Characterization of recombinant tick anticoagulant peptide. A highly selective inhibitor of blood coagulation factor Xa.

M P Neeper,L Waxman,D E Smith,C A Schulman,M Sardana,R W Ellis,L W Schaffer,P K Siegl,G P Vlasuk

doi:10.1016/s0021-9258(18)38226-7

Abstract

Tick anticoagulant peptide (TAP) is a potent, highly selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D. E., Arcuri, K. E., and Vlasuk, G. P. (1990) Science, 248, 593-596). Further detailed studies pertaining to the in vitro and in vivo evaluation of TAP require quantities of the inhibitor which cannot be isolated from ticks. To overcome this limitation we describe here the characterization of recombinant TAP (rTAP) secreted by Saccharomyces cerevisiae. Expression of rTAP was obtained using a chimeric gene containing a fusion between sequences encoding the secretory preproleader of the yeast mating pheromone alpha-factor and a synthetic sequence encoding the 60-amino acid inhibitor under the transcriptional control of a galactose-inducible promoter. Recombinant S. cerevisiae were found to secrete biologically active rTAP into the extracellular medium at levels of 0.1-0.15 g/liter. The secreted inhibitor was purified to homogeneity and found to be indistinguishable from the native inhibitor with respect to several criteria, including primary structure, amino acid composition, and electrophoretic mobility. In addition, purified rTAP and native TAP exhibited similar stoichiometric inhibition of factor Xa in vitro. The in vivo efficacy of rTAP was demonstrated using a model of low grade disseminated intravascular coagulation where the purified inhibitor was shown to significantly inhibit thromboplastin-induced fibrinopeptide A generation following an infusion into conscious rhesus monkeys. The availability of rTAP will allow a detailed evaluation of the in vitro and in vivo properties of this highly specific and potent factor Xa inhibitor.

Highlights

An in-depth investigation into the interaction of Tick anticoagulant peptide (TAP) with factor Xa (fXa) in vitro, as well as the pharmacological potential of this inhibitor in viuo, requires quantities of material that cannot be isolated from its natural source
We have demonstrated conclusively that TAP can be secreted efficiently in high yield using a chimeric gene encoding the preproleader sequence of the a-mating factor fused to a synthetic gene encoding TAP
The purified recombinant inhibitor appeared to be indistinguishable from native TAP with respect to several criteria including the in vitro potency of fXa inhibition

Summary

Introduction

Further detailed studies pertaining to the in vitro and in vivo evaluation of TAP require quantities of the inhibitor which cannot be isolated from ticks. To overcome this limitation we describe here the characterization of recombinant TAP (rTAP) secreted by Saccharomyces cerevisiae. Recombinant S. cerevisiae were found to secrete biologically active rTAP into the extracellular medium at levels of 0.1-0.15 g/liter. Purified rTAP and native TAP exhibited similar stoichiometric inhibition of factor Xa in vitro. The availability of rTAP will allow a detailed evaluation of the in vitro and in vivo properties of this highly specific and potent factor.

Methods

Results

Conclusion