Biochem. J. (2003) 369
(635–642) (Printed in Great Britain)
Group D prothrombin activators from snake venom are structural homologues of mammalian blood coagulation factor Xa
Veena S. RAO* , Jeremiah S. JOSEPH† and R. Manjunatha KINI*‡1
*Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 119260, †Genome Institute of Singapore, Singapore 117528, and ‡Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, U.S.A.
Procoagulant venoms of several Australian elapids contain proteinases that specifically activate prothrombin; among these, Group D activators are functionally similar to coagulation factor Xa (FXa). Structural information on this class of prothrombin activators will contribute significantly towards understanding the mechanism of FXa-mediated prothrombin activation. Here we present the purification of Group D prothrombin activators from three Australian snake venoms (Hoplocephalus stephensi, Notechis scutatus scutatus and Notechis ater niger) using a single-step method, and their N-terminal sequences. The N-terminal sequence of the heavy chain of hopsarin D (H. stephensi) revealed that a fully conserved Cys-7 was substituted with a Ser residue. We therefore determined the complete amino acid sequence of hopsarin D. Hopsarin D shows 
70% similarity with FXa and 98% similarity with trocarin D, a Group D prothrombin activator from Tropidechis carinatus. It possesses the characteristic Gla domain, two epidermal growth factor-like domains and a serine proteinase domain. All residues important for catalysis are conserved, as are most regions involved in interactions with factor Va and prothrombin. However, there are some structural differences. Unlike FXa, hopsarin D is glycosylated in both its chains: in light-chain residue 52 and heavy-chain residue 45. The glycosylation on the heavy chain is a large carbohydrate moiety adjacent to the active-site pocket. Overall, hopsarin D is structurally and functionally similar to mammalian coagulation FXa.
Key words: hopsarin D, notanarin D, notecarin D, prothrombinase, serine proteinase.
Abbreviations used: EGF, epidermal growth factor; ESI-MS, electrospray ionization MS; FVa, factor Va; FXa, factor Xa; Gla, g-carboxyglutamic acid; Lys C, lysyl endopeptidase; MALDI-TOF MS, matrix-assisted laser-desorption ionization–time-of-flight MS; RP-HPLC, reversed-phase HPLC; TFA, trifluoroacetic acid.
1To whom correspondence should be addressed, at the National University of Singapore (e-mail dbskinim@nus.edu.sg).
Received 10 June 2002/10 October 2002; accepted 28 October 2002
Published as BJ Immediate Publication 28 October 2002, DOI 10.1042/BJ20020889
The Biochemical Society, London ©
2003
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