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)     

A transitional state of pro-urokinase that has a higher catalytic efficiency against glu-plasminogen than urokinase.

Plasminogen activation by single-chain urokinase-type plasminogen activator or pro-urokinase (pro-UK) is accompanied by the generation of two-chain urokinase (UK) by plasmin which provides a positive feedback. In the present study, the time course of the activation of Glu-plasminogen and of Lys-plasminogen (10 microM) by pro-UK (1.0 nM) was studied. In the presence of native plasminogen (Glu-plasminogen), three distinct phases with different rates of plasmin generation were observed. The initial phase was slow and corresponded to the intrinsic activity of pro-UK as reflected by the activity of a plasmin-resistant mutant (Lys158----Ala). This was followed by a second phase which had the most rapid rate. The third phase had a plasminogen activation rate which was significantly slower than the second and paralleled the rate of activation by UK (1.0 nM). The second phase coincided with the time at which there was only about 50% conversion of pro-UK to UK, whereas the final phase coincided with essentially complete conversion. In the presence of fibrin fragment E-2 (20 microM), previously shown to strongly promote plasminogen activation by pro-UK, the identical phenomenon was observed, but at one-tenth the concentration of pro-UK. The most rapid rate of plasmin generation again coincided with transitional (25-60%) pro-UK to UK conversion. To further examine this phenomenon, the rate of pro-UK to UK conversion was controlled by using kallikrein in the presence of a plasmin inhibitor. In this experiment, the activation of Glu-plasminogen bound to solid-phase fibrin was measured. A similar three-phase sequence was observed, the highest rate of plasmin generation coinciding with about 45% conversion of pro-UK to UK. A mechanism for this transitional state phenomenon was postulated based on the established significantly higher affinity of pro-UK than of UK for Glu-plasminogen. This exceptional property for a proenzyme may enable a transient activity to be generated during the transition from pro-UK to UK corresponding to the more favorable KM of pro-UK and the kcat of UK. This hypothesis was supported by the results from experiments in which Lys-plasminogen was substituted for the Glu form. No transitional state activity was observed, consistent with the relatively high KM of pro-UK against Lys-plasminogen.     

The AH-site of plasminogen and two C-terminal fragments. A weak lysine-binding site preferring ligands not carrying a free carboxylate function.

Glu-plasminogen [native plasminogen (Glu-1-Asn-790)], Lys-plasminogen [plasmin-cleaved fragment of plasminogen (Lys-77-Asn-790)] and miniplasminogen [fragment of plasminogen (Val-440-Asn-790)] were all found to interact specifically with immobilized 6-aminohexyl ligands. The interactions apparently are mediated by a single weak lysine-binding site, termed the AH-site, as seen from the patterns of inhibition obtained from frontal-quantitative-affinity-chromatography experiments with 6-aminohexanoic acid and alpha-N-acetyl-L-lysine methyl ester as competing ligands. The AH-site, in contrast with the strong lysine-binding site of Glu-plasminogen and Lys-plasminogen, may prefer ligands not carrying a free carboxylate function and therefore may interact with lysine side chains of proteins. In Glu-plasminogen the AH-site is present, but is apparently only partially free to react. It is suggested that it participates in an intramolecular complex and that an equilibrium state between two Glu-plasminogen forms exists. It is further suggested that binding of the plasminogens to fibrin is mainly determined by the AH-site.     

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

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

Prostromelysin-1 (proMMP-3) stimulates plasminogen activation by tissue-type plasminogen activator.

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically binds to tissue-type plasminogen activator (t-PA), without however, hydrolyzing the protein. Binding affinity to proMMP-3 is similar to single chain t-PA, two chain t-PA and active site mutagenized t-PA (Ka of 6.3 x 106 to 8.0 x 106 M-1), but is reduced for t-PA lacking the finger and growth factor domains (Ka of 2.0 x 106 M-1). Activation of native Glu-plasminogen by t-PA in the presence of proMMP-3 obeys Michaelis-Menten kinetics; at saturating concentrations of proMMP-3, the catalytic efficiency of two chain t-PA is enhanced 20-fold (kcat/Km of 7.9 x 10-3 vs. 4.1 x 10-4 microM-1.s-1). This is mainly the result of an enhanced affinity of t-PA for its substrate (Km of 1.6 microM vs. 89 microM in the absence of proMMP-3), whereas the kcat is less affected (kcat of 1.3 x 10-2 vs. 3.6 x 10-2 s-1). Activation of Lys-plasminogen by two chain t-PA is stimulated about 13-fold at a saturating concentration of proMMP-3, whereas that of miniplasminogen is virtually unaffected (1.4-fold). Plasminogen activation by single chain t-PA is stimulated about ninefold by proMMP-3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys-plasminogen to proMMP-3 with 18-fold higher affinity (Ka of 22 x 106 M-1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 x 106 M-1) as compared to Glu-plasminogen (Ka of 1.2 x 106 M-1). Plasminogen and t-PA appear to bind to different sites on proMMP-3. These data are compatible with a model in which both plasminogen and t-PA bind to proMMP-3, resulting in a cyclic ternary complex in which t-PA has an enhanced affinity for plasminogen, which may be in a Lys-plasminogen-like conformation. Maximal binding and stimulation require the N-terminal finger and growth factor domains of t-PA and the N-terminal kringle domains of plasminogen.     

Binding of Glu-plasminogen by fibrinogen and byproducts of its proteolysis

The ability of the native form of plasminogen (Glu-plasminogen) to form complexes with fibrinogen and its fragments immobilized on CNBr-agarose was studied. It was found that unlike Lys-plasminogen, the native form of the proenzyme does not bind to fibrinogen agarose. Limited proteolysis of fibrinogen by plasmin involving alpha C-domains results in the appearance of Glu-plasminogen binding sites at fibrinogen surface. The X2 fragment of fibrinogen binds to about 0.5 moles of Glu-plasminogen at an equimolar ratio of the interacting proteins. Under these conditions, the amount of bound Glu-plasminogen does not increase as a result of subsequent hydrolysis of fibrinogen down to end products, fragments E and D. It was found that Glu-plasminogen interacts with both E- and D-fragments of fibrinogen. Similar to Lys-plasminogen, Glu-plasminogen exhibits a high affinity for the E-fragment. The maximal quantity of the bound protein under the given experimental conditions is 2 moles per mole of the immobilized E-fragment. The interaction of Glu-plasminogen with the E-fragment is mediated by the lysine-binding sites of the proenzyme with a high and low affinity [Kd = 1.8.10(-6) and 7.5.10(-5) M, respectively]. Glu-plasminogen, unlike Lys-plasminogen, shows a low affinity for the D-fragment (Kd = 2.10(-5) M). Glu-plasminogen cannot be adsorbed by arginine-binding sites at the DH fragment-agarose.     

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

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

A monoclonal antibody directed against the high-affinity lysine-binding site (LBS) of human plasminogen. Role of LBS in the regulation of fibrinolysis

One of thirty murine monoclonal antibodies, raised by immunization with human plasmin-alpha 2-antiplasmin complex, was found to be directed against the high-affinity lysine-binding site in plasminogen. Indeed, this antibody (MA-HAL) reacted with plasminogen and with a fragment of plasminogen composed of the first three triple-loop structures (LBS I) and was displaced by 6-aminohexanoic acid (50% displacement at 25 microM). In competitive radioimmunoassays the binding of radiolabeled plasminogen to MA-HAL was reduced to 50% with 2.3 microM alpha 2-antiplasmin or 1.3 microM histidine-rich glycoprotein, which corresponds to the known dissociation constants between these ligands and the high-affinity lysine-binding site of plasminogen. MA-HAL did not influence the activation of plasminogen by tissue-type plasminogen activator in the absence of CNBr-digested fibrinogen, but abolished the effect of CNBr-digested fibrinogen on the Michaelis constant of the reaction. MA-HAL reduced the reaction rate between plasmin and alpha 2-antiplasmin by a factor 20 and abolished the binding of plasminogen to fibrin. These results indicate that MA-HAL specifically binds to and masks the high-affinity lysine-binding site of plasminogen. It therefore is a useful tool for the investigation of the role of this structure in the regulation of fibrinolysis, both at the level of fibrin-stimulated activation of plasminogen and of the inhibition of generated plasmin.     

Vampire bat salivary plasminogen activator exhibits a strict and fastidious requirement for polymeric fibrin as its cofactor, unlike human tissue-type plasminogen activator. A kinetic analysis.

The vampire bat salivary plasminogen activator (BatPA) is virtually inactive toward Glu-plasminogen in the absence of a fibrin-like cofactor, unlike human tissue-type plasminogen activator (tPA) (the kcat/Km values were 4 and 470 M-1 s-1, respectively). In the presence of fibrin II, tPA and BatPA activated Glu-plasminogen with comparable catalytic efficiencies (158,000 and 174,000 M-1 s-1, respectively). BatPA's cofactor requirement was partially satisfied by polymeric fibrin I (54,000 M-1 s-1), but monomeric fibrin I was virtually ineffective (970 M-1 s-1). By comparison, a variety of monomeric and polymeric fibrin-like species markedly enhanced tPA-mediated activation of Glu-plasminogen. Fragment X polymer was 2-fold better but 9-fold worse as cofactor for tPA and BatPA, respectively, relative to fibrin II. Fibrinogen, devoid of plasminogen, was a 10-fold better cofactor for tPA than fibrinogen rigorously depleted of plasminogen, Factor XIII, and fibronectin; the enhanced stimulatory effect of the less-purified fibrinogen was apparently due to the presence of Factor XIII. By contrast, the two fibrinogen preparations were equally poor cofactors of BatPA-mediated activation of Glu-plasminogen. BatPA possessed only 23 and 4% of the catalytic efficiencies of tPA and two-chain tPA, respectively, in hydrolyzing the chromogenic substrate Spectrozyme tPA. However in the presence of fibrin II, BatPA and tPA exhibited similar kcat/Km values for the hydrolysis of Spectrozyme tPA. Our data revealed that BatPA, unlike tPA, displayed a strict and fastidious requirement for polymeric fibrin I or II. Consequently, BatPA may preferentially promote plasmin generation during a narrow temporal window of fibrin formation and dissolution.     

Features of the interaction of Glu- and Lys-forms of plasminogen with native and partially hydrolyzed fibrin]

Glu- and Lys-plasminogen interaction with native and desAABB-fibrin obtained from fibrinogen partially hydrolyzed by plasmin was studied. It was found that native fibrin adsorbs 6 times more Lys-plasminogen as compared to the native form of the proenzyme. The range of the Lys-plasminogen binding does not change, if part of the fibrinogen molecules hydrolyze down to X-fragments. At the same time, the appearance in the system of 1% Xi-fragments leads to a 6-fold increase in the Glu-plasminogen binding. The amount of adsorbed Glu-plasminogen reaches the level of Lys-plasminogen adsorption both in the native and partially hydrolyzed fibrin. It was found that kringle K 1-3 or 6-aminohexanoic acid at saturating for high-affinity lysine-binding sites concentrations do not influence the Glu-plasminogen binding to native fibrin but inhibit it when the partially purified form is used. It is assumed that the manyfold increase of the Glu-plasminogen binding to partially hydrolyzed fibrin is due to the alteration of the proenzyme conformation at the initial steps of fibrin hydrolysis during the formation of Xi fragments.     

Kinetics of glu- and lys-plasminogen activation by the tissue activator in a fibrin clot

Using a modified procedure for measuring the time of fibrin clot lysis, the kinetics of Glu- and Lys-plasminogen activation by the tissue activator was studied. Within the plasminogen concentration range of 0.4-100 nM the rate of activation of both protein forms obeys the Michaelis-Menten kinetics. At Lys-plasminogen concentration equimolar to that of fibrin, the rate of activation of the former decreases down to that of Glu-plasminogen activation. The kinetic constants for Glu- and Lys-plasminogen activation (Km) are equal to 0.055 and 0.013 microM; k = 0.19 and 0.21 s-1, respectively. The Km values for fibrin-bound Glu- and Lys-plasminogen are equal to 0.25 nM and 8 nM, respectively (k = 0.08 and 0.26 s-1, respectively). It is assumed that the tissue activator exhibits a higher affinity for the Glu-plasminogen--fibrin complex than for the Lys-plasminogen-fibrin complex.     

Physical and chemical properties of the NH2-terminal glutamic acid and lysine forms of human plasminogen and their derived plasmins with an NH2-terminal lysine heavy (A) chain.

Comparative physical and chemical data are described for the human NH2-terminal Glu-plasminogen and Lys-plasminogen forms in order to determine the exact relationship between these two types of the zymogen. The molecular weights of Glu-plasminogen and Lys-plasminogen were similar and were determined to be 83, 800 plus or minus 4, 500 and 82, 400 plus or minus 3, 300, respectively, by sedimentation equilibrium methods. The molecular weights were identical in dodecyl sulfate solutions, approximately 83, 000, by sedimentation equilibrium methods. The sedimentation coefficients, s-020, w of Glu-plasminogen and Lys-plasminogen were determined to be 5.0 S, and 4.4 S, respectively. These two plasminogen forms had different partial specific volumes, and calculations of the frictional coefficients from sedimentation coefficients and molecular weights indicated conformation differences. Glu-plasminogen appeared to be larger in size than Lys-plasminogen in acrylamide gel-dodecyl sulfate electrophoresis. The amino acid compositions of Glu-plasminogen and Lys-plasminogen, and their major isolated isoelectric forms, were found to be similar, but several amino acid residues (glutamic acid, alanine, isoleucine, phenylalanine, and lysine) were found to be significantly higher in the Glu-plasminogen forms. The derived plasmins from both the Glu- and Lys-plasminogens with an nh2-terminal Lys- heavy (A) chain were found to have identical molecular weights of 76, 500 plus or minus 2, 500, and sedimentation coefficients, s-020, w of 4.3 S.     

Actin accelerates plasmin generation by tissue plasminogen activator.

Actin has been found to bind to plasmin's kringle regions, thereby inhibiting its enzymatic activity in a noncompetitive manner. We, therefore, examined its effect upon the conversion of plasminogen to plasmin by tissue plasminogen activator. Actin stimulated plasmin generation from both Glu- and Lys-plasminogen, lowering the Km for activation of Glu-plasminogen into the low micromolar range. Accelerated plasmin generation did not occur in the presence of epsilon-amino caproic acid or if actin was exposed to acetic anhydride, an agent known to acetylate lysine residues. Actin binds to tissue plasminogen activator (t-Pa) (Kd = 0.55 microM), at least partially via lysine-binding sites. Actin's stimulation of plasmin generation from Glu-plasminogen was inhibited by the addition of aprotinin and was restored by the substitution of plasmin-treated actin, indicating the operation of a plasmin-dependent positive feedback mechanism. Native actin binds to Lys-plasminogen, and promotes its conversion to plasmin even in the presence of aprotinin, indicating that plasmin's cleavage of either actin or plasminogen leads to further plasmin generation. Plasmin-treated actin binds Glu-plasminogen and t-PA simultaneously, thereby raising the local concentration of t-PA and plasminogen. Together, but not separately, actin and t-PA prolong the thrombin time of plasma through the generation of plasmin and fibrinogen degradation products. Actin-stimulated plasmin generation may be responsible for some of the changes found in peripheral blood following tissue injury and sepsis.     

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

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

A monoclonal antibody specific for Lys-plasminogen. Application to the study of the activation pathways of plasminogen in vivo

Human plasminogen, a glycoprotein with NH2-terminal Glu, is rapidly converted by traces of plasmin to proteolytic derivatives with NH2-terminal Met 68, Lys 77, or Val 78 ("Lys-plasminogen"), which are much more readily activated to plasmin than is Glu-plasminogen. It has, therefore, been proposed that physiological activation of Glu-plasminogen occurs mainly via Lys-plasminogen intermediates (Wiman, B., and Wallén, P. (1973) Eur. J. Biochem. 36, 25-31). In the present study we have characterized a murine monoclonal antibody (LPm1) directed against an epitope exposed in Lys-plasminogen but not in Glu-plasminogen. The antibody was secreted by a hybridoma obtained by fusion of mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells of a mouse immunized with purified Lys-plasmin-alpha 2-antiplasmin complex. Coupling of the alpha-amino groups of Lys-plasminogen with phenylisothiocyanate resulted in complete loss of immunoreactivity for LPm1, which was, however, fully restored by cleavage of the derivatized NH2-terminal amino acid. After a second cycle, immunoreactivity was not restored, indicating that the LPm1 antibody-binding site depends on the presence of Lys 77 and/or Val 78 as NH2-terminal amino acids. The immunoreactivity of Lys-plasminogen with LPm1 is abolished by reduction of the protein, suggesting that conversion of Glu-plasminogen to Lys-plasminogen is associated with a conformational alteration exposing the epitope for the LPm1 monoclonal antibody. In order to investigate the pathways of plasminogen activation in vivo, total plasmin-alpha 2-antiplasmin and Lys-plasmin-alpha 2-antiplasmin complexes were measured with sandwich-type micro enzyme-linked immunosorbent assays. Therefore, microtiter plates were coated with monoclonal antibodies against alpha 2-antiplasmin, and bound antigen was quantitated with horseradish peroxidase-conjugated LPm1 or a monoclonal antibody reacting equally well with Glu-plasmin as with Lys-plasmin. In 25 healthy subjects the plasmin-alpha 2-antiplasmin levels in plasma were undetectable (less than 0.1 nM). Infusion of tissue-type plasminogen activator in patients with thromboembolic disease resulted in generation of high concentrations of Glu-plasmin-alpha 2-antiplasmin complex (620 +/- 150 nM, n = 7) whereas neither Lys-plasmin-alpha 2-antiplasmin complex nor Lys-plasminogen were consistently detected. It is, therefore, concluded that activation of the fibrinolytic system in vivo occurs by direct cleavage of the Arg 560-Val 561 bond in Glu-plasminogen and not via formation of the Lys-plasminogen intermediates.     

Fragment E-2 from fibrin substantially enhances pro-urokinase-induced Glu-plasminogen activation. A kinetic study using the plasmin-resistant mutant pro-urokinase Ala-158-rpro-UK.

In a previous study, it was shown that fibrin fragment E-2 selectively promotes the activation of plasminogen by pro-urokinase (pro-UK) [Liu, J., & Gurewich, V. (1991) J. Clin. Invest. 88, 2012-2017]. In this study, the kinetics of this promotion by fragment E-2 was studied. Alanine-158-rpro-UK (A-pro-UK), a recombinant plasmin-resistant mutant, was used in order to avoid interference by UK generation during the reaction. In some experiments, pro-UK was substituted in order to validate the mutant as a surrogate. In the presence of a range of concentrations (0-20 microM) of fragment E-2, a linear promotion of the catalytic efficiency of A-pro-UK against native Glu-plasminogen was seen which was 245.5-fold at the highest concentration of fragment E-2 and 450-fold at the highest ratio of E-2/plasminogen used. The promotion was largely a function of an increase in kcat, since fragment E-2 induced a less than 10-fold reduction in KM (8.50-1.40 microM). In contrast to this ligand, epsilon-aminocaproic acid (EACA) induced a biphasic promotion of the activation of Glu-plasminogen which was only 18-fold at maximum. Fragment E-2 did not promote the activation of Lys-plasminogen, but the catalytic efficiency of A-pro-UK was 19.7-fold greater against the open Lys-form than against the closed Glu- form of plasminogen. Fragment E-2 had no effect on the amidolytic activity of A-pro-UK or pro-UK, suggesting that the promotion of their activities was indirect and related to a fragment E-2-induced conformational change in Glu-plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution crystal structure of apolipoprotein(a) kringle IV type 7: insights into ligand binding

Apolipoprotein(a) [apo(a)] consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively. Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV(1) to KIV(10)) followed by sequences similar to the kringle V and protease domains of plasminogen. The KIV domains of apo(a) differ with respect to their ability to bind lysine or lysine analogs. KIV(10) represents the high-affinity lysine-binding site (LBS) of apo(a); a weak LBS is predicted in each of KIV(5)-KIV(8) and has been directly demonstrated in KIV(7). The present study describes the first crystal structure of apo(a) KIV(7), refined to a resolution of 1.45 A, representing the highest resolution for a kringle structure determined to date. A critical substitution of Tyr-62 in KIV(7) for the corresponding Phe-62 residue in KIV(10), in conjunction with the presence of Arg-35 in KIV(7), results in the formation of a unique network of hydrogen bonds and electrostatic interactions between key LBS residues (Arg-35, Tyr-62, Asp-54) and a peripheral tyrosine residue (Tyr-40). These interactions restrain the flexibility of key LBS residues (Arg-35, Asp-54) and, in turn, reduce their adaptability in accommodating lysine and its analogs. Steric hindrance involving Tyr-62, as well as the elimination of critical ligand-stabilizing interactions within the LBS are also consequences of this interaction network. Thus, these subtle yet critical structural features are responsible for the weak lysine-binding affinity exhibited by KIV(7) relative to that of KIV(10).     

Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator

Single-chain urokinase-type plasminogen activator (scu-PA) may be obtained from conditioned cell culture media (natural scu-PA) or by expression of the cDNA encoding human scu-PA in Escherichia coli (recombinant scu-PA). The activation of Glu-plasminogen by natural and recombinant scu-PA can be described by a sequence of three reactions, each of which obeys Michaelis-Menten kinetics. Initial activation of plasminogen to plasmin by scu-PA (reaction I) occurs with a high affinity (Km below 0.8 microM) for both scu-PAs, while the catalytic rate constant (k2) is 0.017 s-1 for recombinant scu-PA but only 0.0009 s-1 for natural scu-PA. Subsequent conversion of scu-PA to urokinase (two-chain urokinase-type plasminogen activator, tcu-PA) by generated plasmin (reaction II) occurs with a comparable affinity (Km about 5 microM) for natural and recombinant scu-PA and with a k2 of 0.23 s-1 for natural and 1.2 s-1 for recombinant scu-PA. Finally, activation of plasminogen by tcu-PA (reaction III) occurs with low affinity (Km 30-50 microM) but with a high catalytic rate constant (k2 about 5 s-1) for both natural and recombinant tcu-PA. The differences in the kinetic parameters of the activation of plasminogen by natural or recombinant scu-PA are thus mainly due to differences in turnover rate in the first reaction. Indeed, the catalytic rate constant of the first reaction is about 20-times higher for recombinant scu-PA than for natural scu-PA. Thus, surprisingly, the artificial, unglycosylated recombinant scu-PA molecule has a better catalytic efficiency than its natural glycosylated counterpart.     

Lys-plasminogen is a significant intermediate in the activation of Glu-plasminogen during fibrinolysis in vitro.

Plasminogen, the zymogen form of the fibrinolytic enzyme plasmin, is known to undergo plasmin-mediated modification in vitro. The modified form, Lys-plasminogen, is superior to the native Glu-plasminogen in fibrin binding and as a substrate for activation by tissue-type plasminogen activator (t-PA). The present study was undertaken to determine the existence and significance of the Glu- to Lys-plasminogen conversion during t-PA-mediated lysis of plasma clots in vitro. When human plasma was supplemented with exogenous Lys-plasminogen and clotted, a dose-dependent shortening of lysis time was observed. Formation of Lys-plasminogen in situ during fibrinolysis was determined using 131I-Glu-plasminogen-supplemented plasma. By the time of lysis, Lys-plasminogen had accumulated to about 20% of the initial concentration of Glu-plasminogen. Quantitation of activation of both Glu- and Lys-plasminogen as well as the conversion of Glu- to Lys-plasminogen in plasma supplemented with both 131I-Glu-plasminogen and 125I-Lys-plasminogen was accomplished by determining the flux of the isotopically labeled species along three pathways: Glu-plasminogen-->Glu-plasmin, Glu-plasminogen-->Lys-plasminogen, and Lys-plasminogen-->Lys-plasmin. After a brief lag, the Glu-plasminogen activation rate was constant until lysis was achieved, at which point activation ceased. The Lys-plasminogen activation rate also was essentially constant until lysis but was not characterized by a lag phase. The rate of conversion of Glu- to Lys-plasminogen was nonlinear and correlated directly with the rate of fibrinolysis. By the time lysis had occurred, Glu-plasminogen consumption had been distributed equally between direct activation to plasmin and conversion to Lys-plasminogen, and 45% of the plasmin which had been formed was derived from Lys-plasminogen. These results demonstrate both the formation and the subsequent activation of Lys-plasminogen during fibrinolysis. As a result of improved fibrin binding and activation of Lys-plasminogen compared to Glu-plasminogen, the formation of Lys-plasminogen within a clot constitutes a positive feedback mechanism that can further stimulate the activation of plasminogen by t-PA as fibrinolysis progresses.     

The cell-binding domains of plasminogen and their function in plasma

Plasminogen binding sites are expressed by a wide variety of cell types and serve to promote fibrinolysis and local proteolysis. In this study, the recognition specificity of cells for plasminogen has been examined, primarily using platelets as models. Analyses with plasminogen fragments implicated residues 79-337 (or 353), comprising the first three kringles of plasminogen, as a primary recognition site for plasminogen binding to both thrombin-stimulated and nonstimulated platelets. Other regions of plasminogen, namely residues 354-439 and 442-790, can also participate in the interaction, and these other regions contribute differentially to the binding of the ligand to stimulated and nonstimulated platelets. Binding to nucleated cells, with U937 cells serving as the prototype, is dependent upon a recognition specificity similar to that of unstimulated platelets. Binding of Glu-plasminogen, the native form of the molecule, to thrombin-stimulated platelets has been shown previously to require platelet fibrin. By comparing the interaction of Glu-plasminogen and its degradation product, Lys-plasminogen, with thrombin-stimulated platelets, it is concluded that the cell surface uniquely enhances the affinity of Glu-, but not Lys-plasminogen, for fibrin. Finally, we have demonstrated that cellular receptors and interactive sites within plasminogen are available in the plasma environment. Thus, the functions ascribed to cellular plasminogen receptors can occur within a physiologic setting.