Influence of Sugar Chain on Fibrin Affinity of Recombinant t-PA.

Abstract

Tissue plasminogen activator (t-PA) is a serine protease that cleaves plasminogen into its active form, plasmin. The plasmin then subsequently degrades fibrin.1,2) The activity of t-PA is markedly stimulated in the presence of fibrin. 2)Human t-PA produced by recombinant DNA technology is presently available for clinical use in patients with acute myocardial infarction. [3][4][5] Although t-PA is thought to be clinically superior to streptokinase and urokinase because of its specific affinity for fibrin, the rapid clearance of t-PA from circulating blood sometimes requires the administration of high doses to obtain therapeutic blood levels, which can lead to bleeding incidents due to a decrease in plasma fibrinogen levels.6,7) To solve this problem, several types of mutant type t-PAs (mt-PAs) have been developed. [8][9][10][11][12][13][14] Although most of them are superior to wild type t-PA (WT t-PA) in terms of plasma half life, they are inferior to WT t-PA in terms of activity. They are generally produced by modification of the peptide chain, therefore, these modifications cause the loss of activity and fibrin affinity. [15][16][17] t-PA is composed of 5 domains: finger, epidermal growth factor (EGF)-like, kringle 1 and 2, and serine protease domain. All of them are assumed to be necessary for t-PA to exhibit physiological activity, especially the finger (F) and kringle 2 (K2) domains are indispensable for fibrin affinity. 18,19) In contrast, modification of the sugar chain is not expected to affect the function of each domain. t-PA is glycosylated at three sites, Asn117, Asn184, and Asn448. The sugar moiety at Asn117 carries a high mannose structure, whereas those at Asn184 and Asn448 carry complex type oligosaccharides.20) The Asn184 glycosylation site has been shown to be variably glycosylated by steric hindrance between the kringle 1 and 2 domains.21) This partial glycosylation results in t-PA being expressed as two major glycoforms, termed Type I and Type II, which are defined by the presence and absence of the complex type oligosaccharide at Asn184, respectively. Although most studies have been made on the role of the sugar chain in the plasma clearance of t-PA, [22][23][24][25] little is known about the influence of the sugar chains on the fibrin affinity. In this study, we determined the influence of the sugar chain on the fibrin affinity using 4 kinds of t-PAs, which have differing sugar chain structures.It has been reported that the binding of t-PA to fibrin is mediated by two discrete domains, the F and K2 domains. 26,27) Binding mediated through these domains can be distinguished on the basis of competition for fibrin binding by lysine analogues such as e-amino caproic acid (EACA), which eliminates K2 dependent binding, but does not eliminate F dependent binding. We also demonstrated the role of sugar chains in the fibrin affinity mediated via these domains one by one, using EACA in this study. MATERIALS AND METHODSChemicals Recombinant WT t-PA (lot R8803, 1.3 mg/ ml) and Gln117 t-PA (lot GYL013, ...

Potent anti-tumor and prolonged survival effects of E. coli-derived non-glycosylated kringle domain of tissue-type plasminogen activator

“…E. coli-derived TK1-2 (E-TK1-2) is different from Pichia-derived TK1-2 (P-TK1-2) in terms of glycosylation modifications, since this domain has two N-linked glycosylation sites (17). In the case of t-PA, the parent molecule of TK1-2, glycosylation at Asn117 (kringle 1) and Asn184 (kringle 2) affects fibrin affinity and plasma clearance (18)(19)(20). Therefore, we investigated how nonglycosylated E-TK1-2 affects tumor growth in vivo.…”