Comparison of Textilinin-1 with Aprotinin as Serine Protease Inhibitors and as Antifibrinolytic Agents

Abstract

Textilinin-1 (Q8008) was isolated from the venom of the Pseudonaja textilis and has a 47% sequence identity to the antihaemorrhagic therapeutic agent aprotinin. When equimolar concentrations of enzyme and aprotinin were pre-incubated, plasmin was inhibited 100%, plasma kallikrein 58%, and tissue kallikrein 99%. Under the same conditions, textilinin-1 inhibited plasmin 98%, plasma kallikrein 16% and tissue kallikrein 17%. Whole blood clot lysis was inhibited strongly by both aprotinin and textilinin-1, as shown by thrombelastography. At 2 µM inhibitor lysis initiated by t-PA was greater than 99% inhibited by aprotinin (LY60 = 0.4 ± 0.1) whereas textilinin-1, inhibited lysis by 91% (LY60 = 8.9 ± 0.7). The same trend was found with the lysis of euglobulin fractions. From these data textilinin-1 appears to be a more specific plasmin inhibitor than aprotinin but aprotinin inhibits clot lysis to a greater extent.

Common evolution of waprin and kunitz-like toxin families in Australian venomous snakes

“…Recombinant protein samples used in antibody production, immunoblot detection and functional assays were produced by a number of methods. Recombinant Textilinin-1 was produced as an E. coli expression product by BresaGen Ltd. (Australia) as previously described [12]. Recombinant omwaprin-b was initially produced as a bacterial GST-fusion product using the pGEX-6P-1 E. coli expression system (GE Healthcare, Rydalmere, Australia).…”

“…The potential for omwaprin-b to act as an inhibitor of enzymatic activity was examined against a number of key enzymes within the haemostatic system including plasmin, trypsin, tissue plasminogen activator, kallikrein and urokinase. Tests of enzyme inhibition were performed as a chromogenic assay with appropriate controls as previously described [12]. The effects of omwaprin-b upon blood coagulation and fibrinolysis was examined using a thromboelastograph (Haemoscope Corporation, Niles, Illinois) in addition to tests of prothrombin time and activated partial thromboplastin time [27,28].…”

“…Subsequently, a second family of kunitz-type inhibitors were purified, cloned and characterised from the venom of the common brown snake (Pseudonaja textilis) [10,11]. Two of these inhibitors, Textilinin-1 and -2, demonstrated potent inhibition of plasmin and are thought to be responsible for the anti-fibrinolytic properties of this venom [12]. More recently, a new class of small molecule peptides called waprins have been described from snake venom.…”

Molecular Cloning and Characterization of Chymotrypsin Inhibitor and Chitin-Binding Protein Homologs from the Bumblebee Bombus terrestris

“…These protease inhibitors have been categorized into three classes, Kunitz, Kazal, and Bowman-Birk types (Conlon and Kim, 2000;Gebhard et al, 2004;Song et al, 2008). In addition to the well characterized serine protease inhibition functions of proteins such as trypsin and/or chymotrypsin Zhou et al, 2004;He et al, 2008;Choo et al, 2012;Wang et al, 2012), some serine protease inhibitors are known to be involved in various physiological processes such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation (Masci et al, 2000;Flight et al, 2005Flight et al, , 2009Yuan et al, 2008;CorralRodríguez et al, 2009;Millers et al, 2009;Choo et al, 2012).…”

CHAPTER 5. Reptile Venoms as a Platform for Drug Development

“…159 Although recent recommendations have been made to li the suspension of aprotinin, 160 the more specic actions of textilinin-1 make it attractive for drug development. 161,162 Recombinant Q8008 is expressed in Escherichia coli, and its structure, determined by X-ray crystallography, shows a similar overall fold to aprotinin 163,164 (Figure 5.3).…”