A study of the protection of plasmin from antiplasmin inhibition within an intact fibrin clot during the course of clot lysis

Previous work using soluble fibrin surrogates or very dilute fibrin indicate that inhibition of plasmin by antiplasmin is attenuated by fibrin surrogates; however, this phenomenon has not been quantified within intact fibrin clots. Therefore, a novel system was designed to measure plasmin inhibition by antiplasmin in real time within an intact clot during fibrinolysis. This was accomplished by including the plasmin substrate S2251 and a recombinant fluorescent derivative of plasminogen (S741C-fluorescein) into clots formed from purified components. Steady state plasmin levels were estimated from the rates of S2251 hydrolysis, the rates of plasminogen activation were estimated by fluorescence decrease over time, and residual antiplasmin was deduced from residual fluorescence. From these measurements, the second order rate constant could be inferred at any time during fibrinolysis. Immediately after clot formation, the rate constant for inhibition decreased 3-fold from 9.6 x 10(6) m(-1) s(-1) measured in a soluble buffer system to 3.2 x 10(6) m(-1) s(-1) in an intact fibrin clot. As the clot continued to lyse, the rate constant for inhibition continued to decrease by 38-fold at maximum. To determine whether this protection was the result of plasmin exposure of carboxyl-terminal lysine residues, clots were formed in the presence of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). In the presence of TAFIa, the initial protective effect associated with clot formation occurred; however, the secondary protective effect associated with lysine residue exposure was delayed in a TAFIa concentration-dependent manner. This latter effect represents another mechanism whereby TAFIa attenuates fibrinolysis.     

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 kinetic analysis of the tissue plasminogen activator and DSPAalpha1 cofactor activities of untreated and TAFIa-treated soluble fibrin degradation products of varying size

The kinetics of tissue plasminogen activator (t-PA) and DSPAalpha1-catalyzed plasminogen activation using untreated and TAFIa-treated fibrin degradation products (FDPs), ranging in weight average molecular weight (M(w)) from 0.48 x 10(6) to 4.94 x 10(6) g/mol, were modeled according to the steady-state template model. The FDPs served as effective cofactors for both activators. The intrinsic catalytic efficiencies of both t-PA (17.4 x 10(5) m(-1) s(-1)) and DSPAalpha1 (6.0 x 10(5) m(-1) s(-1)) were independent of FDP M(w). The intrinsic catalytic efficiency of t-PA was 12-fold higher than that measured under identical conditions with intact fibrin as the cofactor. At sub-saturating levels of cofactor and substrate, rates were strongly dependent on FDP M(w) with DSPAalpha1 but not t-PA. Loss of activity with decreasing FDP M(w) correlated with loss of finger-dependent binding of the activators to the FDPs. TAFIa treatment of the FDPs resulted in 90- and 215-fold decreases in the catalytic efficiencies of t-PA (0.20 x 10(5) m(-)(1) s(-1)) and DSPAalpha1 (0.028 x 10(5) m(-1) s(-1)), yielding cofactors that were still 30- and 50-fold better than fibrinogen with t-PA and DSPAalpha1, respectively. Our results show that for both activators the products released during fibrinolysis are very effective cofactors for plasminogen activation, and both t-PA and DSPAalpha1 cofactor activity are strongly down-regulated by TAFIa.     

On the kinetics of the reaction between human antiplasmin and a low-molecular-weight form of plasmin.

The reaction between antiplasmin (A) and a low-molecular-weight form of plasmin (P) proceeds in at least two steps: a fast reversible second-order reaction followed by a slower irreversible first-order transition, and may be represented by: P +A k1 in equilibrium k-1 PA k2 leads to PA'. The low-Mr plasmin, which is obtained by limited elastase digestion, is composed of an intact B chain and a small A chain lacking the lysine-binding sites. The k1 of the reaction is (6.5 +/- 0.5) x 10(5) M-1 s-1 which is 30--60 times smaller than that for normal plasmin and antiplasmin. The dissociation constant of the first step is 1.9 x 10(-9) M which is 10 times higher than for normal plasmin and antiplasmin. The rate constant of the second step is (4.2 +/- 0.2) x 10(-3) s-1 for both normal and low-Mr plasmin. Low Mr plasmin which has substrate bound to its active site does not react or reacts only very slowly with antiplasmin. The reaction rate, however, is only slightly influenced by 6-aminohexanoic acid in concentrations up to 1 mM which decrease the reaction rate of normal plasmin approximately 50-fold. The findings further indicate that the lysine-binding site(s) of plasmin are of great importance for the rate of its reaction with antiplasmin.     

On the biological significance of the specific interaction between fibrin,plasminogen and antiplasmin

The influence of antiplasmin on the interaction between fibrin and plasminogen was studied in plasma and in a purified system. The amount of plasminogen bound to fibrin was quantitated using trace amounts of ¹²⁵I-labeled Glu-plasminogen (plasminogen with NH₂-terminal glutamic acid) or ¹²⁵I-labeled Lys-plasminogen (NH₂-terminal lysine).When whole plasma was clotted, 5.2% of Glu-plasminogen was associated with the fibrin clot. In plasma clotted in the presence of 20 mM 6-amino-hexanoic acid only 1.4% of the plasminogen was bound to fibrin, indicating that about 4% of the plasma plasminogen specifically binds to fibrin. With Lys-plasminogen these values were approximately twice as high.When antiplasmin-depleted plasma was used, only slightly higher amounts of both types of plasminogen were associated with the fibrin. The adsorbed plasminogen was not significantly eluted with plasma or with purified antiplasmin at physiological concentrations.These findings indicate that antiplasmin does not play a significant role in the inhibition of the binding of plasminogen to fibrin or the dissociation of the plasminogen · fibrin complex.These observations in conjunction with previous findings on the kinetics of the plasmin-antiplasmin reaction suggest that the lysine-binding site of plasminogen, which is responsible both for its interaction with fibrin and its interaction with antiplasmin, plays an important role in the very fast neutralization of plasmin formed in circulating blood and serves to attach plasminogen to fibrin and thereby sequestrate plasmin formed in loco from circulating antiplasmin.     

Features of the interaction between alpha2-antiplasmin and plasminogen/plasmin

The kinetic of plasmin, Va1442-plasmin, Lys530-plasmin inhibition reaction by alpha 2-antiplasmin as well as interaction of the inhibitor with different derivatives of the plasminogen and its fragments were studied. It was shown that plasmin, mini- and micro-plasmin activity decreased by 97, 88 and 85%, respectively, for equimolar ratio 1:1 of the inhibitor. The value of the inhibition reached its maximum in 1-2, 5-10 and 10-15 min, respectively. The constants of the complex formation rate were 1.4 x 10(6); 1.7 x 10(5) and 6.2 x 10(4) M-1s-1 for the plasmin, mini- and micro-plasmin with alpha 2-antiplasmin, respectively. Both 10(-2) M 6-aminohexanoic acid and 10(-1) M arginine reduced the complex formation rate between plasmin, mini-plasmin and alpha 2-antiplasmin to the value of the rate reaction between micro-plasmin and inhibitor. alpha 2-Antiplasmin bound with all investigated derivatives and fragments of plasminogen. The amount of inhibitor decreased in the series: plasmin, kringle 1-3, kringle 4, mini-plasminogen, micro-plasminogen. The kringle 1-4 and kringle 5 were determined to control the rate of reaction between enzyme and inhibitor, being not necessary for the inhibition. The comparison of the inhibitor interaction with DPP-plasmin, mini-plasminogen and micro-plasminogen displayed the possibility of the additional region existence in catalytic domain. This region participated in the complex with alpha 2-antiplasmin formation. It is supposed that the multisite interaction between plasmin and alpha 2-antiplasmin provides for the specificity and efficiency the inhibitor action.     

The intrinsic threshold of the fibrinolytic system is modulated by basic carboxypeptidases, but the magnitude of the antifibrinolytic effect of activated thrombin-activable fibrinolysis inhibitor is masked by its instability

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is intrinsically unstable, a property that complicates the study of its role in regulating fibrinolysis. To investigate the effect of basic carboxypeptidases on fibrinolysis under conditions of constant carboxypeptidase activity, we employed pancreatic carboxypeptidase B (CPB), a homologous, stable basic carboxypeptidase, as a surrogate for TAFIa. Clots formed from TAFI-depleted plasma or from purified components were supplemented with tissue-type plasminogen activator and either CPB or TAFIa. The clot lysis data indicate that the down-regulation of fibrinolysis mediated by basic carboxypeptidases involves a threshold mechanism. At carboxypeptidase concentrations above the threshold, plasminogen activation is maintained in a fully down-regulated state; experiments in plasma showed that fibrinolysis is essentially halted by saturating concentrations of TAFIa and that fibrinolysis can be prolonged more than 45-fold by a stable carboxypeptidase. The threshold carboxypeptidase concentration was dependent on tissue-type plasminogen activator and antiplasmin concentrations, indicating that the threshold is determined by the steady-state plasmin concentration. Although obvious with CPB, the threshold was masked by the intrinsic instability of TAFIa and became apparent only when the effect of TAFIa was investigated over the picomolar concentration range. Because of the threshold effect and the instability of TAFIa, exponential increases in TAFIa concentration generate linear increases in lysis time. A model relating lysis time to TAFIa concentration, TAFIa half-life, and the threshold concentration of TAFIa is provided. The threshold effect has potentially important implications regarding the role of TAFIa and the regulation of clot lysis in vivo.     

On the molecular interactions between plasminogen-staphylokinase, alpha 2-antiplasmin and fibrin.

The molecular interactions between the plasminogen-staphylokinase complex, alpha 2-antiplasmin and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by alpha 2-antiplasmin. The second-order rate constant for the inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin was 2.7 +/- 0.3.10(6) M-1 s-1 (mean +/- S.D.; n = 7). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low-Mr plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by alpha 2-antiplasmin was about 30-fold lower (9.3 +/- 0.7.10(4) M-1 s-1, mean +/- S.D.; n = 4) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone D-Val-Phe-Lys-Ch2Cl is 9-fold less efficient than that of plasmin (k2/Ki of 700 M-1 s-1 versus 6300 M-1 s-1). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by alpha 2-antiplasmin by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by alpha 2-antiplasmin thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma.     

Relationship of Plasminogen Activator to Fibrin

THERE are two principal theories of the mechanism of thrombus dissolution by the fibrinolytic system. Alkjaersig et al.¹ suggested that as fibrin polymerizes, plasminogen is adsorbed preferentially to the fibrin and is available in large quantities within a thrombus which is comparatively free of antiplasmin. When an activator enters the circulation it diffuses into the clot converting the plasminogen to plasmin in situ and so promotes lysis. Ambrus and Markus², however, proposed that when plasmin forms in the circulation naturally or during infusion of an activator it is normally bound to the excess antiplasmin present in blood. They suggested that this plasmin/antiplasmin complex is reversible and dissociates in the presence of fibrin, its preferred substrate, so allowing the plasmin to bring about fibrin dissolution by “external lysis”. Neither of these theories, however, is supported by an observed phenomena.     

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)     

Plasmin-mediated activation and inactivation of thrombin-activatable fibrinolysis inhibitor

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates the fibrin cofactor function of tissue-type plasminogen activator-mediated plasmin formation and subsequently fibrin degradation. In the present study, we focused on the role of plasmin in the regulation of TAFIa activity. Upon incubation with plasmin, TAFIa activity was generated, which was unstable at 37 degrees C. Analysis of the cleavage pattern showed that TAFI was cleaved at Arg(92), releasing the activation peptide from the 35.8-kDa catalytic domain. The presence of the 35.8-kDa fragment paralleled the time course of generation and loss of TAFIa activity. This suggested that, in the presence of plasmin, TAFIa is probably inactivated by proteolysis rather than by conformational instability. TAFI was also cleaved at Arg(302), Lys(327), and Arg(330), resulting in a approximately 44.3-kDa fragment and several smaller fragments. The 44.3-kDa fragment is no longer activatable since it lacks part of the catalytic center. We concluded that plasmin can cleave at several sites in TAFI and that this contributes to the regulation of TAFI and TAFIa.     

An elastase-dependent pathway of plasminogen activation

In reaction mixtures containing Glu-plasminogen, alpha 2-antiplasmin, and tissue plasminogen activator or urokinase, either pancreatic or leukocyte elastase enhances the rate of plasminogen activation by 2 or more orders of magnitude. This effect is the consequence of several reactions. (a) In concentrations on the order of 100 nM, elastase degrades plasminogen within 10 min to yield des-kringle1-4-plasminogen (mini-plasminogen), which is 10-fold more efficient than Glu-plasminogen as a substrate for plasminogen activators. Des-kringle1-4-plasminogen is insensitive to cofactor activities of fibrin(ogen) fragments or an endothelial cell cofactor. (b) Des-kringle1-4-plasmin is one-tenth as sensitive as plasmin to inhibition by alpha 2-antiplasmin: k" = 10(6) M-1 s-1 versus 10(7) M-1 s-1. (c) alpha 2-Antiplasmin is disabled efficiently by elastase, with a k" of 20,000 M-1 s-1. The elastase-dependent reactions are not influenced by 6-aminohexanoate. In diluted (10-fold) blood plasma, the capacity of endogenous inhibitors to block plasmin expression is suppressed by 30 microM elastase. It is proposed that elastases provide an alternative pathway for Glu-plasminogen activation and a mechanism for controlling initiation of fibrinolysis by urokinase-type plasminogen activators.     

The effect of fibrin structure on fibrinolysis.

Fibrin structure contributes to the regulation of the fibrinolytic rate. As the fibrin fiber size is decreased, the fibrinolytic rate also decreases. Fibrin structure was altered by either changing the ratio of thrombin to fibrinogen, i.e. altering the assembly rate or by adding a fibrin assembly inhibitor, iopamidol. Changes in the fibrinolytic rate were followed by measuring the time dependence of the decrease in the fiber mass/length ratio during fibrinolysis. A measure of the overall fibrinolytic rate was determined from the decrease in the mass/length ratio versus time. An 8-fold reduction in the fibrinolytic rate was seen on decreasing the mass/length ratio from 2.7 x 10(12) daltons/cm to 0.5 x 10(12) daltons/cm. It is shown that thin fibrin fibers have a decreased rate of conversion of plasminogen to plasmin by tissue plasminogen activator and that thin fibrin fibers are lysed more slowly than thick fibrin fibers.     

Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.     

Pro-urokinase-type plasminogen activator is a substrate for hepsin

Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1') peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 x 10(5) m(-1) s(-1)) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 +/- 1.9 nm uPA h(-1), which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.     

Effects of extracellular DNA on plasminogen activation and fibrinolysis

The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1-5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α(2)-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1-1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4-5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α(2)-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0-20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.     

Tissue plasminogen activator binds to human vascular smooth muscle cells by a novel mechanism. Evidence for a reciprocal linkage between inhibition of catalytic activity and cellular binding.

Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator (tPA) specifically, saturably, and with relatively high affinity (K(d) 25 nM), and this binding potentiates the activation of cell-associated plasminogen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322). We have observed that this binding can be efficiently competed by DFP-inactivated tPA and S478A-tPA but not by tPA inactivated with H-D-Phe-Pro-Arg-chloromethyl ketone (PPACK). VSMC-bound tPA also exhibited a markedly reduced inhibition by PPACK, displaying biphasic kinetics with second-order rate constants of 7. 5 x 10(3) M(-1) s(-1) and 0.48 x 10(3) M(-1) s(-1), compared with 7. 2 x 10(3) M(-1) s(-1) in the solution phase. By contrast, tPA binding to fibrin was competed equally well by all forms of tPA, and its inhibition was unaltered. These effects were shown to extend to the physiological tPA inhibitor, plasminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 complex did not compete tPA binding to VSMC, and the inhibition of bound tPA was reduced by 30-fold. The behavior of the various forms of tPA bound to VSMC correlated with conformational changes in tPA detected by CD spectroscopy. These data suggest that tPA binds to its specific high affinity site on VSMC by a novel mechanism involving the serine protease domain of tPA and distinct from its binding to fibrin. Furthermore, reciprocally linked conformational changes in tPA appear to have functionally significant effects on both the interaction of tPA with its VSMC binding site and the susceptibility of bound tPA to inhibition.     

A functional assay for measuring activated thrombin-activatable fibrinolysis inhibitor in plasma

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase found in plasma that is activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. The active carboxypeptidase, TAFIa, attenuates fibrinolysis by removing newly exposed carboxy-terminal lysine residues on fibrin. The half-maximal effect of TAFIa on clot lysis occurs at 1 nM and the maximal effect occurs at 20 nM. Since the circulating concentration of the procarboxypeptidase is approximately 75 nM, only a small portion needs to be activated to have a significant effect on clot lysis. Several assays to measure total plasma TAFI levels and plasma TAFIa levels after it is fully activated exist. However, no currently available assay is sufficiently sensitive and specific to measure endogenous TAFIa in plasma. We have devised a new sensitive and specific assay for TAFIa in plasma that is based on physiologic function. This assay is based on the fact that TAFIa decreases the cofactor activity of high-molecular-weight fibrin degradation products in the stimulation of plasminogen cleavage in a concentration-dependent fashion. With this assay, we can measure TAFIa concentrations as low as 10 pM in plasma and it is not affected by variability in other hemostatic factors. This assay is reliable and repeatable with intra- and interassay variabilities of 6.5 and 6.1%, respectively.     

Role of plasminogen, plasmin, and plasminogen activators in the migration of fibroblasts into plasma clots

Human diploid fibroblasts were seeded onto or into plasma clots and different aspects of cell adhesion and migration were measured. The roles of plasminogen activators and plasmin were studied by either the removal of plasminogen from plasma prior to clotting or by the addition of 10 mM epsilon-aminocaproic acid, which brings about an inhibition of plasmin in this system. When cells were seeded onto the surface of plasma clots, rates of attachment, spreading, and migration were unaffected by plasminogen depletion or plasmin inhibition. In contrast, when cells were seeded into plasma clots, then, although the rates of cells spreading were unaffected, cell migration was abolished by plasminogen depletion or by plasmin inhibition. When cells were seeded onto the surface of plasma clots and the rate of migration into the clots was measured, there was an absolute requirement for plasmin activity; while fibroblasts migrated rapidly into the fibrin lattice of control clots, in the case of plasminogen-depleted clots, cells failed to penetrate the lattice. Focussing through a plasma clot revealed that fibroblasts do not migrate through the fibrin lattice but instead, localized areas of fibrinolysis are generated and cells migrate over the surface of the area of lysis.