In vivo clearance of tissue plasminogen activator: The complex role of sites of glycosylation and level of sialylation

The involvement of a high mannose oligosaccharide in the clearance of tissue plasminogen activator (t-PA) has been reported previously. Removal of this oligosaccharide by mutation of amino acid #117 from asparagine (Asn) to glutamine (Gln) generates a t-PA variant designated LAt-PA which was shown to clear significantly more slowly than the native molecule. Here we report clearance studies in rabbits with LAt-PA produced in mouse C127 cells under differing growth conditions which indicate that the half-life of the variant is additionally influenced by the degree of sialylation of the remaining complex oligosaccharides. The data suggest that, in addition to the mannose receptor, the asialoglycoprotein receptor is also involved in the hepatic clearance of LAt-PA from the circulation. In addition, t-PA has been shown to exist in two major glycoforms termed Type I and Type II, which are defined by the presence or absence of a complex oligosaccharide on Asn 184 respectively. We also separated the individual glycoforms of LAt-PA and determined their monosaccharide composition. The glycoform with a complex oligosaccharide at Asn 184 has an extended half-life in rabbits compared to the glycoform lacking this oligosaccharide.