Regulation with alpha-2-antiplasmin of Glu-plasminogen activation by tissue activator on fibrin

Interaction of tissue plasminogen activator with alpha-2-antiplasmin and its influence on tissue activator binding to fibrin was studied. Alpha-2-Antiplasmin decreases the binding of tissue activator to fibrin by 20%. The inhibitor formed a complex with tissue plasminogen activator (Kd 78.2 nM) and had no effect on amidolytic activity of the activator. The tissue activator binding to alpha-2-antiplasmin decreases by 20-35% in the presence of 6-aminohexanoic acid. It indicates that not only kringle 2 of the tissue activator molecule takes part in complex formation with alpha-2-antiplasmin, but also other activator domains. Two models were proposed to explain the alpha-2-antiplasmin effect on the Glu-plasminogen activation by tissue activator on fibrin. In the first place, the inhibitor binds to fibrin in the site where the activator complex is localized. It can create steric hindrances for the proenzyme interaction with its activator on fibrin. In the second place, alpha-2-antiplasmin in a complex with tissue plasminogen activator can bring to a change in the activator conformation and a decrease of its functional activity.     

Binding of alpha-2-antiplasmin with fibrinogen/fibrin and their fragments independent of factor XIII

Possible interaction of alpha-2-antiplasmin with fibrinogen, fibrin and their fragments independent of factor XIII as well as the inhibitor effect on the Glu-plasminogen activation by tissue activator were studied. It was shown that alpha-2-antiplasmin is adsorbed on desAA- and desAABBfibrin films (Kd 69.0 +/- 1.0 nM 68.6 +/- 5.3 nM, respectively). Glu-Plasminogen has no effect on the inhibitor binding with desAABBfibrin. Alpha-2-antiplasmin shows strong affinity for fibrin D-dimer (Kd 65.0 +/- 4.0 nM) and D-fragment of fibrinogen (Kd 119.0 +/- 21.0 nM), but it does not interact with E-fragment. The inhibitor inside the fibrin clot decreases 10 times the activation rate of Glu-plasminogen by the tissue activator both is the presence and without factor XIII at physiological ratio of Glu-plasminogen, tissue activator, fibrin and alpha-2-antiplasmin. Thus we have shown that fibrinogen/fibrin binds alpha-2-antiplasmin independent of the factor XIII. Binding sites of the inhibitor are localized in D-fragment of fibrinogen and/or fibrin D-dimer. Alpha-2-antiplasmin inhibits the Glu-plasminogen activation by tissue activator on fibrin.     

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.     

Cross-linking of plasminogen activator inhibitor 2 and alpha 2-antiplasmin to fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen Aalpha chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysine 303 of the Aalpha chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-alphaC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aalpha chain, respectively. PAI-2 or alpha(2)-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aalpha chain. alpha(2)-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the Aalpha chain. Therefore, PAI-2 and alpha(2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis.     

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.     

Plasminogen activation by tissue activator is accelerated in the presence of fibrin(ogen) cyanogen bromide fragment FCB-2

Fibrin, in contrast to fibrinogen, strongly accelerates the plasminogen activation by extrinsic activator (tissue-type plasminogen activator, t-PA). However, when fibrin and fibrinogen are digested with cyanogen bromide, both digests potentiate the t-PA-mediated plasminogen activation equally well. In this report, evidence is presented that this potentiating activity resides in CNBr fragment FCB-2 (= Ho1-DSK) and that a polymeric structure such as fibrin is not a prerequisite for the potentiation.     

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.     

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.     

Plasminogen activation by a tissue activator and effector properties of fibrinogen-N-terminal disulfide (N-DSK) fibrin complex

Fibrinogen-NDSK complex is a model of protofibril having some features of the fibrin polymer structure. This complex has been studied for its ability to stimulate the plasminogen activation by t-PA. The fibrinogen-NDSK complex have increased the rate of plasminogen activation by t-PA as compared to fibrinogen or NDSK taken separately. This acceleration had slow and fast phases. Lys-plasminogen was activated more effectively as compared to glu-plasminogen. The kinetic parameters of glu- and lys-plasminogen activation at fast phase were: Km--0.18 and 0.015 mu/M, Kkat--0.27 and 0.06 s-1, respectively. Fibrinogen X2--fragments, deprived of alpha C-domains and NH2-end peptides of bB-chains, formed complexes with NDSK, which however did not stimulate the plasminogen activation by t-PA. These findings have shown that the fibrinogen-NDSK complex is an effective stimulator of the plasminogen activation by t-PA. The activating ability of the complex may be due to structures formed in the course of fibrinogen and NDSK polymerization as a result of alpha C-domain interaction.     

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

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

Kinetic characteristics of the activation of various structural forms of plasminogen by tissue activator in the presence of fibrin

It was shown that activation of two native plasminogen and miniplasminogen forms by the tissue activator in the presence of fibrin obeys the Michaelis-Menten kinetics. The kinetic parameters of activation of both plasminogen native forms differ insignificantly. For miniplasminogen whose molecule contains no lysine-binding sites, a marked decrease of activation power was observed. The Km value of activator for miniplasminogen is 10 times that of plasminogen form I and 20 times that of plasminogen form II. The kcat/Km value of activator for miniplasminogen is 7 times less than that of plasminogen form I and by one order of magnitude more than that of plasminogen form II. These results testify to the importance of lysine-binding sites in the native plasminogen molecule during the activation of fibrinolysis by the major physiological activator.     

On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid

The binding of tissue-type plasminogen activator (t-PA) to fibrin is mediated both by its finger domain and by its kringle-2 domain. In this report, we investigate the relative affinities of these domains for lysine. Human recombinant t-PA deletion-mutant proteins were prepared and their ability to bind to lysine-Sepharose was investigated. Mutants containing the kringle-2 domain bound to lysine-Sepharose, whereas mutants lacking this domain but containing the finger domain, the epidermal growth factor domain or the kringle-1 domain did not bind to lysine-Sepharose. Mutant proteins containing the kringle-2 domain could be specifically eluted from lysine-Sepharose with epsilon-amino caproic acid. This lysine derivative also abolished fibrin binding by the kringle-2 domain but had no effect on the fibrin-binding property of the finger domain. Thus, a lysine-binding site is involved in the interaction of the kringle-2 domain with fibrin but not in the interaction of the finger domain with fibrin. The implications of the nature of these two distinct interactions of t-PA with fibrin on plasminogen activation by t-PA will be discussed.     

Influence of various structural domains of fibrinogen and fibrin on the potentiation of plasminogen activation by recombinant tissue plasminogen activator

Fibrinogen, fibrin, and related fragments have varying stimulatory effects on the initial rate of the activation of human plasminogen ([Glu¹]Pg) by recombinant tissue plasminogen activator (rt-PA). A detailed analysis of this enhancement was undertaken using various purified and complexed forms of the known domains of fibrin(ogen) with a view to gaining additional knowledge regarding the substructures of fibrinogen and fibrin that are important for their stimulatory capacities. Both arvin-mediated fibrin, as well as fibrinogen fragments generated as a result of its cleavage with CNBr, stimulate the activation in a biphasic manner, most likely as a result of changes in the promoter molecule accompanying the denaturation processes that are normally employed to either solubilize or generate these particular promoters. Using purified fibrinogen and fibrin fragments, it was found that fragment E, which binds to [Glu¹]Pg, does not enhance the activation reaction, while fragment D₁ has a potentiating effect. This suggests that the binding of [Glu¹]Pg to fibrin(ogen) alone is not, in itself, sufficient for stimulation of activation to occur, but that the rt-PA-fibrin(ogen) interaction is fundamental to this same process. All purified and mixtures of fragments containing the fragment D domain (e.g., D₂E, X-oligomer, fragment X) stimulate the reaction to a greater degree than fibrinogen and fragment D₁. It is concluded that the fibrinogen D domain is asine qua non for the enhancement reaction, while structures containing the E domain had a symbiotic effect on enhancement.On study leave from the National Institute for Biological Standards and Control, South Mimms, HERTS EN6 3QG, England.     

On the structure of the stable complex between plasmin and alpha-2-antiplasmin

Rabbits were immunized with a conjugate of leukotriene (LT) C₄ and bovine serum albumin prepared by coupling the single free amino group of the hapten to the protein using gluteraldehyde. Binding of [³H]LTC₄ to the antibodies obtained is inhibited by 50% with 1.5 ng LTC₄. The relative cross-reaction of LTD₄ is 16% and of LTC₄-methyl ester 3.6%. The validity of the radioimmunoassay was demonstrated by comparison with bioassay using the isolated guinea pig ileum. Using the radioimmunoassay it could be shown that endogenous LTC₄ is released in a dose-dependent manner by human polymorphonuclear leucocytes stimulated with the divalent cation ionophore A23187.     

Serum antithrombin III and alpha-2-antiplasmin concentration in patients with lung carcinoma

Serum antithrombin III and alpha-2-plasmin inhibitor concentrations has been evaluated in 26 patients with lung carcinoma. We observed a twofold decrease in antithrombin III level and no differences between test and control groups in alpha-2-plasmin inhibitor concentrations. Evidently, the decreased antithrombin III level may reflect its consumption because of enhanced plasma thrombin activity, whereas normal alpha-2-plasmin inhibitor may result in no induction of secondary fibrinolysis followed by the stimulation of the coagulation system or no conditions for primary increased fibrinolysis. It seems possible, that the antithrombin III level in the serum may at least partly reflect the tendency for hypercoagulability and spreading of cancer.     

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.     

Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes

Tissue kallikrein and factor Xa were found to activate tissue plasminogen activator (t-PA) at a rate comparable with that of plasmin. During the activation reaction, the single-chain molecule was converted into a two-chain form. A slight t-PA activating activity was also found in plasma kallikrein. Other activated coagulation factors, factor XIIa, factor XIa, factor IXa, factor VIIa, thrombin and activated protein C had no effect on t-PA activation. t-PA was also activated by a tissue kallikrein-like enzyme that was isolated from the culture medium of melanoma cells. These results indicate that tissue kallikrein and factor Xa may participate in the extrinsic pathway of human fibrinolysis.     

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.     

Effect of streptokinase on the interaction of alpha-2-antiplasmin with different sites of plasmin molecule

Interaction of streptokinase and alpha-2-antiplasmin with plasmin and plasminogen fragments was compared. Binding sites on the enzyme become half-saturated, streptokinase and alpha-2-antiplasmin concentration being 8.5 and 30 nM, respectively. 6-Aminohexanoic acid in concentration of 20 mM reduces the adsorption of streptokinase and and alpha-2-antiplasmin by 20 and 60%, respectively. From all the investigated fragments, streptokinase shows the greatest affinity for mini-plasminogen and alpha-2-antiplasmin for kringles 1-3. Both proteins in the presence of 20 mM 6-aminohexanoic acid do not bind with kringle domains. Arginine dose 0.1 M does not influence streptokinase adsorption on mini-plasminogen and decreases the value of alpha-2-antiplasmin binding with mini-plasminogen by 50%. The data obtained indicate that plasminogen molecule has the sites of the highest affinity for streptokinase on the serine-proteinase domain, however for alpha-2-antiplasmin it is in the kringles 1-3. Streptokinase with equimolar quantity in respect of alpha-2-antiplasmin inhibits the adsorption of alpha-2-antiplasmin on the plasmin by 70% and in the presence of 6-aminohexanoic acid it is inhibited completely. Addition of streptokinase also increases the influence of increasing concentration of the acid. Inhibiting influence of streptokinase decreases, and that of 6-aminohexanoic acid increases, when plasmin is modified with diisopropylfluorophosphate in its active centre. At the same time maximum inhibition of streptokinase adsorption on the plasmin at different concentrations of alpha-2-antiplasmin and 6-aminohexanoic acid accounts for only 20%. We suppose that in the process of complex formation streptokinase competes with alpha-2-antiplasmin for the binding sites on the catalytic domain of the plasmin. Partial or complete blocking of the plasmin active centre contact zone by streptokinase effectively protects it from inhibition by alpha-2-antiplasmin.