Comparative in vitro study of the kinetics of various reactions involved in the process of thrombolysis initiated by streptokinase (SK) and staphylokinase (STA) was carried out. It was shown that at the interaction of an equimolar ratio of plasminogen (Pg) with SK or STA the rate of formation and the specific esterase activity of the complex plasmin (Pm) · SK are higher than those of the complex Pm · STA. The catalytic efficiency (kcat/Km) of hydrolysis of the chromogenic plasmin substrates by Pm · SK complex was 2 times higher than by Pm · STA complex. In the absence of fibrin catalytic efficiency (kPg/K(Pg)) of activation of Glu-plasminogen and Lys-plasminogen glycoform II by Pm · SK complex was higher than by Pm · STA complex, but the pres- ence of fibrin increased kPg/K(Pg)) activation of both plasminogens by Pm · STA complex significantly stronger than by Pm · SK complex due to the decrease in K(Pg)). In contrast to STA (15.5 kDa), SK molecule (47 kDa) creates significant steric hindrances for the interaction of plasmin in Pm · SK complex with protein inhibi- tors. In addition, SK caused greater fibrinogen degradation than STA. It is 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 plas- ma, by Pm · STA complex are significantly higher than those by Pm · SK complex. It was revealed that the species specificity of STA and S K 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 fell in the series: human > dog > rabbit for SK and the dog > human > rabbit for STA. The results show that in the purified system SK is a more effective activator of plasminogen than STA. In the system con- taining 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 recyclization in the presence of α2AP.
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 frequency of venous and arterial thromboses and plasminogen level were investigated in 78 patients with antiphospholipid syndrome (APS), 35 of whom with systemic lupus erythematosus (SLE+APS) and 43 - with primary APS (PAPS). The levels and genotype of plasminogen activator inhibitor type 1 (PAI-1) were determined in 45 patients with APS, of whom 21 patients with SLE+APS and 24 patients with PAPS. A control group included 10 healthy individuals without autoimmune disease signs and thromboses on period of investigation and in past history. It was 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 ELIZA method, which detects active, latent and bound with plasminogen activator PAI-1, were opposed with frequency of thromboses in APS patients. Correlation between the high and increased levels of PAI-1 and high positive aPL levels was found for one third of 43 patients with APS and thrombosis. One of the possible mechanisms of this interconnection was considered. It was shown that arterial and, to a more extent, venous thromboses are associated with the 4G/5G polymorphism of PAI-1 gene and high plasma level of the inhibitor in 79% of APS patients. At the presence of the 4G allele patients with SLE+APS had higher PAI-1 levels than patients with PAPS. The obtained results show that measuring the levels of plasminogen and PAI-1 as well as the 4G/5G polymorphism of PAI-1 gene which is associated with thromboses may have the practical significance for identification of high risk of thrombosis in APS patients.
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 microM), 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 microM) had no effect, while 2 microM 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.
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