Staphylokinase as a plasminogen activator component in recombinant fusion proteins.

The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.

Inducible nitric oxide synthase-deficient mice have enhanced leukocyte-endothelium interactions in endotoxemia.

Nitric oxide (NO) from constitutive NO synthase (NOS) has been postulated to be a homeostatic regulator of leukocyte-endothelial cell interactions. By contrast, the inducible NO synthase (iNOS) isoform has been invoked as a potential pathogenic enzyme in numerous inflammatory diseases. The objective of this study was to determine whether the iNOS isoform is also capable of functioning as a regulator of leukocyte recruitment. Mice received endotoxin (LPS, 30 microg/kg, i.v.); 2-4 h later, intravital microscopy was used to examine leukocyte rolling and adhesion in postcapillary venules of the cremaster muscle and the sinusoids and postsinusoidal venules of the hepatic microcirculation. Leukocyte recruitment into the lung was also examined. RT-PCR confirmed that this treatment induced iNOS mRNA expression in wild-type mice as early as 2 h after LPS treatment. Between 2 and 4 h after LPS administration, the number of rolling and adherent leukocytes in cremasteric postcapillary venules and of adherent cells in liver postsinusoidal venules of iNOS-deficient mice were significantly higher than in wild-type mice. Leukocyte accumulation in the lung (measured by myeloperoxidase assay) was also significantly elevated in iNOS-deficient animals. These effects could not be attributed to differences in systemic blood pressure, shear rates, circulating leukocyte numbers, or baseline levels of rolling and adhesion because these parameters were not different between the two groups. To establish whether the differences in leukocyte recruitment were related to the leukocytes per se, perfusion of iNOS+/+ or iNOS-/- septic blood over purified E-selectin (using parallel plate flow chambers) revealed much larger recruitment of iNOS-/- leukocytes. These results suggest that iNOS induced in response to LPS releases NO that is capable of reducing leukocyte accumulation by affecting leukocytes directly and raises the possibility that induction of iNOS is a homeostatic regulator for leukocyte recruitment.