Pharmacokinetic and distribution analysis of variant forms of tissue-type plasminogen activator with prolonged clearance in rat.
Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy. Because of its rapid half-life (T1/2) of approximately five minutes, intravenous (IV) infusion of large doses (approximately 100 mg) are required in patients treated for myocardial infarction. To identify the determinant(s) on t-PA responsible for such rapid clearance, metabolically labeled forms of recombinant t-PA were analyzed in rats following IV administration. The following seven forms of t-PA were tested: (a) natural or glycosylated wild-type t-PA; (b) nonglycosylated wild-type t-PA; (c) delta F t-PA, which lacks the fibronectin fingerlike domain; (d) delta E t-PA, which lacks the epidermal growth factor (EGF) domain; (e) delta FE t-PA, which lacks both the finger and EGF domains; (f) delta FE3X t-PA, a form of delta FE t-PA in which Asn-linked glycosylation is prevented at all known glycosylation sites (Asn-117, 184, and 448; replaced by Gln); and (f) delta FE1X t-PA, a form of delta FE t-PA in which high-mannose-type glycosylation is prevented at Asn-117. Both glycosylated and nonglycosylated wild-type t-PA cleared in an exponential biphasic manner, with an initial alpha-phase T1/2 of 0.8 and 1.9 minutes, respectively. This result demonstrates that carbohydrate is not the primary mediator of the rapid clearance of t-PA. The liver was the primary organ responsible for uptake of these molecules. All other proteins tested, except for delta E t-PA, demonstrated primarily monophasic clearance patterns with T1/2 ranging between 12 and 27 minutes, and reduced uptake in the liver. delta E t-PA however, cleared in a biphasic manner with an alpha-phase T1/2 of 2.1 minutes. Results presented suggest that the clearance of t-PA is mediated by two distinct mechanisms. The primary determinant(s) responsible for modulating the rapid clearance of t-PA appears to be resident within the polypeptide sequence encoding the finger and/or EGF domains, with emphasis on the finger domain. A second and less significant contribution to clearance is defined by the presence and type of glycosylation.
Larsen
G R
GR
Genetics Institute, Cambridge, MA 02140.
Metzger
M
M
Henson
K
K
Blue
Y
Y
Horgan
P
P
D000818:Animals; D001216:Asparagine; D002236:Carbohydrate Conformation; D002460:Cell Line; D002872:Chromosome Deletion; D006224:Cricetinae; D005260:Female; D014644:Genetic Variation; D006031:Glycosylation; D008297:Male; D008657:Metabolic Clearance Rate; D010053:Ovary; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000097:blood / Q000493:pharmacokinetics*; D010959:Tissue Plasminogen Activator / Q000097:blood / Q000235:genetics / Q000493:pharmacokinetics*
D011994:Recombinant Proteins; D001216:Asparagine; D010959:Tissue Plasminogen Activator