L.I. Moukhametova

Synergy of in vitro Thrombolytic Action of Combinations of Recombinant Staphylokinase and Single-Chain Urokinase-Type Plasminogen Activator

Kinetics of lysis of human plasma clots immersed in plasma were studied in vitro at 37°C under the influence of recombinant staphylokinase, single-chain urokinase-type plasminogen activator (scu-PA), and their simultaneous and consecutive combinations. Staphylokinase and scu-PA caused concentration- and time-dependent lysis of the clots; 32 nM staphylokinase and 75 nM scu-PA separately caused 50% lysis in 4 h. At these equally effective concentrations staphylokinase in 4 h induced a significantly lesser exhaustion of the plasma plasminogen, α2-antiplasmin, and fibrinogen than scu-PA. Combinations of staphylokinase (<30 nM) and scu-PA (<75 nM) rendered synergic thrombolytic action on the clots. The synergy of thrombolytic action was more pronounced on the simultaneous addition of the two agents than on their consecutive addition, scu-PA 30 min after staphylokinase. In 4 h after the addition, staphylokinase (25 nM) or scu-PA (15 nM) induced 24% and 2% lysis, respectively, whereas the simultaneous and consecutive combination of the same concentrations of these agents induced 58% and 50% lysis, respectively. The simultaneous combination of 15 nM staphylokinase and 15 nM scu-PA resulted in maximal 3.8-fold increase in the thrombolytic effect as compared to the expected total effect of the individual agents. Synergic combinations of the two agents caused lesser exhaustion of plasma plasminogen, α2-antiplasmin, and fibrinogen as compared with the expected total effect of these agents used separately. Thus, simultaneous and consecutive combinations of staphylokinase and scu-PA in a relatively narrow range of their concentrations possessed synergistic fibrinselective thrombolytic action on the plasma clot in vitro.

Prolonged plasma clot lysis induced by acyl-derivatives of urokinase in vitro

Two acyl-derivatives of urokinase, p-trimethylam inocinnamoyl-urokinase (TMAC-Uk) and p-guanidinobenzoyl-urokinase (GB-Uk), reactivating with different rates (kreac were 6×10−4/s and 6×10−5/s, respectively) were prepared. In comparison with free urokinase, acyl-activators were more stable in human plasma, and their stability increased with the decrease in the reactivation rate. Plasma clotlysis induced by all three agents was time- and dose-dependent, but acyl-activators caused a more prolonged fibrinolysis and lengthened lag-phase than free urokinase. Slowly reactivating GB-Uk induced the most long-lasting clotlysis, whereas free urokinase was more effective for the first 3 h. A combination of GB-Uk with low dose urokinase promoted the long-lasting clotlysis with the shortened lag-phase.

Biospecific properties of lysine-containing polyelectrolyte coatings for blood contacting devices

Biospecific properties of thromboresistant bilayer and multilayer coatings based on polyelectrolyte complexes of modified copolymer of N-vinylpyrrolidone and maleic acid (VPMA) with chitosan, amphiphilic chitosan or albumin were investigated. VPMA contained an affinity ligand towards plasminogen, α-amino coupled lysine residues. Polyethylene and polystyrene surfaces were investigated before and after their covering by protective polyelectrolyte coatings. The specific adsorption of plasminogen (precursor of the fibrinolytic enzyme plasmin) from its solutions and from human plasma was investigated using these model systems. It was found that all coatings with the outer contact layer of the lysine-containing affinity polymer exhibited affinity towards plasminogen. However, multilayer polyelectrolyte coatings were more efficient than the bilayer coatings with a single layer application and the affinity polymer coatings without interlayer. The decrease in the degree of thrombogenicity of the materials modified by polyelectrolyte coatings has been demonstrated in vitro and ex vivo. Employment of the proposed modification of surfaces will improve hemocompatibility of medical devices.

Diffusional transport of urokinase and acyl-urokinase into fibrin and plasma clots: time of contact and fibrinolytic response in vitro

The effect of short-time contacts (2—20 min) of fibrin and plasma clots with solutions of urokinase and acyl-urokinase in a buffer or human blood plasma on the degree and duration of fibrinolysis was studied in vitro. Both plasminogen activators readily diffuse into clots in these time intervals and initiate prolonged dose-dependent lysis of washed fibrin clots devoid of plasma inhibitors. In a plasma clot—plasma system containing inhibitors, the fibrinolytic action of urokinase that penetrated is rapidly suppressed. Acyl-urokinase that penetrated into plasma clots supports prolonged thrombolysis in the absence of an activator in the surrounding plasma due to its resistance to the action of inhibitors and slow reactivation (kdeac = 6·10–5 s–1).