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.     

The effect of kringles K1-3, K4 and K5 on lysis of fibrin clots caused by the activation of Glu- and Lys-plasminogen by a tissue activator

Kringles K1-3, K4 and K5 are studied for their effect on tissue plasminogen activator-induced fibrin clot lysis in the presence of Glu- and Lys-plasminogen. It is established that kringles K4 and K5 inhibit fibrinolysis of Glu-plasminogen, and K1-3--that of Lys-plasminogen. The role of plasminogen molecule kringles in the plasminogen interaction with fibrin polymer is discussed.     

Plasminogen binding by alpha 2-antiplasmin and histidine-rich glycoprotein does not inhibit plasminogen activation at the surface of fibrin.

alpha 2-antiplasmin (alpha 2-AP) exerts its inhibitory effect on fibrinolysis by rapidly inhibiting the plasmin evolved; in addition, it has been suggested that interference with the binding of plasminogen to fibrin, a function shared with histidine-rich glycoprotein (HRGP), may also be significant in inhibition of fibrinolysis. To elucidate if plasminogen binding by these two alpha 2-globulins may decrease the generation of plasmin by tissue-type plasminogen activator (t-PA) at the surface of fibrin, a system mimicking the fibrin/plasma interface was used. Attempts were made to differentiate the plasminogen binding from the plasmin inhibitory function of alpha 2-AP. The activation of human Glu-plasminogen (native plasminogen with NH2-terminal glutamic acid) by fibrin-bound t-PA was performed in a plasma environment using either normal plasma, alpha 2-AP- or HRGP-depleted plasmas supplemented with increasing amounts of the lacking protein, or in a reconstituted system with purified plasminogen and various concentrations of alpha 2-AP and HRGP. The activation of Glu-plasminogen in alpha 2-AP-depleted plasma containing a normal concentration of HRGP produced a time-dependent increase in the generation of plasmin. The addition of 1 microM-alpha 2-AP to this plasma prevented the formation of Lys-derivatives and produced a marked decrease (42%) in the number of plasminogen-binding sites. In contrast, the addition of 1.5 microM-HRGP to HRGP-depleted plasma containing a normal amount of alpha 2-AP produced only a modest (17%) decrease in the amount of plasmin(ogen) bound. Moreover, in a purified system the amount of plasminogen-binding sites and thereby of plasmin generated at the surface of fibrin in the presence of both alpha-2 globulins was similar to the amount generated in the presence of alpha 2-AP alone. These results indicate clearly that the formation of reversible complexes between plasminogen and alpha 2-AP does not interfere with the binding and activation of plasminogen at the fibrin surface. In contrast, the inhibition of plasmin by alpha 2-AP decreases importantly the number of plasminogen-binding sites (carboxyl-terminal lysines) and inhibits thereby the accelerated phase of fibrinolysis. It can be concluded that interference of the binding of plasminogen to fibrin by alpha 2-AP during plasminogen activation, does not play a significant role in inhibition of fibrinolysis, and that the plasminogen-binding effect of HRGP, if any, is obscured by the important inhibitory effect of alpha 2-AP.     

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

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

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.     

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

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

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.     

Mechanism of action of θ-amino acids on plasminogen activation and fibrinolysis induced by staphylokinase

Stimulation of Lys-plasminogen (Lys-Pg) and Glu-plasminogen (Glu-Pg) activation under the action of staphylokinase and Glu-Pg activation under the action of preformed plasmin-staphylokinase activator complex (Pm-STA) by low concentrations and inhibition by high concentrations of omega-amino acids (>90-140 mM) were found. Maximal stimulation of the activation was observed at concentrations of L-lysine, 6-aminohexanoic acid (6-AHA), and trans-(4-aminomethyl)cyclohexanecarboxylic acid 8.0, 2.0, and 0.8 mM, respectively. In contrast, the Lys-Pg activation rate by Pm-STA complex sharply decreased when concentrations of omega-amino acids exceeded the above-mentioned values. It was found that formation of Pm-STA complex from a mixture of equimolar concentrations of staphylokinase and Glu-Pg or Lys-Pg is stimulated by low concentrations (maximal at 10 mM) of 6-AHA. Negligible increase in the specific activities of plasmin and Pm-STA complex was detected at higher concentrations of 6-AHA (to maximal at 70 and 50 mM, respectively). Inhibitory effects of omega-amino acids on the rate of fibrinolysis induced by staphylokinase, Pm-STA complex, and plasmin were compared. It was found that inhibition of staphylokinase-induced fibrinolysis by omega-amino acids includes blocking of the reactions of Pm-STA complex formation, plasminogen activation by this complex, and lysis of fibrin by forming plasmin as a result of displacement of plasminogen and plasmin from the fibrin surface. Thus, the slow stage of Pm-STA complex formation plays an important role in the mechanism of action of omega-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to alpha-->beta change of Glu-Pg conformation, stimulation of Pm-STA complex formation leads to increase in Glu-Pg activation rate in the presence of low concentrations of omega-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by omega-amino acids is responsible for appearance of long lag phases on curves of fibrinolysis induced by staphylokinase.     

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

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

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.     

Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin

The binding of human Glu- and Lys-plasminogens to intact fibrin clots, to lysine-Sepharose, and to fibrin cleaved by plasmin was quantitatively characterized. On intact fibrin clots, there was one strong binding site for Glu-plasminogen with a dissociation constant, Kd, of 25 microM and one strong binding site for Lys-plasminogen with a Kd of 7.9 microM. In both cases, the number of plasminogen binding sites per fibrin monomer was 1. Also, a much weaker binding site for Glu-plasminogen was observed with a Kd of about 350 microM. Limited digestion of fibrin by plasmin created additional binding sites for plasminogen with Kd values similar to the binding of plasminogen to lysine-Sepharose. This was predictable given the observations that plasminogen binds to lysine-Sepharose and can be eluted with epsilon-aminocaproic acid [Deutsch, D.G., & Mertz, E.T. (1970) Science (Washington, D.C.) 170, 1095-1096] and that plasmin preferentially cleaves fibrin at the carboxy side of lysyl residues [Weinstein, M.J., & Doolittle, R.F. (1972) Biochim. Biophys. Acta 258, 577-590], because the structures of the lysyl moiety in lysine-Sepharose and of epsilon-aminocaproic acid are identical with the structure of a COOH-terminal lysyl residue created by plasmin cleavage of fibrin. The Kd for the binding of Glu-plasminogen to lysine-Sepharose was 43 microM and for fibrin partially cleaved by plasmin 48 microM. The Kd for the binding of Lys-plasminogen to lysine-Sepharose was 30 microM. With fibrin partially cleaved by plasmin, there were two types of binding sites for Lys-plasminogen, one with a Kd of 7.6 microM and the other with a Kd of 44 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Binding of plasminogen to extracellular matrix

We have previously demonstrated that plasminogen immobilized on various surfaces forms a substrate for efficient conversion to plasmin by tissue plasminogen activator (t-PA) (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, R. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human plasminogen to the extracellular matrix synthesized in vitro by cultured endothelial cell monolayers. The binding was specific, saturable at plasma plasminogen concentrations, reversible, and lysine-binding site-dependent. Functional studies demonstrated that matrix immobilized plasminogen was a much better substrate for t-PA than was fluid phase plasminogen as shown by a 100-fold decrease in Km. Activation of plasminogen by t-PA and urokinase on the matrix was equally efficient. The plasmin generated on the matrix, in marked contrast to fluid phase, was protected from its fast-acting inhibitor, alpha 2-plasmin inhibitor. Matrix-associated plasmin converted bound Glu- into Lys-plasminogen, which in turn is more rapidly activated to plasmin by t-PA. The extracellular matrix not only binds and localizes plasminogen but also improves plasminogen activation kinetics and prolongs plasmin activity in the subendothelial microenvironment.     

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.     

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.     

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

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

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.     

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

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

Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator

The activity of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) is stimulated by heparin. Heparin binds tightly to t-PA, u-PA, and plasminogen and decreases the usual stimulatory effect of fibrin on t-PA activity. In the present study we have found that low molecular weight heparin (LMW-heparin) preparations obtained by nitrous acid depolymerization or heparinase treatment of standard heparin have different properties with respect to their interaction with the fibrinolytic system. LMW-heparin prepared by either method does not stimulate plasmin formation by t-PA. However, these preparations of heparin still efficiently accelerate the inhibition of thrombin by antithrombin III. Binding data show that LMW-heparin does not bind t-PA and Glu-plasminogen and only binds very weakly to Lys-plasminogen. These results illustrate that it is possible to selectively destroy the fibrinolytic stimulating properties of heparin while leaving the classical anticoagulant characteristics intact.     

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

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