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.     

Oxidative degradation of rat mast-cell heparin proteoglycan.

The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.     

Tissue-type plasminogen activator and its substrate Glu-plasminogen share common binding sites in limited plasmin-digested fibrin

The enzyme tissue-type plasminogen activator (t-PA) and its substrate Glu-plasminogen can both bind to fibrin. The assembly of these three components results in about a 1000-fold acceleration of the conversion of Glu-plasminogen into plasmin. Fibrin binding of t-PA is mediated both by its finger (F) domain and its kringle-2 domain. Fibrin binding of Glu-plasminogen involves its kringle structures (K1-K5). It has been suggested that particular kringles contain lysine-binding sites and/or aminohexyl-binding sites, exhibiting affinity for specific carboxyl-terminal lysines and intrachain lysines, respectively. We investigated the possibility that t-PA and Glu-plasminogen kringles share common binding sites in fibrin, limitedly digested with plasmin. For that purpose we performed competition experiments, using conditions that exclude plasmin formation, with Glu-plasminogen and either t-PA or two deletion mutants, lacking the F domain (t-PA del.F) or lacking the K2 domain (t-PA del.K2). Our data show that fibrin binding of t-PA, mediated by the F domain, is independent of Glu-plasminogen binding. In contrast, partial inhibition by Glu-plasminogen of t-PA K2 domain-mediated fibrin binding is observed that is dependent on carboxyl-terminal lysines, exposed in fibrin upon limited plasmin digestion. Half-maximal competition of fibrin binding of both t-PA and t-PA del.F is obtained at 3.3 microM Glu-plasminogen. The difference between this value and the apparent dissociation constant of Glu-plasminogen binding to limitedly digested fibrin (12.1 microM) under these conditions is attributed to multiple, simultaneous interactions, each having a separate affinity. It is concluded that t-PA and Glu-plasminogen can bind to the same carboxyl-terminal lysines in limitedly digested fibrin, whereas binding sites composed of intrachain lysines are unique both for the K2 domain of t-PA and the Glu-plasminogen kringles.     

Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.     

Inhibitory effect of angiostatins on activity of the plasminogen/plasminogen activator system

Angiostatins, kringle-containing fragments of plasminogen, are potent inhibitors of angiogenesis. Effects of three angiostatin forms, K1–3, K1–4, and K1-4.5 (0–2 μM), on the rate of native Glu-plasminogen activation by its physiological activators in the absence or presence of soluble fibrin were investigated in vitro. Angiostatins did not affect the intrinsic amidolytic activities of plasmin and plasminogen activators of tissue type (tPA) and urokinase type (single-chain scuPA and two-chain tcuPA), but inhibited conversion of plasminogen to plasmin in a dose-dependent manner. All three angiostatins suppressed Glu-plasminogen activation by tcuPA independently of the presence of fibrin, and the inhibitory effect increased in the order: K1-3 < K1-4 < K1-4.5. The inhibitory effects of angiostatins on the scuPA activator activity were lower and further decreased in the presence of fibrin. Angiostatin K1-3 (up to 2 μM) had no effect, while 2 μM angiostatins K1-4 and K1-4.5 inhibited the fibrin-stimulated Glu-plasminogen activation by tPA by 50 and 100%, respectively. The difference in effects of the three angiostatins on the Glu-plasminogen activation by scuPA, tcuPA, and tPA in the absence or presence of fibrin is due to the differences in angiostatin structures, mechanisms of action, and fibrin-specificity of plasminogen activators, as well as due to the influence of fibrin on the Glu-plasminogen conformation. Angiostatins in vivo, which mimic plasminogen-binding activity, can inhibit plasminogen activation stimulated by various proteins (including fibrin) of extracellular matrix, thereby blocking cell migration and angiogenesis. The data of this work indicate that the inhibition of Glu-plasminogen activation under the action of physiological plasminogen activators by angiostatins can be implicated in the complex mechanism of their antiangiogenic and antitumor action.     

Activation of fibrin-bound plasminogen by pro-urokinase and its complementariness with that by tissue plasminogen activator

Single chain urokinase (SC-UK) is a precursor of 55 kd two-chain UK (TC-UK). Treatment with catalytic proportions of plasmin or kallikrein converts SC-UK to TC-UK as a consequence of cleavage of its Lys158-Ile159 peptide bond. This plasmin-mediated activation of SC-UK induces a positive feedback secondary reaction and complicates measurement of its activity against its natural substrate, Glu-plasminogen. The fibrin-selective effect of pro-UK-induced clot lysis is not related to fibrin binding. Rather, a conformational change in Glu-plasminogen, conferred when it binds to certain carboxy-terminal lysine residues on fibrin, has been implicated in this mechanism. This is complementary to t-PA. Fibrin-bound t-PA was found to exclusively activate plasminogen bound to certain internal lysine residues. Their complementariness is believed to explain their synergism in fibrinolysis.     

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.     

Biological control of tissue plasminogen activator-mediated fibrinolysis

Fibrinolysis, the body's ability to degrade fibrin, is an integrated part of hemostasis. Overactivity in the fibrinolytic system causes bleeding and underactivity causes thrombosis. Tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), alpha 2-antiplasmin (alpha 2-AP) and plasminogen are definitely involved in fibrinolysis because: (1) these components can be assigned a fibrinolytic role in purified systems, i.e. in vitro, and (2) abnormal structural variants and abnormal levels of these components give rise to bleeding or to thrombosis. The biological control of tPA-mediated fibrinolysis is both cellular and humoral. The cellular regulation compasses synthesis of tPA and PAI-1 and release/uptake of these components. The humoral regulation involves: (1) the reaction between tPA and PAI-1; (2) the fibrin-stimulated plasminogen activation; (3) the reaction between plasmin and alpha 2-AP and (4) plasmin degradation of fibrin. The highly developed biological control of tPA-mediated fibrinolysis is indicative of its physiological importance.     

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)     

Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis

This study deals with the effect of fibrin on the transformation of Glu-plasminogen to Glu-plasmin during fibrinolysis. It focuses particularly on changes in fibrin effector function caused by plasmin-catalysed fibrin degradation. Conversion of 125I-labelled Glu-plasminogen to Glu-plasmin was catalysed by urokinase or tissue plasminogen activator, in the presence of different preparations of progressively degraded fibrin. Plasmin catalysis of Glu-plasminogen and the fibrin (derivative) effector was inhibited by aprotinin. The presence of intact fibrin enhanced the rate of Glu-plasmin formation catalysed by tissue plasminogen activator, but not by urokinase. The presence of initially plasmin-cleaved fibrin, however, increased the rates of Glu-plasmin formation with both activators, as compared to those found with intact fibrin. The rate enhancements induced by initial plasmin degradation of the fibrin effector were associated with an increase in its affinity to both Glu-plasminogen and tissue plasminogen activator, suggesting causal relationships. The weak binding of urokinase was unaffected by fibrin degradation, indicating that effector function was solely exerted on the Glu-plasminogen moiety of urokinase-activated systems. Further degradation of fibrin decreased the stimulating effect on Glu-plasmin formation. This decrease occurred at an earlier stage of degradation with tissue plasminogen activator than with urokinase, indicating that greater integrity of the fibrin effector is necessary for its optimal interaction with the tissue plasminogen activator than with Glu-plasminogen. Concentrations of tranexamic acid that saturate low-affinity lysine-binding sites nearly completely dissociated the binding of Glu-plasminogen to degraded fibrin, but not to intact fibrin. In analogy with the binding of lysine analogues to these sites, the conformation of Glu-plasminogen may be altered by binding to degraded fibrin, thus giving rise to the increased activation rate.     

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.     

Kinetics of in vitro fibrinolysis by plasmin and a plasmin-streptokinase equimolar complex]

Kinetics of fibrinolysis by plasmin and plasmin streptokinase complex have been studied using fibrin gels formed from purified fibrin and human blood plasma. The gels were placed into buffer or blood plasma. The contributions of plasminogen and alpha 2-antiplasmin present or absent in both phases to the kinetics of fibrinolysis were quantitatively estimated. In the complex catalyzed fibrinolysis, plasminogen activation reaction dominated whereas in plasmin-catalyzed fibrinolysis, the inhibitor involved reaction, suppressing the process, prevailed.     

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.     

Inhibition of the process of Glu-plasminogen activation by tissue activator with fibrin, DDE-complex, and D-dimer using alpha-2-antiplasmin

The alpha-2-antiplasmin influence on the Glu-plasminogen activation by tissue activator both on fibrin and fibrin(ogen) fragments was investigated. The kinetics of activation was studied and velocity of this process in the absence and presence of the inhibitor was calculated. It was established that alpha-2-antiplasmin decreased the velocity of Glu-plasminogen activation on desAABBfibrin, DDE-complex and DD-dimer and did no influence upon proenzyme activation on fibrinogen fragment--Ho1-DSK. In the presence of fibrin plasminogen activation linear related to the amount added tissue activator in limit concentration from 5 before 50 units/ml. It was shown that alpha-2-antiplasmin reduced the activation velocity with used concentration of tissue activator. Fibrin hydrolysis by plasmin, forming on its surface during the plasminogen activation by tissue activator, was also inhibited with alpha-2-antiplasmin. The obtained results are explained by the influence of the inhibitor on formation of the triple complex between plasminogen, tissue activator and fibrin, and competition of the alpha-2-antiplasmin for lysine-binding sites of tissue activator kringle 2 or for binding sites of the activator on fibrin.     

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.     

Interaction of one-chain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin

Tissue-type plasminogen activator (t-PA) plays a central role in fibrinolysis in vivo. Although it is known to bind to fibrin, the dissociation constant (Kd) and number of moles bound per mole of fibrin monomer (n) have never been measured directly. In this study, the binding of both the one-chain form and the two-chain form of recombinant, human t-PA to fibrin was measured. Although more one-chain t-PA than two-chain t-PA is bound to fibrin, the Kd's and n's were within experimental error of each other. Significantly more t-PA is bound to clots made from fibrinogen which has been digested with plasmin than to clots made from intact fibrinogen. The additional binding was shown to be due to the formation of new set(s) of binding site(s) with dissociation constants that are 2-4 orders of magnitude tighter than the binding site present on clots made from intact fibrinogen. epsilon-Aminocaproic acid was capable of competing for the loose binding site present on both intact and degraded fibrin but had little effect on the binding of t-PA to the new site(s) formed by plasmin digestion. This increase in binding caused by plasmin-mediated proteolysis of fibrin suggests a possible mechanism for a positive regulation capable of accelerating fibrinolysis.     

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.     

Binding and activation of plasminogen on the platelet surface

A mechanism by which platelets might participate in fibrinolysis by binding plasminogen and influencing its activation has been examined. Binding of radioiodinated human Glu-plasminogen to washed human platelets was time-dependent and was enhanced 3-9-fold by stimulation of platelets with thrombin but not with ADP. The interaction with both stimulated and unstimulated cells was specific, saturable, divalent ion-independent, and reversible. The platelet-bound ligand had the molecular weight of plasminogen, and no conversion to plasmin was detected. Scatchard analyses provided evidence for a single class of plasminogen-binding sites on both stimulated and unstimulated cells. The Kd for thrombin-stimulated platelets was 2.6 +/- 1.3 microM, and 190,000 +/- 45,000 molecules were bound per cell, whereas unstimulated platelets bound 37,000 +/- 10,500 molecules/cell with a Kd of 1.9 +/- 0.15 microM. Plasminogen binding was inhibited in a dose-dependent manner by omega-aminocarboxylic acids at concentrations consistent with a requirement for an unoccupied high affinity lysine-binding site for plasminogen binding to the cells. When platelet-bound plasminogen was incubated with tissue plasminogen activator, urokinase, or streptokinase, gel analysis established that plasmin was preferentially associated with the platelet relative to the supernatant. Plasminogen and plasmin interacted with thrombin-stimulated platelets with similar binding characteristics, and there was no evidence for a binding site for plasmin which did not also bind plasminogen. Therefore, the results suggest that plasminogen activation is enhanced on the cell surface. In sum, these results indicate that platelets bind plasminogen at physiologic zymogen concentrations and this interaction may serve to localize and promote plasminogen activation.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.