Polyamidoamine (PAMAM) dendrimers are used in medicine for systemic drug delivery. The safety assessment of biomaterials, which come into contact with blood components and tissues is especially important. For example, PAMAM-NH2 dendrimers G4–G7 cause platelet and fibrinogen aggregation. We have studied the effect of the PAMAM-COOH dendrimers G1.5–G3.5 on key components of the fibrinolysis system. The dendrimers have not affected the plasmin activity. However, the amidolytic activities of tissue activators of plasminogen (tPA) and urokinase (uPA) have decreased markedly with increasing the dendrimer concentration and generation. The peak intensity at 340 nm of Glu-plasminogen fluorescence in the absence and presence of 0.15 M NaCl has decreased with increasing the dendrimer concentration, thus indicating the change in the microenvironment of the Trp residues and molecular conformation of the open and closed Glu-plasminogen forms. A comparison of the fluorescence quenching constants suggests that the anionic dendrimer and chloride ion simultaneously bind to Glu-plasminogen and each of these ligands exerts its independent effects through the separate binding sites. With increasing the dendrimer concentration, the rate of the tPA- and uPA-induced activation of Glu-plasminogen has dramatically decreased due probably to the formation of dendrimer*plasminogen complexes with the increasing dendrimer content, which makes the plasminogen bond increasingly unavailable for the cleavage by the activators. The rate of tPA- and uPA-induced lysis of the plasma clot in plasma has moderately decreased with increasing the dendrimer concentration due probably to the reduced effect of the dendrimer on the fibrin-bound plasminogen. Thus, the nature of the inhibitory action of the anionic PAMAM dendrimers on the Glu-plasminogen activation and tPA- and uPA-induced thrombolysis consists of a noticeable inactivation of the activators and a significant change in plasminogen conformation. The PAMAM-COOH dendrimers in circulation can affect other physiological and pathological processes, which are associated with an important role of the plasminogen/plasmin system.

For elucidation of the mechanisms of IgG binding with human plasminogen in prostate cancer patients, we propose an original ELISA on polystyrene plates with immobilized heavy and light plasminogen chains. The level of IgG bound to plasminogen heavy chain in the serum of prostate cancer patients significantly exceeded that in healthy volunteers. IgG treated with plasmin more actively (by more than 2 times) bound plasminogen heavy chain than intact IgG. These findings indicate the involvement of lysine-binding sites of plasminogen heavy chain in the interaction with the C-terminal lysine of IgG and their fragments. ROC analysis of ELISA data showed significant differences between serum samples from patients with prostate cancer and benign prostatic hyperplasia. It is hypothesized that IgG in the tumor region undergo proteolysis and their products appear in the circulation.

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 (IgG Pm-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 IgG Pm-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 IgG Pm-t . A preliminary treatment of IgG samples with carboxypeptidase В (CPB) inhibited the binding of IgG Pm-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 IgG Pm-t in the specific binding to the lysine-binding sites of Pg. The study of the effects of intact IgG and IgG Pm-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. IgG Pm-t accelerated the generation of plasmin from Pg. In this case, the stimulatory effect of IgG Pm-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 IgG Pm-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.

Covalent conjugates of streptokinase (SK) with polyamidoamine (PAMAM) dendrimers G1.5, G2.5, and G3.5 (SK–G1.5, SK–G2.5, and SK–G3.5) with the protein–polymer molar ratios of (1: 1), (1: 5), and (1: 10) were obtained and their properties were studied as compared to the properties of free SK. It was shown that the initial rates of formation of the modified Pm. SK complex, activation of plasminogen, and lysis of the plasma clot under the action of SK–dendrimer conjugates decreased with increasing number of bound dendrimers (from 1 to 10) and increased with increasing dendrimer generation (from G1.5 up to G3.5). Conjugates SK–G3.5 (1: 1) and (1: 5) were the most active compared to other conjugates. It was found that the catalytic efficiency of plasminogen activation (kPg/KPg) by conjugates SK–G3.5 (1: 1) (0.15 μM–1 min–1) and SK–G3.5 (1: 5) (0.12 μM–1 min–1) was comparable to the efficiency of free SK (0.18 μM–1 min–1). Probably, small in size, soft, and easily deformable dendrimers G1.5 and G2.5 are able to penetrate into the internal shielded cavities of the native SK molecule and there modify amino groups that are important for the effective formation of the Pm · SK complex. By contrast, the larger and more rigid molecule of dendrimer G3.5 modifies, mainly, exposed lysine residues in the SK molecule, without affecting the latent internal lysines. Conjugates SK–G3.5 (1: 1) and (1: 5), which had the maximum activator activity, retained up to 85% of thrombolytic activity compared to the activity of free SK. In addition, due to modification of the exposed lysines—most sensitive to proteolysis in the SK molecule—with dendrimer G3.5, which has the highest density of negative charge on its surface, SK–G3.5 (1: 1) and (1: 5) conjugates were more stable in plasma and caused less exhaustion of plasma levels of plasminogen, α2-antiplasmin, and fibrinogen than free SK in vitro. Thus, thrombolytic activity of the SK–dendrimer conjugates depends on the degree of modification of the amino groups of SK, size, stiffness, and density of the negative charge on the surface of the PAMAM dendrimer. Conjugates SK–G3.5 (1: 1) and (1: 5) are potential candidates for the development of a new thrombolytic agent.

•Thrombolytic activity of SK-PAMAM conjugates depends on dendrimer generation and modification degree of SK amino groups.•SK-dendrimers conjugates cause less fibrinogenolysis than free SK.•Compared with SK-G1.5 and SK-G2.5 conjugates, SK-G3.5 conjugates have the highest thrombolytic activity.

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.

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 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 (k cat/K m) 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 (k Pg/K Pg) 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 k Pg/K Pg of activation of both plasminogens by Pm•STA complex much more than by Pm•SK complex due to a decrease in K Pg 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.