R.B. Aisina

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.

Properties of the Urokinase-Type Plasminogen Activator Modified with Phenylglyoxal

A chemical modification of single-chain urokinase-type plasminogen activator (scu-PA) with phenylglyoxal under mild conditions resulted in scu-PA derivatives with various numbers of the modified Arg residues. The study of properties of the resulting derivatives demonstrated that the modification of 4–12 Arg residues did not cause any loss of the activator, fibrinolytic, and potential amidase activities of the activator. The scu-PA with four modified Arg residues was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the R178RHRGGS184 cluster, which was located in the superficial loop of the scu-PA globule and was shown to interact with a complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of scu-PA and the two-chain urokinase-type plasminogen activator for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator.

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 θ-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-amino-methyl)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 θ-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 θ-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 θ-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 θ-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to α→β 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 θ-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by θ-amino acids is responsible for appearance of long lag phases on curves of fibrinolysis induced by staphylokinase.

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.

Polymorphism of the plasminogen activator inhibitor type 1 gene, plasminogen level and thromboses in patients with the antiphospholipid syndrome

The frequency of venous and arterial thromboses and plasminogen level have been investigated in 78 patients with the antiphospholipid syndrome (APS), including 35 patients with systemic lupus erythematosus (SLE + APS) and 43 patients with primary APS (PAPS). The levels and genotypes of plasminogen activator inhibitor type 1 (PAI-1) were determined in 45 patients with APS (21 patients with SLE + APS and 24 patients with PAPS). A control group included 10 individuals without autoimmune disease signs and thromboses during the observation period and in anamnesis. It has been shown for the first time that for one third of 67 patients with APS and thromboses, high-positive levels of antiphospholipid antibodies (aPL) are associated with low plasminogen levels. The levels of PAI-1 antigen measured by the ELISA method, which detects active, latent and bound to plasminogen activator PAI-1, were compared with frequency of thromboses in APS patients. In one third of 43 patients with APS and thromboses the high and increased levels of PAI-1 were associated with high-positive aPL levels. One of possible mechanisms of this interrelationship was considered. It was shown that arterial and, to a greater extent, venous thromboses are associated with the 4G/5G polymorphism of the PAI-1 gene and high plasma level of the inhibitor in 79% of APS patients. In the presence of the 4G allele SLE + APS patients had higher PAI-1 levels than PAPS patients. The data obtained show that measuring the levels of plasminogen and PAI-1 as well as the 4G/5G polymorphism of the PAI-1 gene associated with thromboses may have the practical importance for identification of high risk of thrombosis in APS patients.

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.

Mechanism of the inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5, a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis, on kinetic parameters (kPg and KPg) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect on the kPg value, but increases the value of KPg plasminogen activation by urokinase. A decrease in the kPg value and an increase in the KPg value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is a competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA with a mixed type. Such an influence of angiostatin on the kinetic constants of the plasminogen activation by urokinase suggests that angiostatin dose-dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In the case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with the formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex, which leads to a decrease in kPg and an increase in KPg of the plasminogen activation. Inhibition constants by angiostatin (Ki) of plasminogen-activator activities of uPA and tPA determined by the Dixon method were found to be 0.59 ± 0.04 and 0.12 ± 0.05 μM, respectively.