Fibrin and plasminogen structures essential to stimulation of plasmin formation by tissue-type plasminogen activator

Plasminogen activation catalysed by tissue-type plasminogen activator (t-PA) has been examined in the course of concomitant fibrin formation and degradation. Plasmin generation has been measured by the spectrophotometric method of Petersen et al. (Biochem. J. 225 (1985) 149-158), modified so as to allow for light scattering caused by polymerized fibrin. Glu1-, Lys77- and Val442-plasminogen are activated in the presence of fibrinogen, des A- and des AB-fibrin and the rate of plasmin formation is found to be greatly enhanced by both des A- and des AB-fibrin polymer. Plasmin formation from Glu1- and Lys77-plasminogen yields a sigmoidal curve, whereas a linear increase is obtained with Val442-plasminogen. The rate of plasmin formation from Glu1- and Lys77-plasminogen declines in parallel with decreasing turbidity of the fibrin polymer effector. In order to study the effect of polymerization, this has been inhibited by the synthetic polymerization site analogue Gly-Pro-Arg-Pro, by fibrinogen fragment D1 or by prior methylene blue-dependent photooxidation of the fibrinogen used. Inhibition of polymerization by Gly-Pro-Arg-Pro reduces plasmin generation to the low rate observed in the presence of fibrinogen. Antipolymerization with fragment D1 or photooxidation has the same effect on Glu1-plasminogen activation, but only partially reduces and delays the stimulatory effect on Lys77- and Val442-plasminogen activation. The results suggest that protofibril formation (and probably also gelation) of fibrin following fibrinopeptide release is essential to its stimulatory effect. The gradual increase and subsequent decline in the rate of plasmin formation from Glu1- or Lys77-plasminogen during fibrinolysis may be explained by sequential exposure, modification and destruction of different t-PA and plasminogen binding sites in fibrin polymer.     

Zymogen-activation kinetics. Modulatory effects of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid and poly-D-lysine on plasminogen activation.

The kinetics of plasminogen activation catalysed by urokinase and tissue-type plasminogen activator were investigated. Kinetic measurements are performed by means of a specific chromogenic peptide substrate for plasmin, D-valyl-L-leucyl-L-lysine 4-nitroanilide. Two methods are proposed for the analysis of the resulting progress curve of nitroaniline formation in terms of zymogen-activation kinetics: a graphical transformation of the parabolic curve and transformation of the curve for nitroaniline production into a linear progress curve by the addition of a specific inhibitor of plasmin, bovine pancreatic trypsin inhibitor. The two methods give similar results, suggesting that the reaction between activator and plasminogen is a simple second-order reaction at least at plasminogen concentrations up to about 10 microM. The kinetics of both Glu1-plasminogen (residues 1-790) and Lys77-plasminogen (residues 77-790) activation were investigated. The results confirm previous observations showing that trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at relatively low concentrations enhances the activation rate of Glu1-plasminogen but not that of Lys77-plasminogen. At higher concentrations both Glu1- and Lys77-plasminogen activation are inhibited. The concentration interval for the inhibition of urokinase-catalysed reactions is shown to be very different from that of the tissue-plasminogen activator system. Evidence is presented indicating that binding to the active site of urokinase (KD = 2.0 mM) is responsible for the inhibition of the urokinase system, binding to the active site of tissue-plasminogen activator is approx. 100-fold weaker, and inhibition of the tissue-plasminogen activator system, when monitored by plasmin activity, is mainly due to plasmin inhibition. Poly-D-lysine (Mr 160 000) causes a marked enhancement of plasminogen activation catalysed by tissue-plasminogen activator but not by urokinase. Bell-shaped curves of enhancement as a function of the logarithm of poly-D-lysine concentration are obtained for both Glu1- and Lys77-plasminogen activation, with a maximal effect at about 10 mg/litre. The enhancement of Glu1-plasminogen activation exerted by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is additive to that of poly-D-lysine, whereas poly-D-lysine-induced enhancement of Lys77-plasminogen activation is abolished by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Analogies are drawn up between the effector functions of poly-D-lysine and fibrin on the catalytic activity of tissue-plasminogen activator.     

The role of fragment X polymers in the fibrin enhancement of tissue plasminogen activator-catalyzed plasmin formation

When thrombin-mediated fibrin formation and tissue plasminogen activator (t-PA)-mediated fibrinolysis proceed in dynamic interaction, desA-(desB beta 1-42)-fragment X polymers are shown to be the predominant fibrin derivatives present during the rapid second phase of Glu1- and Lys78-plasminogen activation. To further investigate the effect of this intermediate, a method was developed for the production and purification of fibrinogen-derived desA-(desB beta 1-42)-fragment X, deprived of both COOH-terminal A alpha-chains, but still capable of thrombin-mediated polymerization. DesA-(desB beta 1-42)-fragment X polymer was compared to intact fibrin with regard to its stimulatory effect on Glu1-, Lys78-, and Val443-plasminogen activation, and its binding of Glu1- and Lys78-plasminogen. Pure fragment X polymer gave rise to a biphasic activation pattern like that of fibrin, demonstrating similar kinetics of rapid phase activation. The dissociation constant for the binding of plasminogen to the effector decreases by a factor of 14, and the stoichiometry increases by a factor of 2 upon plasmin-catalyzed cleavage of both native Glu1- to Lys78-plasminogen, and fibrin to fragment X polymer. We conclude that desA-fibrin protofibril formation is sufficient to initiate fibrin enhancement of t-PA-catalyzed plasminogen activation, and that optimal stimulation depends on further plasmin-mediated modification of the fibrin effector to desA-fragment X-related moieties. Optimal stimulation is dependent on the presence of the kringle 1-4 domains of plasminogen and probably results from altered and increased binding of both plasminogen and t-PA to the modified effector.     

The binding of plasminogen fragments to cultured human umbilical vein endothelial cells.

Glu-plasminogen, kringle 1-5, kringle 1-3, and miniplasminogen exhibited strong binding to human umbilical vein endothelial cells (HUVEC). On the other hand, no significant binding was obtained with microplasminogen and kringle 4. Kringle 1-5 and miniplasminogen, which both contained kringle 5, specifically inhibited the binding of plasminogen to HUVEC while kringle 1-3 did not. The results implied plasminogen molecule contained at least two binding sites, with which it interacted HUVEC. The stronger binding site was located in kringle 5 and the weaker one was in kringle 1-3. Kringle 4 and the active site domain exhibited no significant binding to HUVEC. The interaction of plasminogen with HUVEC is mainly through binding site on kringle 5.     

Isolation and characterization of microplasminogen. A low molecular weight form of plasminogen

A functionally active human microplasminogen without kringle structures was produced by incubation of plasminogen with urokinase-free plasmin at an alkaline pH. The microplasminogen was purified by affinity chromatography on lysine- and soybean trypsin inhibitor-Sepharose and by chromofocusing. Human plasminogen is specifically cleaved at Arg529-Lys530 by plasmin to form microplasminogen, which consists of a single polypeptide of 261 residues from the COOH-terminal portion of native plasminogen. It has an Mr of 28,617, calculated from the sequence, which is consistent with the molecular weight determined by sodium dodecyl sulfate gel electrophoresis. Microplasminogen is a slightly basic protein and is eluted from a chromofocusing column at pH 8.3. It can be activated by urokinase and streptokinase to a catalytically active microplasmin. The specific amidolytic activity of microplasmin is about three times higher than Lys77-plasmin on a weight basis and is about the same on a molar basis. The activation of microplasminogen by streptokinase is slower than that of either Glu-plasminogen or Lys77-plasminogen. On the other hand, the activation of microplasminogen by urokinase is faster than that of either of the latter. The Arg560-Val561 bond is cleaved during activation of both microplasminogen and native plasminogen.     

The course and prerequisites of Lys-plasminogen formation during fibrinolysis

Plasmin-catalyzed modification of the native plasma zymogen Glu1-plasminogen to its more reactive Lys78 form has been shown to be enhanced in the presence of fibrin. The aim of the present work has been to characterize the influence of fibrinopeptide release, fibrin polymerization, and plasmin cleavage of fibrin on the rate of Lys78-plasminogen formation. 125I-Labeled Glu1- to Lys78-plasminogen conversion was catalyzed by performed Lys78-plasmin, or by plasmin generated during plasminogen activation with tissue plasminogen activator or urokinase. The two forms of plasminogen were quantitated following separation by polyacrylamide gel electrophoresis in acetic acid/urea. Plasmin generated by plasminogen activator was monitored by a fixed-time amidolytic assay. The rate of Lys78-plasminogen formation was correlated, in separate experiments, to the simultaneous, plasmin-catalyzed cleavage of 125I-labeled fibrinogen or fibrin to fragments X, Y, and D. The radiolabeled components were quantitated after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results show that the formation of both bathroxobin-catalyzed des-A-fibrin and thrombin-catalyzed des-AB-fibrin leads to marked stimulation of Lys78-plasminogen formation, whereas inhibition of fibrin polymerization, with Gly-Pro-Arg-Pro, abolishes the stimulatory effect. The rate of Lys78-plasminogen formation varies markedly in the course of fibrinolysis. The apparent second-order rate constant of the reaction undergoes a transient increase upon transformation of fibrin to des-A(B) fragment X polymer and decreases about 10-fold to the level observed during fibrinogenolysis upon further degradation to soluble fragments Y and D.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A monoclonal antibody to the epsilon-aminocaproic acid binding site on the kringle 4 region of human plasminogen that accelerates the activation of Glu1-plasminogen by urokinase

A monoclonal antibody, 10-F-1, previously shown [V. A. Ploplis, H. S. Cummings, and F. J. Castellino (1982) Biochemistry 21, 5891-5897] to interact with a particular epsilon-aminocaproic acid (EACA)3 binding site on the kringle 4 (K4) region of human Glu1-plasminogen (Glu1-Pg), has been employed to assess the contribution of this particular EACA site toward the enhancement, by EACA and its analogs, of the urokinase (UK)-catalyzed activation of Glu1-Pg. As is the case with EACA-like compounds, the presence of antibody 10-F-1 accelerates the activation of Glu1-Pg by UK, but does not enhance the similar activation of Lys77-plasminogen. In the presence of concentrations of antibody 10-F-1 which saturate its binding site on Glu1-Pg, the Km of Glu1-Pg activation by UK is raised from 1.4 +/- 0.2 microM, a value obtained in the absence of antibody, to 17.0 +/- 2.0 microM. On the other hand, the kcat for this activation, 0.038 +/- 0.005 s-1, is elevated to 2.45 +/- 0.2 s-1 at saturating concentrations of antibody 10-F-1. The kcat/Km for activation under these conditions is 0.027 s-1 microM-1 in the absence of antibody, and 0.144 s-1 microM-1 in the presence of saturating levels of antibody 10-F-1. This demonstrates that the interaction of this antibody with its epitope results in a fivefold stimulation of the activation rate of Glu1-Pg by UK. The availability of antibody 10-F-1 allows for a specific means of probing the function of one of the four to five thermodynamically equivalent weak EACA sites on human plasminogen. From this particular study, it is concluded that the weak binding site for EACA on the K4 domain of Glu1-Pg is either in-part or in-whole responsible for the enhancing effect of EACA on human Glu1-Pg activation by UK.     

Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator

The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin.     

Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance

Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.     

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro.

The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.     

Enzymatic properties of single-chain and two-chain forms of a Lys158----Glu158 mutant of urokinase-type plasminogen activator

Recombinant single-chain urokinase-type plasminogen activator (rscu-PA), in which the plasmin-sensitive peptide bond Lys158-Ile159 is destroyed by site-specific mutagenesis of Lys158 to Glu (rscu-PA-Glu158), is quantitatively converted to two-chain urokinase-type plasminogen activator (rtcu-PA-Glu158) by treatment with endoproteinase Glu-C (Staphylococcus aureus V8 proteinase). The catalytic efficiency (k2/Km) of rscu-PA-Glu158 for the activation of plasminogen is 20 times lower (0.0001 microM-1 s-1) than that of rscu-PA (0.002 microM-1 s-1). In contrast, rtcu-PA-Glu158 has very similar properties to rtcu-PA obtained by digestion of rscu-PA with plasmin, including binding to benzamidine-Sepharose, catalytic efficiency for the activation of plasminogen (0.035 microM-1 s-1 versus 0.046 microM-1 s-1) and fibrinolytic activity in an in vitro plasma clot lysis system. It is concluded that the amino acid in position 158 is a main determinant of the functional properties of single-chain urokinase-type plasminogen activator but not of the two-chain form.     

Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin.

Human tissue-type plasminogen activator (t-PA) catalyses the conversion of inactive plasminogen into active plasmin, the main fibrinolytic enzyme. This process is confined to the fibrin surface by specific binding of t-PA to fibrin and stimulation of its activity by fibrin. Tissue-type plasminogen activator contains five domains designated finger, growth factor, kringle 1, kringle 2 and protease. The involvement of the domains in fibrin specificity was investigated with a set of variant proteins lacking one or more domains. Variant proteins were produced by expression in Chinese hamster ovary cells of plasmids containing part of the coding sequence for the activator. It was found that kringle 2 domain only is involved in stimulation of activity by fibrin. In the absence of plasminogen and at low concentration of fibrin, binding of t-PA is mainly due to the finger domain, while at high fibrin concentrations also kringle 2 is involved in fibrin binding. In the presence of plasminogen, fibrin binding of the kringle 2 region of t-PA also becomes important at low fibrin concentrations.     

Effectors of the activation of human [Glu1]plasminogen by human tissue plasminogen activator

The activation of human [Glu1]plasminogen [( Glu1]Pg) by human recombinant (rec) two-chain tissue plasminogen activator (t-PA) is inhibited by Cl-, at physiological concentrations, and stimulated by epsilon-aminocaproic acid (EACA), as well as fibrin(ogen). Chloride functions as a result of its binding to [Glu1]Pg, with a Ki of approximately 9.0 mM, thereby rendering [Glu1]Pg a less effective substrate for two-chain rec-t-PA. EACA stimulates the activation in Cl-(-)containing solutions, with a Ka of approximately 4.0 mM, primarily by reversal of the Cl-(-)inhibitory effect. Fibrinogen appears to exert its stimulatory properties mainly through effects on the enzyme, two-chain rec-t-PA, with a Ka of approximately 3.7 microM in activation systems containing physiological levels of Cl-. Analysis of the results of this paper reveals that normal plasma components, Cl- and fibrinogen, exert major regulatory roles on the ability of [Glu1]Pg to be activated by two-chain rec-t-PA, in in vitro systems. The presence of Cl- inhibits the stimulation of [Glu1]Pg activation that would normally occur in the presence of fibrinogen, a result of possible importance to the observation that some degree of systemic fibrinogenolysis accompanies therapeutic use of tissue plasminogen activator.     

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.     

Kinetic analysis of covalent hybrid plasminogen activators: effect of CNBr-degraded fibrinogen on kinetic parameters of Glu1-plasminogen activation

The kinetic parameters of three activator species of Glu1-plasminogen (Glu1-Plg) were compared in their reaction at pH 7.4 and 37 degrees C, in the presence and absence of CNBr-digested fibrinogen (CNBr-Fg). The urokinase- (u-PA-) derived covalent hybrid activator PlnA-u-PAB had an apparent Michaelis constant (Kplg) of 7.44 microM, a catalytic rate constant (kplg) of 51.1 min-1, and a second-order rate constant (kplg/Kplg) of 6.87 microM-1 min-1. The tissue plasminogen activator (t-PA) derived covalent hybrid activator PlnA-t-PAB was characterized by a Kplg of 3.33 microM, a kplg of 1.03 min-1, and a kplg/Kplg of 0.309 microM-1 min-1. The kplg/Kplg values for the parent u-PA and t-PA activators were 6- and 16-fold higher than the respective hybrids, mainly due to an approximately 10-fold increase in the apparent Kplg for the hybrids. In the presence of CNBr-Fg, the increase of the kplg/Kplg values for u-PA and its hybrid was 1.1-fold, but for t-PA and its hybrid, the increases were 7- and 12-fold, respectively. In both the absence and presence of CNBr-Fg, activator t-PAB had an apparent Kplg of 19.1 and 27.6 microM and a kplg of 2.9 and 5.0 min-1, respectively. The increase in the kplg/Kplg value with CNBr-Fg was 1.2-fold. The streptokinase- (SK-) derived activators Glu1-plasmin.SK (Glu1-Pln.SK), Val442-Pln.SK, and Val561-Pln.SK had apparent Kplg values of 0.458, 0.268, and 0.121 microM and kplg values of 20.0, 126.0, and 63.3 min-1, respectively. In the presence of CNBr-Fg, the first two activators showed an approximately 1.4-fold increase and the last showed a 1.4-fold decrease in their kplg/Kplg values. The catalytic efficiency (kplg/Kplg) of the various activator species fell in the decreasing order SK greater than u-PA greater than t-PA, in either the presence or absence of CNBr-Fg. CNBr-Fg enhanced significantly the activities of only two activators, t-PA and PlnA-t-PAB.     

Functional characterization of monoclonal antibodies directed against fibrin binding domains of tissue-type plasminogen activator

Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes.     

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.     

Fibrin structures during tissue-type plasminogen activator-mediated fibrinolysis studied by laser light scattering: relation to fibrin enhancement of plasminogen activation

The aim was to relate fibrin structure and the stimulatory effect of fibrin on plasminogen activation during t-PA-mediated fibrinolysis using Lys⁷⁸-plasminogen as activator substrate. Structural studies were undertaken by static and dynamic laser light scattering, cryo transmission electron microscopy and by the measurement of conversion of fibrin to X-, Y- and D-fragments. The kinetics of plasmin formation were monitored by measurement of the rate of pNA-release from Val-LeuLys-pNA. The process of fibrin formation and degradation comprised three phases. In the first phase, protofibrils with an average length of about 10 times that of fibrinogen were formed. The duration of this phase decreased with increasing t-PA concentration. The second phase was characterized by a sudden elongation and lateral aggregation of fibrin fibers, most pronounced at low levels of t-PA, and by formation of fragment X-polymer. The third phase was dominated by fragmentation of fibers and by formation of Y- and D-fragments: Plasmin degraded the fibers from within, resulting in the formation of long loose bundles, which subsequently disintegrated into thin filaments with a length of less than 10 and a mass per length close to one relative to fibrinogen. Plasmin generation at high t-PA concentrations sets in just prior to (and at low t-PA concentrations shortly after) the onset of the rapid second phase of elongation and lateral aggregation of fibrin fibers. The maximal rate of plasmin formation per mol t-PA was the same at all concentrations of activator and was achieved close to the time of the peak level of fragment X-polymer. Plasmin formation ceased after formation of substantial amounts of Y- and D-fragments. At this stage the length was between 300 and 3 and the mass per length close to 1, both relative to fibrinogen. In conclusion our results indicate that (1) formation of short fibrin protofibrils is the minimal requirement for the onset of the stimulatory effect of fibrin on plasminogen activation by t-PA, (2) formation of fragment X protofibrils is sufficient to induce optimal stimulation of plasminogen activation, and (3) plasmin degrades laterally aggregated fibrin fibers from within, resulting in the conversion of the fibers into long loose bundles, which later disintegrate into thin filaments.Abbreviations t-PA tissue-type plasminogen activator - Lys⁷⁸-plasminogen plasmin-modified plasminogen, mainly with NH₂-terminal lysine (residues 78-791, residue numbering according to Forsgren et al. 1987) - Val-Leu-Lys-pNA H-D-valyl-L-leucyl-L-lysine-4-nitroanilide - Phe-Pip-Arg-pNA H-D-phenylalanyl-L-arginine-4-nitroanilide - pNA p-nitroanilide - SDS sodium dodecyl sulphate The present work has been supported by the Danish Natural Science Research Council and the Danish Agricultural and Veterinary Research CouncilDeceased on August 2, 1991 Correspondence to: R. Bauer     

Requirement of zymogen modification for activation of porcine plasminogen.

In physiological salt solutions, porcine plasminogen is refractory to activation by urokinase or trypsin and to proteolysis at Lys77 by plasmin or trypsin. Plasminogen becomes a substrate for urokinase (at Arg560), plasmin (at Lys77), and trypsin (at both bonds) if chloride ion is removed or if 6-aminohexanoate (2.5 mmol/L) is added. Irrespective of salts, activation of des(1-77)plasminogen is as efficient as activation of des(kringle1-4)plasminogen and is inhibited 50% by 2.5 mmol/L 6-aminohexanoate. In solutions lacking chloride or containing 6-aminohexanoate, plasminogen, des(1-77)plasminogen, and des(kringle1-4)plasminogen show no tendency to saturate urokinase in physiologically relevant concentrations (10 mumol/L). The findings are interpreted as indicating that plasminogen requires modification, either by proteolysis or by ligands, for activation.