Structure and Function of Trypsin-Loaded Fibrinolytic Liposomes

Protease encapsulation and its targeted release in thrombi may contribute to the reduction of haemorrhagic complications of thrombolysis. We aimed to prepare sterically stabilized trypsin-loaded liposomes (SSLT) and characterize their structure and fibrinolytic efficiency. Hydrogenated soybean phosphatidylcholine-basedSSLTwere prepared and their structure was studied by transmission electron microscopy combined with freeze fracture (FF-TEM), Fourier transform infrared spectroscopy (FT-IR), and small-angle X-ray scattering (SAXS). Fibrinolytic activity was examined at 45, 37, or 24°C on fibrin or plasma clots with turbidimetric and permeation-driven lysis assays. Trypsin was shown to be attached to the inner surface of vesicles (SAXS and FF-TEM) close to the lipid hydrophilic/hydrophobic interface (FT-IR). The thermosensitivity ofSSLTwas evidenced by enhanced fibrinolysis at 45°C: time to reduce the maximal turbidity to 20% decreased by 8.6% compared to 37°C and fibrin degradation product concentration in the permeation lysis assay was 2-fold to 5-fold higher than that at 24°C.SSLTexerted its fibrinolytic action on fibrin clots under both static and dynamic conditions, whereas plasma clot dissolution was observed only in the permeation-driven assay. The improved fibrinolytic efficiency ofSSLTunder dynamic conditions suggests that they may serve as a novel therapeutic candidate for dissolution of intravascular thrombi, which are typically exposed to permeation forces.

The Binding of Diiodosalicylate and Flufenamate to the Plasma Antiactivator: Analysis of Chemical Fibrinolysis

SummaryThe fibrinolytic effect of diiodosalicylate was expressed as apparent units urokinase. The dissociation constant of the urokinase-inhibitor complex is increased 19 times in the presence of 10-2 M diiodosalicylate. The binding of diiodosalicylate to the antiactivator was estimated and the increase in free urokinase in the presence of diiodosalicylate was calculated. With 10-2 M diiodosalicylate present only 4% of the inhibitor remains free to bind urokinase. Therefore, the fibrinolytic action of diiodosalicylate can be explained on the basis of its binding to the inhibitor and the release of free urokinase.