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

Filippovich, I., Sorokina, N., Pierre, L. S., Flight, S., de Jersey, J., Perry, N., Masci, P. P. and Lavin, M. F. (2005) Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity. British Journal of Haematology, 131 2: 237-246. doi:10.1111/j.1365-2141.2005.05744.x


Author Filippovich, I.
Sorokina, N.
Pierre, L. S.
Flight, S.
de Jersey, J.
Perry, N.
Masci, P. P.
Lavin, M. F.
Title Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity
Journal name British Journal of Haematology   Check publisher's open access policy
ISSN 0007-1048
Publication date 2005-01-01
Sub-type Article (original research)
DOI 10.1111/j.1365-2141.2005.05744.x
Volume 131
Issue 2
Start page 237
End page 246
Total pages 10
Editor F. Cotter
I. Hann
J. Reilly
Place of publication Oxford
Publisher Blackwell Publishing Ltd
Language eng
Subject C1
321206 Preventive Medicine
730220 Injury control
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 gamma-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 Ca2+. 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.
Keyword Hematology
Pseudonaja Textilis
Factor Xa-like Protease
Cloning
Chimeric Constructs
Blood Coagulation
Coagulation-factor-xa
Hamster Ovary Cells
Snake-venom
Pseutarin-c
Structural Similarity
Taipan Snake
Brown Snake
Purification
Proteins
Subunit
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collections: Faculty of Science Publications
Excellence in Research Australia (ERA) - Collection
2006 Higher Education Research Data Collection
 
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Citation counts: TR Web of Science Citation Count  Cited 18 times in Thomson Reuters Web of Science Article | Citations
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Created: Wed, 15 Aug 2007, 15:50:55 EST