Regina Heinzel-Wieland

Antileukoprotease: An Endogenous Protein in the Innate Mucosal Defense against Fungi

Previous studies have suggested that endogenous protease inhibitors may participate in the mucosal host defense. Antileukoprotease (ALP) is an important protease inhibitor found on various mucosal surfaces, including those of the respiratory and genital tracts. This study reports on the antimicrobial activity of recombinant (r) ALP toward the human fungal pathogens Aspergillus fumigatus and Candida albicans. rALP expressed pronounced fungicidal activity toward metabolically active A. fumigatus conidia and C. albicans yeast cells; however, metabolically quiescent A. fumigatus conidia were totally resistant. In contrast with the protease inhibitory activity of rALP, the fungicidal activity was localized primarily in the NH2-terminal domain. On a molar base, the fungicidal activity of rALP was comparable with that of human defensins and lysozyme. In addition, rALP caused inhibition of C. albicans yeast cell growth. By exhibiting antifungal activity, ALP may play an important role in the innate mucosal defense against human pathogenic fungi.

Thrombin inhibitory and clot-specific fibrinolytic activities of the urokinase variant, M23 (rscu-PA-40 kDa/Hir)

The recombinant bifunctional urokinase variant, M23 (rscu-PA-40 kDA/Hir), comprising the kringle and protease domain of single-chain urokinase-type plasminogen activator and a C-terminal fragment of hirudin in one single-chain molecule, was evaluated for its thrombin-inhibitory and fibrinolytic properties in vitro and in vivo. M23 inhibited thrombin-activated coagulation of human blood and thrombin-induced aggregation of human platelet rich plasma in a concentration-dependent manner. The ADP-induced aggregation of human platelet rich plasma was not influenced by M23. In contrast, recombinant single-chain urokinase-type plasminogen activator (saruplase) inhibited neither blood coagulation nor platelet rich plasma aggregation. M23 and saruplase both lysed radiolabelled human thrombi immersed in human plasma (Chandler Loop system) with equal potency. However, there was a significantly lower systemic generation of plasmin (measured as consumption of alpha 2-antiplasmin) by M23 compared to saruplase. In anaesthetized non-heparinized rabbits, experimental femoral artery thrombosis was treated with intravenous bolus injections of M23 or saruplase (6 mg/kg, each). Thrombolytic restoration of arterial blood perfusion was significantly higher in M23- than in saruplase-treated rabbits. Plasma fibrinogen concentrations were decreased markedly in saruplase-treated animals, but remained at significantly higher levels in M23-treated rabbits. In conclusion, the bifunctional molecule, M23, showed thrombin inhibitory and fibrinolytic properties in human in vitro systems and exerted superior thrombolytic effects to saruplase in rabbit femoral artery thrombosis. In vitro and in vivo data indicate that the fibrinolytic activity of M23 is highly clot-specific.