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.

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.