L.I. Mukhametova

Structure and function of plasminogen/plasmin system

The main physiological function of plasmin is blood clot fibrinolysis and restoration of normal blood flow. To date, however, it became apparent that in addition to thrombolysis, the plasminogen/plasmin system plays an important physiological and pathological role in a number of other essential processes: degradation of the extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, and tumor cell migration. This review focuses on structural features of plasminogen, regulation of its activation by physiological plasminogen activators, inhibitors of plasmin, and plasminogen activators, and the role of plasminogen binding to fibrin, cellular receptors, and extracellular ligands in various functions performed by plasmin thus formed.

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.

Properties of streptokinase incorporated into polyethylene glycol microcapsules

Thrombolytic therapy with high doses of streptokinase (SK), which are required due to its rapid clearance from the bloodstream, is accompanied by side effects. In this work, the SK was incorporated into water-soluble polyethylene glycol (PEG) microcapsules with the double emulsification method in order to increase its lifetime in bloodstream and decrease side effects. Four preparations of SK*PEG-microcapsules with a high degree of the SK enclosure (∼90–91%) and total retention of fibrinolytic activity were produced under varying emulsification conditions (PEG molecular mass 20 or 40 kDa and PEG/SK ratio 12 or 8 mg of PEG/1000 IU SK). SK was released from the PEG-microcapsules at different rates: the time of complete release varied from 45 to 90 min (pH 7.4, 37°C). Comparative in vitro study of thrombolytic activity and side effects of the SK in a free and encapsulated state was conducted. It was found that the rate of human plasma clot lysis under the action of encapsulated SK preparations is equal (with exception of a short lag-period) to the rate of lysis induced by the free SK, provided that the doses were equal (500 IU/mL). Furthermore, the SK*PEG-microcapsules caused the reduced exhaustion of plasminogen and fibrinogen in plasma when compared with the free SK.

Thrombolytic and fibrinogenolytic properties of bioconjugate streptokinase-polyamidoamine dendrimers in vitro

• Thrombolytic activity of SK-PAMAM conjugates depends on dendrimer generation and modification degree of SK amino groups.

Streptokinase and staphylokinase: Differences in the kinetics and mechanism of their interaction with plasminogen, inhibitors, and fibrin

The comparative in vitro study of the kinetics of various reactions involved in the process of thrombolysis initiated by streptokinase (SK) and staphylokinase (STA) has been carried out. It has been shown that upon the interaction of plasminogen (Pg) with SK or STA in equimolar quantities, the formation rate and the specific esterase activity of the complex plasmin (Pm) with SK (Pm•SK) is higher than those of the complex Pm•STA. The catalytic efficiency (kcat/Km) of hydrolysis of the chromogenic plasmin substrates by Pm•SK complex is 2 times higher than by Pm•STA complex. In the absence of fibrin, the catalytic efficiency (kPg/KPg) of activation of Glu-plasminogen and Lys-plasminogen glycoform II by Pm•SK complex is higher than by Pm•STA complex, but the presence of fibrin increases kPg/KPg of activation of both plasminogens by Pm•STA complex much more than by Pm•SK complex due to a decrease in KPg In contrast to STA (15.5 kDa), an SK molecule (47 kDa) creates remarkable steric hindrances for the interaction of plasmin in Pm•SK complex with protein inhibitors. In addition, SK causes higher fibrinogen degradation in plasma than STA. It has been shown that Pm•SK and Pm•STA complexes lyse fibrin clots in buffer with similar rates, while the rate of lysis of plasma clots, immersed in plasma, by Pm•STA complex is remarkably higher than in the case of Pm•SK complex. It has been revealed that the species specificity of STA and SK is determined mainly by the rate of formation and the efficiency of Pm•SK and Pm•STA complexes in the activation of autologous plasminogen. The lysis efficiency of plasma clots of mammals falls in the series: human > dog > rabbit for SK and dog > human > rabbit for STA. The results show that in the purified system SK is a more effective plasminogen activator than STA. In the system containing fibrin and α2-AP, the activator and fibrinolytic activities of STA are higher than those of SK, due to the increased stability in plasma and fibrin specificity of STA, the fast reaction of the complex Pm•STA with α2AP, and the ability of the STA to recycling in the presence of α2AP.

Streptokinase-polyethylene glycol conjugates with increased stability and reduced side effects

Covalent SK-PEG2 and SK-PEG5 conjugates with various degrees of modification of the protein amino groups were obtained by variation of the duration of streptokinase (SK) incubation with activated polyethylene glycol (M 2 and 5 kDa, PEG2 and PEG5); their properties were studied in comparison with the properties of unmodified SK in vitro. SK-PEG2 and SK-PEG5 conjugates with the highest stability in plasma retaining 80% of initial fibrinolytic activity were formed at modification degrees of 54 and 52%, respectively. Interaction of the conjugates with equimolar plasminogen resulted in the formation of plasmin (Pm) activator complexes Pm·SK-PEG2 and Pm·SK-PEG5 with the maximum amidase activity being the same as that of Pm complex with native SK. Catalytic efficiency of plasminogen activation (kPg/KPg) was found to be slightly higher (2.84 min−1 μM−1) in case of Pm·SK-PEG2 complex and slightly lower, in case of the Pm·SK-PEG5 complex (1.17 min−1 μM−1), if compared to that of the unmodified complex Pm·SK (2.1 min−1 μM−1). Investigation of lysis kinetics of human plasma clot and depletion of plasminogen and fibrinogen plasma levels under the effect of equal doses of SK in free and conjugated forms demonstrated that SK-PEG2 and SK-PEG5 conjugates possess high thrombolytic activity (89 and 72% to the activity of free SK, respectively) and cause 3.5–4-fold lower side effects than free SK. The SK-PEG2 and SK-PEG5 conjugates with increased stability in plasma and reduced side effects may be used in therapy of thrombotic disorders.

Autoantibodies to Plasminogen and Their Role in Tumor Diseases

Plasma level of IgG autoantibodies to plasminogen was measured by ELISA in patients with benign prostatic hyperplasia (n=25), prostatic cancer (n=17), lung cancer (n=15), and healthy volunteers (n=44). High levels of IgG to plasminogen were found in 2 (12%) of 17 healthy women, in 1 (3.6%) of 27 specimens in a healthy man, in 17 (68%) of 25 specimens in prostatic cancer, in 10 (59%) of 17 specimens in lung cancer, and in 5 (30%) of 15 specimens in benign prostatic hyperplasia. Comparison of plasma levels of anti-plasminogen IgG by affinity chromatography showed 3-fold higher levels in patients with prostatic cancer vs. healthy men.

The in vitro cross-effects of inhibitors of renin-angiotensin and fibrinolytic systems on the key enzymes of these systems

The effects of hypotensive agents (captopril, enalaprilat, and lisinopril) on the activities of components of the fibrinolytic system (FS) and the effects of antifibrinolytic agents (6-aminohexanoic acid (6-AHA) and tranexamic acid (t-AMCHA)) on the activities of angiotensin converting enzyme (ACE) were studied in vitro. Enalaprilat did not affect the FS activity. Captopril considerably inhibited the amidase activities of urokinase (u-PA), tissue plasminogen activator (t-PA), and plasmin ([I]50 (2.0−2.6) ± 0.1 mM), and the activation of Glu-plasminogen by t-PA and u-PA ([I]50 (1.50−1.80) ± 0.06 mM), which may be due to the presence of a mercapto group in the inhibitor molecule. Lisinopril did not affect the amidase activities of FS enzymes, but stimulated Glu-plasminogen activation by u-PA and inhibited activation fibrin-bound Glu-plasminogen by t-PA ([I]50 (12.0 ± 0.5) mM). Presumably, these effects can be explained by the presence in lisinopril of a Lys side residue, whose binding to lysine-binding Glu-plasminogen centers resulted, on the one hand, in the transformation from its closed conformation to a semi-open one and, on the other hand, in its desorption from fibrin. Unspecific inhibition of the activity of ACE, a key enzyme of the renin-angiotensin system, in the presence of 6-AHA and t-AMCHA ([I]50 10.0 ± 0.5 and 7.5 ± 0.4 mM, respectively) was found. A decrease in the ACE activity along with the growth of the fibrin monomer concentration was revealed. The data demonstrate that, along with endogenous mediated interaction between FS and RAS, relations based on the direct interactions of exogenous inhibitors of one system affecting the activities of components of another system can take place.

Effect of Specific Cleavage of Immunoglobulin G by Plasmin on the Binding and Activation of Plasminogen

A method of ELISA for measuring the binding of different samples of immunoglobulin (IgG) and its fragments to human plasminogen (Pg) has been developed. Instead of plasminogen, the heavy chain of plasminogen (Pg-H) containing five ligand-binding kringle domains, immobilized on the surface of the plate, was used in this method as a detector. It was found that IgG treated with plasmin (IgGPm-t) binds to the immobilized Pg-H 2.84 times more strongly than intact IgG. Both IgG samples showed a weak nonspecific binding to the immobilized light chain of plasminogen (Pg-L). It was shown that 0.2 M L-lysine inhibits the binding of IgGPm-t and does not affect the nonspecific binding of intact IgG to the immobilized Pg-H, indicating the involvement of lysine-binding regions of Pg-H in binding to IgGPm-t. A preliminary treatment of IgG samples with carboxypeptidase В (CPB) inhibited the binding of IgGPm-t and did not affect the nonspecific binding of intact IgG to the immobilized Pg-H, which indicates a key role of the С-terminal lysine of IgGPm-t in the specific binding to the lysine-binding sites of Pg. The study of the effects of intact IgG and IgGPm-t on the rate of activation of Glu- and Lys-forms of Pg (Glu-Pg and Lys-Pg) by a tissue activator of Pg (tPA) and urokinase (uPA) in buffer showed that intact IgG completely inhibited the activation of Glu-Pg and Lys-Pg with both tPA and uPA. Presumably, the inhibitory effect of intact IgG is due to steric hindrances that it creates for protein–protein interactions of the activators with the zymogen. IgGPm-t accelerated the generation of plasmin from Pg. In this case, the stimulatory effect of IgGPm-t on the activation of Glu-Pg under the action of tPA was ∼25% higher than on the activation of Lys-Pg, which is explained by more significant conformational changes in the Glu-Pg molecule compared with the Lys-Pg molecule after their binding to IgGPm-t. The results suggest that the specific cleavage of IgG by plasmin may be one of the ways by which the plasminogen/plasmin system is involved in various physiological and pathological processes.

PO-17 - Identification of IgG bound to plasminogen in oncologic diseases

INTRODUCTION The binding of plasminogen (Pg) to cell receptors and extracellular ligands facilitates its activation to plasmin, which stimulates the extracellular matrix degradation, neoangiogenesis and tumor invasion. Plasmin can also degrade IgG thereby exposing C-terminal lysine residues. Previously, we have found IgG specifically bounded to Pg in the plasma of patients with malignant tumors. AIM To identify IgG degraded by plasmin in the plasma of cancer patients. MATERIALS AND METHODS Methods of ELISA were used for comparative research of levels of IgG bound to Pg in plasma of patients with the prostate cancer (PC, n=25) and lung cancer (LC, n=17). Plasma of healthy donors (n=29) was used as control. All patients signed informed consent for participation in this study. Affinity chromatography on Pg-sepharose was used for the quantification of IgG. Carboxypeptidase was used for remove of C-terminal lysine residues of the IgG. The program ATTESTAT was used for nonparametric analysis. RESULTS The frequency of occurence of elevated levels of IgG to Pg in plasma was detected in 68% of patients with PC, 59% of patients with LC and only 12% of healthy women and 10% of healthy men. The quantification of antibodies in plasma samples showed that the quantity of IgG to Pg in patients with PC was 27% from the total amount of IgG and in healthy men - 9%. Treatment of diluted plasma samples with carboxypeptidase B abolished the elevated levels of IgG to Pg, as well as the specific activity of the purified IgG to Pg-sepharose. CONCLUSIONS C-terminal lysine residues which are formed as a result of degradation of native IgG with plasmin can bind to lysine binding sites on the kringle domains of Pg. Increased levels of these degraded IgG can be marker at cancer.