Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity

Abstract

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that c-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca 2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.

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

“…1C, spots 21, 23, and 32) corresponding to the NH 2 -terminal region of the light chain. In the case of Factor Va-like and Factor Xa-like proteins it is unlikely that multiple cDNA isoforms exist because we have isolated full-length cDNAs for these genes and found only two isoforms for Factor Xa-like and a single species for Factor Va-like (32,33). In the present study, the use of mass spectrometry failed to distinguish between these isoforms due to limited tryptic peptide sequence coverage.…”

Proteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles

“…The predicted peptides are most similar to therian serine protease inhibitors SERPINA4 and SERPINA11. Serine protease inhibitors are found in the venoms of snakes, where they play key roles in blood coagulation (39,40). They have also been identified in sea anemone (41) and wasp (42) venoms.…”

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

“…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]. Anti-bacterial effects of omwaprin-b were further examined using the gram positive bacterial strains Staphylococcus aureus, Staphylococcus epidermis and Streptococcus epidermis in addition to the gram negative strains Escherichia coli and Pseudomonas aeruginosa according to the protocols described by Nair et al [17].…”