogen activators not only hydrolyse a specific arginine-valine bond in plasminogen, but may also cleave other proteins such as fibronectin. We studied the substrate specificity, particularly the preference for arginyl over lysyl peptide bonds, of tissue-type plasminogen activator (t-PA) as well as of two-chain urokinase-type plasminogen activator (u-PA). The arginine/lysine preference was determined with three pairs of tripeptidyl-p-nitroanilide substrates having either arginine or lysine in the P1 position and varied from 5.2 to 14.1 for u-PA and from 55.6 to 99.8 for t-PA. It was concluded that both t-PA and u-PA preferred arginyl to lysyl peptide bonds. However, u-PA had a significantly lower arginine/lysine preference than t-PA, indicating that u-PA represents a less specific proteinase. This may point to functions of u-PA other than plasminogen activation, which involve cleavage of lysyl bonds.     

Kinetic analysis of the effects of heparin and lipoproteins on tissue plasminogen activator mediated plasminogen activation

Heparin sulfate and the less sulfated glycosaminoglycan heparan sulfate enhance human plasminogen (Pg) conversion to plasmin by tissue-type plasminogen activator (t-PA). Kinetic studies indicate that both heparin and heparan increase the kcat of t-PA-mediated Pg activation by 25- and 3.5-fold, respectively. The Km of plasmin formation is unaltered by the presence of either heparin or heparan. Both heparin and heparan stimulate the activity of t-PA by interacting with the finger domain of t-PA, with association constants of 1 microM and 200 nM, respectively. Additionally, the lipoproteins lipoprotein(a) [Lp(a)] and low-density lipoprotein (LDL) inhibit the heparin enhancement of Pg activation. Lp(a) is a competitive inhibitor and LDL is a mixed inhibitor of t-PA-mediated Pg activation, with inhibition constants of 30 and 70 nM, respectively. The inhibition constants correspond to physiologic concentrations of these lipoproteins. These data suggest that heparin, heparan, and lipoproteins may play an important in vivo role in regulating cell surface associated activation of the fibrinolytic system.     

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.     

Ca(2+) -induced binding of anticoagulation factor II from the venom of Agkistrodon acutus with factor IX

Anticoagulation factor II (ACF II), a coagulation factor X- binding protein from the venom of Agkistrodon acutus has both anticoagulant and hypotensive activities. Previous studies show that ACF II binds specifically with activated factor X (FXa) in a Ca(2+) -dependent manner and inhibits intrinsic coagulation pathway. In this study, the inhibition of extrinsic coagulation pathway by ACF II was measured in vivo by prothrombin time assay and the binding of ACF II to factor IX (FIX) was investigated by native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR). The results indicate that ACF II also inhibits extrinsic coagulation pathway, but does not inhibit thrombin activity. ACF II also binds with FIX with high binding affinity in a Ca(2+) -dependent manner and their maximal binding occurs at about 0.1 mM Ca(2+) . ACF II has similar binding affinity to FIX and FX as determined by SPR. Ca(2+) has a slight effect on the secondary structure of FIX as determined by circular dichroism spectroscopy. Ca(2+) ions are required to maintain in vivo function of FIX Gla domain for its recognition of ACF II. However, Ca(2+) at high concentrations (>0.1 mM) inhibits the binding of ACF II to FIX. Ca(2+) functions as a switch for the binding between ACF II and FIX. ACF II extends activated partial thromboplastin time more strongly than prothrombin time, suggesting that the binding of ACF II with FIX may play a dominant role in the anticoagulation of ACF II in vivo.     

Heparin oligosaccharides enhance tissue-type plasminogen activator: a correlation between oligosaccharide length and stimulation of plasminogen activation

The rate of plasminogen (Pg) activation by tissue-type Pg activator (t-PA) is enhanced by heparin-derived oligosaccharides. Kinetic analysis of the effects of heparin oligosaccharides, ranging in size from di- to dodecasaccharides, on Pg activation demonstrates that stimulation of the reaction is dependent on the size of the heparin oligosaccharides. Di- and tetrasaccharides enhance the activation through 2-fold increases in kcat and 4-fold decreases in Km. Hexasaccharide and larger oligosaccharides stimulate the reaction by increasing the kcat by as much as 4-fold, but do not affect the Km. Previous experiments have shown that lipoprotein(a) [Lp(a)] inhibits Pg activation by t-PA, but only in the presence of a template which enhances t-PA activity such as fibrinogen fragments or intact heparin. Similiarly, Lp(a) inhibits the enhancement of t-PA activity by the larger heparin oligosaccharides but has no effect on t-PA activity in the presence of di- and tetrasaccharides. The results of this study when considered with our previous observations (Edelberg & Pizzo, 1990) suggest that the enhancement in Pg activation by the smaller oligosaccharides is mediated exclusively via binding to t-PA while the larger oligosaccharides may interact with both t-PA and Pg. Furthermore, studies of Pg activation in the presence of both heparin oligosaccharides and fibrinogen fragments demonstrate that t-PA is stimulated preferentially by fibrinogen fragments.     

Mechanism of factor Va inactivation by plasmin. Loss of A2 and A3 domains from a Ca2+-dependent complex of fragments bound to phospholipid

The coagulation cofactor Va (FVa) is a noncovalent heterodimer consisting of a heavy chain (FVaH) and a light chain (FVaL). Previously, the fibrinolytic effector plasmin (Pn) has been shown to inhibit FVa function. To understand this mechanism, the fragmentation profile of human FVa by Pn and the noncovalent association of the derived fragments were determined in the presence of Ca(2+) using anionic phospholipid (aPL)-coated microtiter wells and large (1 microm) aPL micelles as affinity matrices. Following Pn inactivation of aPL-bound FVa, a total of 16 fragments were observed and their NH(2) termini sequenced. These had apparent molecular weights and starting residues as follows (single letter abbreviation is used): 50(L1766), 48(L1766), 43(Q1828), 40(Q1828), 30(S1546), 12(T1657), and 7(S1546) kDa from FVaL; and 65(A1), 50(A1), 45(A1), 34(S349), 30(L94), 30(M110), and 3 small <5(W457, W457, and K365) kDa from FVaH. Of these, 50(L1766), 48(1766), 43(Q1828), and 40(Q1828) spanning the C1/C2 domains, and 30(L94), but not the similar 30(M110), positioned within the A1 domain remained associated with aPL. These were detected antigenically during Pn- or tissue plasminogen activator-mediated lysis of fibrin clot formed in plasma. Chelation by EDTA dissociated the 30(L94)-kDa fragment, which was observed to associate with intact FVaL upon recalcification, indicating that the Leu-94 to Lys-109 region of the A1 domain plays a critical role in the FVaL and FVaH Ca(2+)-dependent association. By using domain-specific monoclonal antibodies and an assay for thrombin generation, loss of FVa prothrombinase function was coincident with proteolysis at sites in the A2 and A3 domains resulting in their dissociation. Inactivation of FV or FVa by Pn was independent of the thrombophilic R506Q mutation. These results identify the molecular composition of Pn-cleaved FVa that remains bound to membrane as largely A1-C1/C2 in the presence of Ca(2+) and suggest that Pn inhibits FVa by a process involving A2 and A3 domain dissociation.     

Effects of human fibrinogen and its cleavage products on activation of human plasminogen by streptokinase

The influence of human fibrinogen (Fg) and its terminal plasminolytic digestion products, fragment D and fragment E, on the kinetics of activation of human plasminogen (Pg) by catalytic levels of streptokinase (SK) has been investigated. Both Fg and fragment D enhanced the rates of activation of human Glu1-Pg, Lys77-Pg, and Val442-Pg. Fragment E was refractive in this regard. In the case of Glu1-Pg, the Km for activation by SK, 0.4 microM, was not affected by the presence of Fg or fragment D. The kcat for this same reaction, 0.12 s-1, was elevated to 0.3 s-1 at saturating levels of these effector molecules. On the other hand, the Km for activation of Lys77-Pg, 0.5 microM, was decreased to 0.09 microM, whereas the kcat, 0.33 s-1, was not altered in the presence of saturating concentrations of Fg or fragment D. In the case of Val442-Pg, the Km for this same activation, 2.0 microM, was lowered to 0.4 microM and 0.25 microM in the presence of Fg and fragment D, respectively. The kcat for this process, 1.0 s-1, was unchanged in the presence of these agents. The concentrations of Fg (KFg) and fragment D (KFD) that led to half-maximal stimulation of the activation rates were determined. For Fg with Glu1-Pg, Lys77-Pg, and Val442-Pg, the KFg values were 0.08 microM, 0.14 microM, and 0.17 microM, respectively. The KFD values for these same plasminogens were 0.25 microM, 2.0 microM, and 1.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmin converts factor X from coagulation zymogen to fibrinolysis cofactor

Known anticoagulant pathways have been shown to exclusively inhibit blood coagulation cofactors and enzymes. In the current work, we first investigated the possibility of a novel anticoagulant mechanism that functions at the level of zymogen inactivation. Utilizing both clotting and chromogenic assays, the fibrinolysis protease plasmin was found to irreversibly inhibit the pivotal function of factor X (FX) in coagulation. This was due to cleavage at several sites, the location of which were altered by association of FX with procoagulant phospholipid (proPL). The final products were approximately 28 and approximately 47 kDa for proPL-bound and unbound FX, respectively, which did not have analogues when activated FX (FXa) was cleaved instead. We next investigated whether the FX derivatives could interact with the plasmin precursor plasminogen, and we found that plasmin exposed a binding site only on proPL-bound FX. The highest apparent affinity was for the 28-kDa fragment, which was identified as the light subunit disulfide linked to a small fragment of the heavy subunit (Met-296 to approximately Lys-330). After cleavage by plasmin, proPL-bound FX furthermore was observed to accelerate plasmin generation by tissue plasminogen activator. Thus, a feedback mechanism localized by proPL is suggested in which plasmin simultaneously inhibits FX clotting function and converts proPL-bound FX into a fibrinolysis cofactor. These data also provide the first evidence for an anticoagulant mechanism aimed directly at the zymogen FX.     

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.     

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.     

Interactions of plasminogen and tissue plasminogen activator (t-PA) with amphoterin. Enhancement of t-PA-catalyzed plasminogen activation by amphoterin

The heparin-binding p30 protein amphoterin is proposed to mediate adhesive interactions of the advancing plasma membrane in migrating and differentiating cells. Since the NH2-terminal part of amphoterin is exceptionally rich in lysine residues, we have studied its interactions with plasminogen and tissue plasminogen activator (t-PA). On immunostaining of N18 neuroblastoma cells, amphoterin and t-PA showed a close co-localization in the filopodia of the leading membrane and in the substrate-attached material. In purified systems, both t-PA and plasminogen bound to immobilized amphoterin, and their binding was inhibited by the lysine analogue epsilon-aminocaproic acid. Plasminogen bound to immobilized amphoterin was activated by t-PA, and this resulted in effective degradation of the immobilized amphoterin. Correspondingly, amphoterin-bound t-PA activated plasminogen. In solution amphoterin accelerated t-PA-catalyzed plasminogen activation maximally 46-fold. The results indicate that t-PA and plasminogen form through their lysine-binding sites a complex with amphoterin, which results in acceleration of plasminogen activation and effective degradation of amphoterin. We suggest that local acceleration of t-PA-catalyzed plasminogen activation by amphoterin at the leading membrane enhances the penetration of growing cytoplasmic processes through extracellular materials during cell migration, differentiation and regeneration. The amphoterin-mediated adhesion at the leading membrane may be transient in nature, because the protein also enhances its own breakdown by accelerating t-PA-catalyzed plasminogen activation.     

The elusive role of the potential factor X cation-binding exosite-1 in substrate and inhibitor interactions

A number of studies suggest that blood-clotting factor X (FX) uses secondary site(s) to interact (as a substrate) with its activators. Numerous pieces of evidence also imply that, within prothrombinase (as an enzyme), activated FX (FXa) uses exosite(s) for cofactor Va and/or prothrombin recognition. Similarly, FXa exosite(s) seem to govern interaction with inhibitors. An obvious difference between FXa and thrombin resides within a region called exosite-1: positively charged in thrombin and clearly of opposite polarity in FXa. To investigate the role of this potential cation-binding exosite, we prepared a series of mutants within loops 34-40 and 70-80 of FX. Overall, the mutations induced relatively subtle, non-synergistic modulation. The potential exosite was dispensable for FX activation and is unlikely to constitute a critical region for factor Va binding, albeit it is clearly important for prothrombin activation. Our data also implicate loop 34-40 of FXa in the interaction with the tissue factor pathway inhibitor, in prevention of plasminogen activator inhibitor-1 binding, and in tempering inhibition by heparin-activated antithrombin. Compared with FX, mutants with reduced electrostatic potential potentiated thrombin production in FX-depleted plasma, whereas mutants with inverted electrostatic potential impeded clotting. Despite the definite consequences observed, disruption of the potential cation-binding exosite of FX had rather weak effects, far from what would be expected if this region was as crucial as in thrombin.     

Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis

Modification of glutamic and aspartic acid residues of tissue-type plasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide leads to a decrease in affinity for lysine and fibrin, to a decrease of plasminogen activation activity in the presence of a fibrin mimic, but leaves amidolytic activity and plasminogen activation without fibrin mimic unaffected. Experiments with kringle-2 ligands and a deletion mutant of t-PA (K2P) suggests that glutamic or aspartic acid residues in K2 of t-PA are involved in stimulation of activity, lysine binding and fibrin binding. Mutant t-PA molecules were constructed by site-directed mutagenesis in which one or two of the five aspartic or glutamic acid residues in K2 were changed to asparagine or glutamine respectively. Mutation of Asp236 and/or Asp238 leads to t-PA molecules with 3- to 4-fold lower specific activity in the presence of fibrin mimic and having no detectable affinity for lysine analogs. However, fibrin binding was not influenced. Mutation of Glu254 also leads to a 3- to 4-fold lower activity, but to a much smaller reduction of lysine or fibrin binding. Residues Asp236 and Asp238 are both essential for binding to lysine derivatives, while Glu254 might be involved but is not essential. Residues Asp236, Asp238 and Glu254 are all three involved in stimulation of activity. Remarkably, mutation of residues Asp236 and/or Asp238 appears not to influence fibrin binding of t-PA whereas that of Glu254 does.     

Plasminogen activation by antiplasminogen monoclonal antibody IV-1C. Properties and mechanism of reaction

In review the results of investigation of plasminogen(Pg) activation by antiplasminogen monoclonal antibody IV-1c have been presented. Antigenic determinant of IV-1c was localized in Val709-Gly718 site of Pg protease domain. IV-1c completely inhibited the Pg activation by streptokinase, but increased the rate of Pg activation by t-PA and urokinase. Catalytic properties of plasmin in complex with IV-1c were studied. It was found that IV-1c induced catalytic activity in Pg-IV-1c complex. It was shown that Pg and IV-1c interacts in complex by two-centre mechanism: IV-1c binds with Pg by paratope and by N-terminal lysine of gamma-chain and Pg binds to IV-1c by one of the lysine binding sites and by V709-G718 site of protease domain. The influence of pH, temperature, 1.5 mM Ca2+, Mg2+, Sr2+, Ba2+, Co2+, Ni2+ cations and 10 mM Cl-, F-, Ac-, SO4(2-), HPO4(2-) anions on lag and fast phases of Pg activation by VI-1c was investigated. It was revealed that Val709-Gly718 site was determining in Pg activation by IV-1c and streptokinase.     

Fibrinogen lysine residue A alpha 157 plays a crucial role in the fibrin-induced acceleration of plasminogen activation, catalyzed by tissue-type plasminogen activator

In previous studies, we have shown that the stretch 148-197 of the fibrinogen A alpha chain plays a crucial role in the acceleration of the tissue-type plasminogen activator (t-PA)-catalyzed plasminogen activation. In this study we have synthesized parts of A alpha 148-197 and analogues thereof. We found that the peptides with sequences identical with A alpha 148-161 and A alpha 149-161 of human fibrinogen accelerate the plasminogen activation by t-PA, whereas the corresponding peptides in which lysine residues A alpha 157 had been replaced by valine or arginine had no accelerating capacity. Furthermore, succinylation of the lysine residue(s) in the synthesized peptides A alpha 148-161 and A alpha 149-161 leads to loss of accelerating action. These findings show that lysine residue A alpha 157 is crucial for the accelerating action of fibrin on the t-PA-catalyzed plasminogen activation.