Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model

Abstract

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom of Pseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant, Ki approximately 10(-11) mol/l), while both of the latter bound less tightly (Ki approximately 10(-9) mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan, Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

Molecular Diversity in Venom from the Australian Brown Snake, Pseudonaja textilis

“…There were notable differences in proteins in the molecular mass range of 5-10 kDa. It has been reported that short and long neurotoxins and textilinins are known to occur in this size range (3,4,9). By reference to Fig.…”

“…These include postsynaptic ␣-neurotoxins (2-4), the presynaptic neurotoxin textilotoxin (5-7), phospholipase A 2 s (PLA 2 s) 1 (6,8); serine protease inhibitors (9,10), and a prothrombin activator (11,12). Fry (13) has written a comprehensive review on the properties of venom components from Australian elapids.…”

“…This is similar to the short-chain ␣-neurotoxins (4) where alternative splicing of the P. textilis short neurotoxin 1 gene (PT sntx1) gives rise to two mRNAs and two neurotoxins. Masci et al (9) identified and characterized serine protease inhibitors from the venom, named them textilinins, and showed that they significantly reduce bleeding in an animal model. More recently, six isoforms of textilinin have been identified in P. textilis venom gland-derived cDNA (10).…”

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 fromPseudonaja textilis textilis

“…For example, cone snails have peptides that act as calcium or sodium channel blockers or bind to the nicotinic acetylcholine receptor (Nielsen et al, 2000). Small proteins or peptides found in snake venoms include the PLA 2 phospholipases (Kwong et al, 1989) and Kunitz-type serine protease inhibitors, such as the venom basic protease inhibitor (Chen et al, 2001) and the textilinins (Masci et al, 2000).…”

“…To date, six textilinins from Pseudonaja textilis textilis, the Australian Common Brown snake, have been identified by cloning from venom-gland cDNA (Masci et al, 2000). All consist of $60 amino-acid residues.…”