Plasminogen activation in diabetes mellitus. Kinetic analysis of plasmin formation using components isolated from the plasma of diabetic donors

Two components of the fibrinolytic system, plasminogen and the vascular plasminogen activator, have been isolated to apparent homogeneity from the post-venous occlusion plasma of three diabetic patients (hemoglobin A1C greater than 7%) and of one nondiabetic control person. Plasminogen activation was studied for each person separately in the absence and presence of CNBr fragments of fibrinogen. Activation of diabetic plasminogen by urokinase was not significantly altered as compared to the activation of control plasminogen. The same was found when diabetic plasminogen was activated by control vascular plasminogen activator in the presence of fibrinogen fragments but only at plasminogen concentrations below 10-30 nM; at higher substrate concentrations, however, plasminogen activation was impaired in a pattern resembling substrate inhibition. Activation of control plasminogen by diabetic vascular plasminogen activator was completely impaired in the absence of fibrinogen fragments. Addition of fibrinogen fragments stimulated plasmin formation by diabetic vascular plasminogen activator resulting in kinetic constants which were similar to the activation of control plasminogen by control vascular plasminogen activator in the absence of fibrinogen fragments (Km = 7.5 microM, kcat = 0.05 S-1). Addition of fibrinogen fragments in controls decreased Km values to less than 0.1 microM. Despite addition of fibrinogen fragments the rate of plasmin formation from diabetic plasminogen by diabetic vascular plasminogen activator isolated from the same diabetic donor was so small that kinetic constants could not be calculated.     

Fibrinogen lysine residue A alpha 157 plays a crucial role in the fibrin-induced acceleration of plasminogen activation, catalyzed by tissue-type plasminogen activator

In previous studies, we have shown that the stretch 148-197 of the fibrinogen A alpha chain plays a crucial role in the acceleration of the tissue-type plasminogen activator (t-PA)-catalyzed plasminogen activation. In this study we have synthesized parts of A alpha 148-197 and analogues thereof. We found that the peptides with sequences identical with A alpha 148-161 and A alpha 149-161 of human fibrinogen accelerate the plasminogen activation by t-PA, whereas the corresponding peptides in which lysine residues A alpha 157 had been replaced by valine or arginine had no accelerating capacity. Furthermore, succinylation of the lysine residue(s) in the synthesized peptides A alpha 148-161 and A alpha 149-161 leads to loss of accelerating action. These findings show that lysine residue A alpha 157 is crucial for the accelerating action of fibrin on the t-PA-catalyzed plasminogen activation.     

Plasminogen activator-anti-human fibrinogen conjugate

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.     

Fibrin‐targeted plasminogen activation by plasminogen activator,PadA, from Streptococcus dysgalactiae

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three‐domain streptokinase (SK) and two‐domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to α2‐antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single‐domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single‐domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single‐domain plasminogen activator that activates bovine plasminogen in a fibrin‐targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin‐specific plasminogen activators for thrombolytic therapy.     

Interaction of plasminogen and fibrin in plasminogen activation

Glu1-, Lys77-, miniplasminogens, kringle 1-3, kringle 1-5A, and kringle 1-5R were able to bind with fibrin, while microplasminogen and kringle 4 did not bind significantly. Kringle 1-5A, but not kringle 1-3, effectively inhibited the binding of Glu1-, Lys77-, and miniplasminogens with fibrin. Miniplasminogen also inhibited the binding of Glu1-plasminogen with fibrin. The binding of kringle 1-3 with fibrin was blocked by mini- or Glu1-plasminogen. It is therefore evident that there are two fibrin-binding domains in plasminogen and that the one in kringle 5 is of higher affinity than that in kringle 1-3. CNBr cleavage products of fibrinogen effectively enhanced the activation of Glu1-, Lys77-, or miniplasminogens, but not microplasminogen, by tissue-type plasminogen activator. Kringle 1-5, but not kringle 1-3, dose-dependently inhibited the enhancement by fibrinogen degradation products of Glu1-plasminogen activation by the activator. Lysine and epsilon-aminocaproic acid could inhibit the binding of plasminogens and plasminogen derivatives with fibrin and block the enhancement effect of fibrinogen degradation products on plasminogen activation. The data clearly illustrate that the binding of plasminogen with fibrin, mainly determined by kringle 5, is essential for effective activation by tissue-type plasminogen activator. However, the presence of kringle 1-4 in the plasminogen molecule is required for the full enhancing effect since the kcat/Km of miniplasminogen activation in the presence of fibrinogen degradation products was 8.2 microM-1 min-1 which is significantly less than 52.0 microM-1 min-1 of Glu1-plasminogen.     

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.     

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

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

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

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

Fibrinolytic and fibrinogenolytic effects of acyl urokinase and urokinase combinations in vitro

The in vitro thrombolytic and side effects of double-strand urokinase-type plasminogen activator (tcu-PA), p-guanidinobenzoyl tcu-PA (GB-tcu-PA), and their combinations were compared. The reversible blocking of the active site stabilizes GB-tcu-PA in human plasma and results in longer thrombolysis and lesser side effects than those of tcu-PA. However, the acylated activator displays a marked lag period of thrombolytic action. GB-tcu-PA and tcu-PA combinations (molar ratios 4 : 1 and 1 : 1) induce a prolonged and effective thrombolysis without the initial lag period at a low level of plasminogen, fibrinogen, and alpha2-antiplasmin depletion in the plasma surrounding the clot.     

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.     

Fibrinogen fragment D is necessary and sufficient to anchor a surface plasminogen-activating complex in Streptococcus pyogenes

In this study, the importance of different domains of the fibrinogen molecule in the binding and assembly of a surface plasminogen (plgn) activator has been analyzed. This was achieved using SELDI technology that enabled dissociation of bound fragments from intact bacteria and accurate distinction between fibrinogen fragments based on their molecular mass. These studies indicate that Streptococcus pyogenes binds directly to human fibrinogen fragment D but not fragment E. The predominant surface proteins binding to fragment D were associated with the mrp gene product. Surface-associated fibrinogen fragment D was capable of anchoring a functional surface plgn activator complex. Taken together, these data indicated that fragment D of fibrinogen is necessary and sufficient to anchor a plgn activator complex on the surface of Streptococcus pyogenes.     

Thrombolysis with tissue plasminogen activator in patients with acute myocardial infarction

Thrombolytic agents are being employed clinically in increasing numbers of patients in the attempt to eliminate occlusive coronary thrombi in patients with evolving myocardial infarction. When administered by the intracoronary route, streptokinase lyses is successful in coronary thrombi in more than two-thirds of patients, but when administered intravenously is successful in only one-third. Since streptokinase is a nonselective plasminogen activator, it induces fibrinogenolysis when administered selectively or systematically with an attendant marked reduction in plasma fibrinogen levels and significant bleeding complications. In contrast, the action of tissue plasminogen activator (t-Pa) is relatively selective for fibrinolysis (as opposed to fibrinogenolysis). It induces coronary thrombolysis in at least 60% of patients when administered either into a coronary ostium or a peripheral vein without producing substantial reductions in circulating fibrinogen. Bleeding complications are modest and usually related to high administered doses and concomitant heparinization, and occur primarily at sites of vascular access. Thus, t-Pa appears to be a promising agent for thrombolytic treatment of patients with evolving acute myocardial infarction.     

Identification of a site in fibrin(ogen) which is involved in the acceleration of plasminogen activation by tissue-type plasminogen activator

The rate of activation of plasminogen by tissue-type plasminogen activator is greatly increased by fibrin, but not by fibrinogen. A possible explanation for this phenomenon could be that conformational changes take place during the transformation of fibrinogen to fibrin which lead to exposure of sites involved in the accelerated plasmin formation. This is also supported by our recent observation that some enzymatically prepared fragments of fibrinogen and fibrin (D EGTA, D-dimer, Y) and also CNBr fragment 2 from fibrinogen have this property. CNBr fragment 2 consists of amino acid residues A alpha (148-207), B beta (191-224) + (225-242) + (243-305) and gamma 95-265, kept together by disulphide bonds. In order to study the localization of a stimulating site within this structure we purified the chain remnants of CNBr fragment 2 after reduction and carboxymethylation, and found that only A alpha 148-207 was stimulating. This was further confirmed by digesting pure A alpha-chains with CNBr and purifying the resulting A alpha-chain fragments. CNBr digests of B beta- and gamma-chains were not stimulatory. The A alpha-chain remnant (residues 111-197) in D EGTA and D-dimer also comprise the major part (residues A alpha 148-197) of the CNBr A alpha-chain fragment. We conclude that a site capable of accelerating the plasminogen activation by tissue-type plasminogen activator preexists in fibrinogen, that this site becomes exposed upon fibrin formation or disruption of fibrinogen by plasmin or CNBr and that this site is within the stretch A alpha 148-197, which is retained in the A alpha-chain remnants of fibrinogen degradation products.     

Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu144 through Leu411) of recombinant single-chain urokinase-type plasminogen activator

The cDNA encoding a low Mr derivative (residues 144-411) of human single-chain urokinase-type plasminogen activator was cloned, the recombinant low Mr single-chain urokinase-type plasminogen activator (rscu-PA-32k) was expressed in Chinese hamster ovary cells, and the translation product was purified to homogeneity from conditioned cell culture medium. rscu-PA-32k is very similar to intact recombinant single-chain urokinase-type plasminogen activator in terms of its very low activity (120 IU/mg) on a chromogenic substrate for urokinase (pyroglutamylglycylarginine p-nitroanilide), its plasminogen-dependent fibrinolytic activity on fibrin plates (specific activity = 170,000 IU/mg), its plasminogen activating potential, and the lack of specific binding to fibrin. In a rabbit jugular vein thrombosis model, comparable thrombolysis was obtained with rscu-PA-32k as compared to low molecular weight two-chain urokinase (50% lysis at 2.1 and 1.6 mg/kg infused over 4 h). Thrombolysis was associated with much less extensive systemic fibrinogen breakdown with rscu-PA-32k than with two-chain urokinase (residual fibrinogen at 50% lysis of 71 and 10%, respectively). It is concluded that the functional properties of rscu-PA-32k, expressed with a high efficiency, are similar to those of its previously characterized natural counterpart.     

Dehydroepiandrosterone therapy in men with angiographically verified coronary heart disease: the effects on plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and fibrinogen plasma concentrations

INTRODUCTION: The aim of this study was to analyze the influence of DHEA therapy on fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations in men with decreased serum DHEA-S levels and angiographically verified coronary heart disease (CHD). MATERIAL AND METHODS: The study included thirty men aged 41-60 years (mean age 52 +/- 0.90 yr) with serum DHEA-S concentration < 2000 mg/l, who were randomized into a double-blind, placebo-controlled, cross-over trial. Subjects completed the 80 days study of 40 days of 150 mg oral DHEA daily or placebo, and next groups were changed after 30 days of wash-out. Fasting early morning blood samples were obtained at baseline and after each treatment to determine serum hormones levels (testosterone, DHEA-S, LH, FSH and estradiol) and also fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations. RESULTS: Administration of DHEA was associated with 4.5-fold increase in DHEA-S levels. Estrogen levels significantly increased after DHEA from 22.1 +/- 0.7 pg/ml to 26.4 +/- 1.6 pg/l (mean +/- SEM; p < 0.05), while testosterone levels did not changed. Fibrinogen concentrations significantly decreased in DHEA group from 4.5 +/- 0.3 g/l to 3.83 +/- 0.2 g/l (p < 0.05 vs. placebo). Changes of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were not statistical significant (respectively: 8.37 +/- 0.4 ng/ml vs. 8.93 +/- 0.5 ng/ml and 82.3 +/- 6.3 ng/ml vs. 92.7 +/- 9.1 ng/ml (mean +/- SEM; NS vs. placebo). Tolerance of the treatment was good and no adverse effects were observed. CONCLUSIONS: DHEA therapy in dose of 150 mg daily during 40 days in men with DHEAS levels < 2000 mg/l and angiographically verified coronary heart disease (CHD) was connected with significant decreasing of fibrinogen concentration and increasing of estradiol levels, and did not influence on plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations.     

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.     

Fibrinogen–Fibrin System Regulators from Bloodsuckers

Thrombin inhibitors from bloodsucking leeches and insects that block conversion of fibrinogen to fibrin are considered. Regulatory mechanisms influencing the fibrinogen–fibrin system in leeches include fibrinogen degradation, inhibition of factor XIIIa, and lysis of fibrin clots. The review also summarizes recent data on plasminogen activator from the vampire bat Desmodus rotundus and a role of fibrin as its cofactor.     

Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator

Two-chain tissue-type plasminogen activator (t-PA), which consists of a heavy chain (Mr congruent to 38,000) and a light chain (Mr congruent to 31,000) connected by a disulfide bridge, was reduced with 2-mercaptoethanol and then air-reoxidized at a low protein concentration and carboxamidomethylated. The two chains were separated by means of zinc chelate-agarose, which was found to bind the light chain selectively. The light chain was fully active on the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine p-nitroanilide (S-2288) and partially active on plasminogen. The plasminogen activator activity of the light chain was, in contrast to that of two-chain t-PA, not stimulated by fibrin or fibrinogen fragments. Fibrin-agarose chromatography of radiolabeled chains showed that only the heavy chain bound to fibrin. These results indicate that the active site-containing light chain in t-PA needs the heavy chain for fibrin stimulation of its plasminogen activator activity.     

Fibrinolytic and fibrinogenolytic effects of acyl urokinase and urokinase combinations in vitro

The in vitro thrombolytic and side effects of double-strand urokinase-type plasminogen activator (tcu-PA), p-guanidinobenzoyltcu-PA (GB-tcu-PA), and their combinations were compared. The reversible blocking of the active site stabilizes GB-tcu-PA in human plasma and results in longer thrombolysis and lesser side effects than those of tcu-PA. However, the acylated activator displays a marked lag period of thrombolytic action. GB-tcu-PA and tcuPA combinations (molar ratios 4: 1 and 1: 1) induce a prolonged and effective thrombolysis without the initial lag period at a low level of plasminogen, fibrinogen, and α₂-antiplasmin depletion in the plasma surrounding the clot.     

Influence of beta-sitosterol on the fibrinolytic potential in rabbits

Long-term treatment of rabbits with beta-sitosterol (40 mg/kg over 3 months) caused an increased fibrinolytic activity in blood, an increased fibrinolytic capacity and an enhanced plasminogen activator activity in tissue of lungs and kidneys. The 3-months lasting beta-sitosterol administration did not influence the content of plasminogen activator inhibitor, plasminogen, alpha 2-antiplasmin, antithrombin III and fibrinogen.