Pharmacokinetic and distribution analysis of variant forms of tissue-type plasminogen activator with prolonged clearance in rat

Abstract

Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy. Because of its rapid half-life (T 1/2 ) of approximately five minutes, intravenous (IV) infusion of large doses (~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) ΔF t-PA, which lacks the fibronectin fingerlike domain; (d) ΔE t-PA, which lacks the epidermal growth factor (EGF) doamin; (e) ΔFE t-PA, which lacks both the finger and EGF domains; (f) ΔFE3X t-PA, a form of ΔFE t-PA in which Asn-linked glycosylation is prevented at all known glycosylation sites (Asn-117, 184, and 448; replaced by Gln); and (f) ΔFE1X t-PA, a form of ΔFE 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 α-phase T 1/2 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 ΔE t-PA, demonstrated primarily monophasic clearance patterns with T 1/2 ranging between 12 and 27 mintues, and reduced uptake in the liver. ΔE t-PA however, cleared in a biphasic manner with an α-phase T 1/2 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.