Slow Clearance of Acylated, Hybrid Thrombolytic Enzymes

Abstract

SummaryTwo hybrid plasminogen activators, plasmin A-chair/t-PA Bchain and plasmin A-chain/u-PA B-chain have been synthestzed and purified in sufficient yield to permit measurement of clearance in small laboratory animals. Each hybrid enzyme was reversibly acylated at the active centre to allow the pharmacokinetic profile to be followed using an activity-based method without interference from plasma inhibitors. The acylated plasmin/u-PA hybrid had a clearance half-life (t½) in guinea pigs of approximately 80 min, whereas acyl u-PA had a t½ of 3 min. The pharmacokinetic profile of the acylated plasmin/t-PA hybrid was measured in guinea pigs, rats and rabbits; the half-lives in all three species were 60–80 min compared to half-lives of acylated, native t-PA that were in the range 0.5–1.0 min. Thus, plasmin A-chaincontaining, acylated hybrid enzymes are cleared some 30- to 100-fold more slowly than the acylated parent activators.

Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159)

“…Thus the results obtained for the hybrids substantiate results previously obtained for the activator coimplex streptokinase-plasminogen and its stabilized pro-enzyme 'APSAC' [anisoylated Lys-plasminogen streptokinase activator complex; Eminase (Beecham)], where high fibrin affinity was also associated with fibrin enhancement of activity (Fears et al, 1985;Cassels et al, 1987). Our observations of considerable fibrin enhancement for the plasmin A-chain hybrids linked to the B-chain of u-PA or t-PA, together with the finding of a slow clearance in vivo (Robinson et al, 1988), suggest that these enzymes deserve further evaluation as novel thrombolytic agents.…”

“…The synthesis and characterization of the plasmin Achain/t-PA B-chain hybrid (apparent Mr 86000) and the plasmin A-chain/u-PA B-chain hybrid (apparent Mr 86000) were described elsewhere (Robinson et al, 1988). t-PA was purified from the recombinant Bowes-melanoma cell line TRBM6 (Browne et al, 1985;Dodd et al, 1986b); the major species had an apparent Mr of 63000.…”

“…We and others recently showed that it was possible to construct hybrid enzymes comprising the A-chain of Lys-plasmin and the B-chain of either t-PA (Ile276-t-PA) or u-PA (Ile'59-u-PA) Nakayama, 1986;Robbins & Tanaka, 1986;Robbins & Boreisha, 1987;Robinson et al 1988). The hybrids bind to fibrin, but less is known about the effect of fibrin on enzyme activity.…”

Binding of plasminogen activators to fibrin: characterization and pharmacological consequences

“…Plasmin A-chain hybrids with the B-chain of t-PA or u-PA possess the fibrin affinity of plasminogen. The initial results of colleagues (1986, 1987), demonstrating fibrin enhancement, have been substantiated (R. Fears, unpublished work) for the hybrids prepared by Robinson et al (1988). The presence of the entire A-chain of plasmin in a complex with u-PA confers greater fibrin affinity than does the truncated sequence comprising Kringles 1-3 (Nakayama et al, 1986).…”

Interaction of a Plasmin A-Chain/t-PA B-Chain Hybrid Enzyme with Plasma Inhibitors In Vivo and In Vitro

“…A part from EM INA SE, these all suffer from the disadvantage that they are cleared rapidly from the circulation and may require continuous infusion to maintain therapeutic levels in the blood. Much current research in our own and other laboratories has thus been directed towards producing novel plasminogen activators with extended pharm acokinetic profiles (1)(2)(3)(4)(5)(6).…”