Comparative Analysis of Prothrombin Activators from the Venom of Australian Elapids
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分子生物学进展 2005年第9期
* The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, Royal Brisbane Hospital, Brisbane, Australia; School of Life Sciences, Queensland University of Technology, Brisbane, Australia; Southern Clinical Division, Faculty of Health Sciences, University of Queensland, Brisbane, Australia; and Comparative Genomics Centre, James Cook University, Townsville, Australia
E-mail: martinL@qimr.edu.au.
Abstract
A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X– and factor V–like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X–like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%–97%). The factor V–like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%–98%). Expression of both the factor X– and factor V–like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.
Key Words: prothrombin activator ? factor X ? factor V ? snake venom ? gene cloning ? Oxyuranus scutellatus
Introduction
Snake venoms are a complex mixture of pharmacologically important protein and polypeptide toxins, many of which are known to affect the mammalian blood hemostatic mechanism via coagulant, anticoagulant, and/or fibrinolytic actions. These venom components are related to each other within several families of venomous snakes, including the Elapidae, the Colubridae, the Viperidae, and the Atractaspididae. Australian venomous snakes, belonging exclusively to the Elapidae family, are among the most toxic in the world (Broad, Sutherland, and Coulter 1979). A unique feature of the procoagulant properties of venoms from Australian elapid snakes is that they are limited to prothrombin activation and contain no thrombin-like enzymes. This is due to the presence of prothrombin activators that contain a component that is structurally and functionally similar to that of mammalian coagulation factor Xa (Chester and Crawford 1982).
One of the key reactions in the mammalian blood coagulation and hemostatic process is the cleavage of the zymogen prothrombin to thrombin which in turn activates fibrinogen to fibrin, eventually resulting in clot formation. The proteolytic cleavage of prothrombin is mediated by the prothrombinase complex, a multimeric complex composed of factor Xa in association with its nonenzymatic cofactor factor Va in the presence of calcium on a negatively charged phospholipid bilayer (Jackson and Nemerson 1980; Davie, Fujikawa, and Kisiel 1991). Human activated factor X is a two-chained serine protease linked via a single disulfide bond, with a 52–amino acid glycopolypeptide that is released upon activation. The presence of a vitamin K–dependent, -carboxyglutamic acid residue–rich region at the N-terminus of the light chain plays a critical role in the binding of phospholipids and calcium ions, while the heavy chain contains the catalytic site residues (Jackson 1984; Persson, Hogg, and Stenflo 1993).
Prothrombin activators from snake venoms are currently classified on the basis of their cofactor requirement (Kini, Morita, and Rosing 2001). Metalloproteinases that convert prothrombin to meizothrombin, such as ecarin A isolated from Echis carinatus, are classified as group A (requiring no cofactors for activity) or group B (requiring calcium only for activity) prothrombin activators (Morita and Iwanaga 1978). Group C prothrombin activators are serine proteases that do not require mammalian coagulation factor Va for the cleavage of prothrombin as they contain their own noncatalytic factor Va–like molecule complexed with a factor Xa–like protein (Rao and Kini 2002). As in the case of the mammalian prothrombinase complex, the factor V–like subunit of the snake prothrombin activator serves to increase proteolytic activity. Examples of group C prothrombin activators include pseutarin C (isolated from the venom of the common brown snake, Pseudonaja textilis) and oscutarin C (present in the venom of the coastal taipan, Oxyuranus scutellatus) (Walker, Owen, and Esmon 1980; Masci, Whitaker, and de Jersey 1988). Similarly, group D prothrombin activators are also serine proteases; however, they require calcium, phospholipids, and mammalian coagulation factor Va for maximal activity and include the proteases trocarin D (from the rough-scaled snake, Tropidechis carinatus), hopsarin D (from the Stephen's banded snake, Hoplocephalus stephensii), and notecarin D (identified in the venom of the mainland tiger snake, Notechis scutatus) (Tans et al. 1985; Marsh, Fyffe, and Bennett 1997; Weinstein, Williams, and White 2001). Interestingly, the prothrombin activators identified from Australian elapid snakes appear to be limited to groups C and D only.
Studies on the prothrombin-activating properties of Australian snake venoms have to date focussed primarily upon purified fractions of the native protein, with emphasis on the fact that they are the only nonmammalian and nonhepatic source of molecules capable of activating prothrombin (Joseph et al. 1999). Recently, however, both the factor X–like and much larger factor V–like nonenzymatic subunits of pseutarin C, the prothrombin activator present in the venom of P. textilis, have been isolated and the genes cloned (Rao, Swarup, and Kini 2003, 2004). Pseutarin C comprises approximately 40% of the total dry weight of the venom of P. textilis and its injection results in massive disseminated intravascular coagulation within the body of its prey, ultimately resulting in death (Masci, Whitaker, and de Jersey 1988). Its factor X–like component demonstrates a similar structure to that of mammalian coagulation factor X with a propeptide region, and light and heavy chains bound by a single disulfide linkage and separated via an activation peptide that is cleaved posttranslationally. Overall, there is 48% amino acid identity with human factor Xa. It has been demonstrated that cleavage of this activation peptide is critical for correct folding of the protease and that activity is dependent on posttranslational glycosylation and -carboxylation (Baugh and Krishnaswamy 1996; Joseph et al. 2003). The nonenzymatic subunit of pseutarin C also contains a domain structure similar to its mammalian counterpart with a typical A1-A2-B-A3-C1-C2 domain architecture, accompanied by a number of unique posttranslational modifications (Rao, Swarup, and Kini 2003).
In this study we describe the cloning, characterization, and comparative analysis of components of both group C and group D prothrombin activators isolated from the venom glands of a number of Australian elapids. Results of the study provide significant information not only on the molecular interactions between factors Va and Xa in the formation of the prothrombinase complex but also on the evolutionary relationships between members of the Australian Elapidae family.
Materials and Methods
RNA Isolation and cDNA Synthesis
Venom glands were excised from Australian elapids, including the coastal taipan (O. scutellatus), inland taipan (Oxyuranus microlepidotus), common brown snake (P. textilis), mainland tiger snake (N. scutatus), rough-scaled snake (T. carinatus), Stephen's banded snake (H. stephensii), red-bellied black snake (Pseudechis porphyriacus), and mulga (Pseudechis australis), collected under National Parks and Wildlife's permit number W4\00261\01\SAA. Snap-frozen glands were homogenized with a polytron, RNA isolated using the Tri Reagent method (Sigma-Aldrich, St. Louis, Mo.), precipitated in isopropanol, washed with 70% ethanol, and resuspended in diethylopyrocarbonate treated water. First-strand cDNA was synthesized from 1 μg of total RNA with an oligo(dT)12–18 primer via reverse transcription with 200 units of Superscript II RNase H– Reverse Transcriptase (Invitrogen, Mt. Waverly, Australia). The final reaction was ethanol precipitated, and cDNA samples were resuspended in sterile water and stored at –20°C.
Factor X–like Protease Precursor Amplification and Cloning
Polymerase chain reaction (PCR) amplification of the full-length coding sequence of the factor X–like protease was performed from all cDNAs isolated from Australian elapids. An approximately 1.4-kb product was amplified with 50 pmol of each of the forward (5'-ATG GCT CCT CAA CTA CTC CTC TG-3') and reverse (5'-TTA GAG CCG ACC AGT GCT TGA CTC-3') primers designed from the recently published P. textilis sequence (AY631238 [GenBank] ). The PCR reaction mix was made to a final volume of 25 μl containing approximately 200 ng of cDNA template, 1 unit of AmpliTaq gold (Applied Biosystems, Foster City, Calif.) in 1 x buffer, 2 mM MgCl2, and 200 μM deoxyribonucleoside triphosphates. All reactions were run with appropriate no template controls, with an initial denaturation at 95°C for 8 min, followed by 30 cycles of 95°C for 30 s, 52°C for 45 s, and 72°C for 2 min, and a final extension of 72°C for 7 min.
The resulting PCR products were analyzed on a 1% tris-acetate EDTA agarose gel, and bands of interest were excised and purified with a QIAEX II Gel Extraction kit according to the manufacturer's protocol (Qiagen, Hilden, Germany). Purified PCR products were cloned via the pGEM?-T vector system (Promega, Madison, Wis.). The ligation mix was ethanol precipitated prior to transformation into electrocompetent dH5 Escherichia coli cells, with recombinant clones selected on LB-ampicillin plates (50 μg/ml) supplemented with isopropyl-beta-D-thiogalactoside and 5-bromo-4-chloro-3-indolyl-bD-galactoside. Multiple clones were then sequenced from each snake with an ABI Big Dye Terminator cycle sequence ready reaction kit (PerkinElmer, Norwalk, Conn.). Sequencing was performed in both the forward and reverse directions, as well as with internal primers where appropriate, and results were assembled and analyzed with BioEdit software (Isis Pharmaceuticals Inc., Carlsbad, Calif.). For phylogenetic analyses, the predicted protein sequences were aligned using ClustalW, subjected to maximum likelikood analysis using MolPhy version 2.3 via the Dayhoff model and in local rearrangement of Neighbor-Joining trees mode (1,000 bootstrap replicates) (Adachi and Hasegawa 1996). In these analyses, platypus (Ornithorhynchus anatinus) (AAG00453 [GenBank] and human (Homo sapiens) (CAI41386 [GenBank] factor X sequences were defined as out-groups.
Factor V–like Protein Amplification and Cloning
PCR amplification of the full-length cDNA transcript for the nonenzymatic factor V–like unit of the snake prothrombin-activating complex was performed in elapids containing group C complexes using primer sequences designed from the previously published P. textilis sequence (AY168281 [GenBank] ). The complete mRNA transcript of 4.4 kb was amplified as two overlapping PCR fragments of 2,271 bp (fragment 1, including start codon) and 2,202 bp (fragment 2, including stop codon). PCR amplification was performed in a fashion similar to that of the factor X–like protease with fragment 1 amplified with forward (5'-ATG GAA GAT ACA GTG TGA GCC C-3') and reverse (5'-CTC TGT CTG TTC CCT GCG TGG C-3') primers, while fragment 2 was amplified with forward (5'-CCA GAC CCA GAA TCG GAT GCG C-3') and reverse (5'-GCC TTA AAA AAC TTC ACA ACC-3') primers. Reaction mixtures were thermocycled at 95°C for 10 min, followed by 37 cycles of 95°C for 30 s, 64°C for 45 s (fragment 1) or 59°C for 45 s (fragment 2), and 72°C for 2 min 30 s, followed by a final extension of 72°C for 7 min. PCR products were then visualized on a 1.25% TAE agarose gel, and the appropriate band was excised, purified, cloned, and sequenced as previously described from P. textilis, O. scutellatus, and O. microlepidotus.
Quantitative PCR Analysis
Transcription of the factor X– and factor V–like prothrombin activator genes were analyzed via quantitative PCR (qPCR). cDNA was synthesized from 1.5 μg of venom gland RNA for each snake as well as liver RNA purified from O. scutellatus. Prior to cDNA synthesis, total RNA was digested with two units of RQ1 DNase-polymerase to remove any contaminating genomic DNA that may affect expression results (Promega). DNase-treated RNA was ethanol precipitated prior to cDNA synthesis, and 1:50 dilution of the cDNA was then used in subsequent qPCR reactions as template. In all cases 7.5 μl of a 2 x Sybr Green PCR master mix was added to 5 pmol of a forward and reverse primer mix and 3 μl of diluted cDNA template in a total volume of 15 μl (Applied Biosystems). A 190-bp fragment of the factor X–like protease was amplified with a forward (5'-GAA GAC CCC TAT CCA GTT CTC-3') and reverse (5'-CAT GCA GGT GTG CCT GTC CAC A-3') primer pair, while a 201-bp section of the factor V–like protein was amplified with forward (5'-CTC AAG GAG CCA CAT CAA TGA C-3') and reverse (5'-GGA TGA TAC GAA TGA ACC TGG AC-3') primers, based on a sequence 100% homologous to all snakes. Each template was run in triplicate, and every run included the amplification of the 60S ribosomal protein housekeeping gene, which was amplified as a 195-bp product with forward (5'-GCA AGC GTA TGA ACA CCA ACC C-3') and reverse (5'-AGA GCA GCT GGG ACG ACC ATT C-3') primers.
Reaction mixtures were thermocycled at 95°C for 10 min, followed by 35 cycles of 95°C for 15 s, 64°C for 15 s, and 72°C for 30 s on a Rotor-Gene 3000 (Corbett Research, Mortlake, Australia). A standard curve of cycle time using a range of known quantities of plasmid as template was also obtained. PCR products were run on a 1% TAE agarose gel to confirm amplification, and the results including cycle time for product formation and calculated transcript concentration as determined by the Rotor-Gene software were exported into Microsoft Excel. Averages of the triplicate samples in duplicate runs were then taken, and the results normalized to that of the housekeeping gene.
Prothrombin Activator Detection in Venom
The presence and quantity of the factor X– and factor V–like components of the snake prothrombinase complex were examined in the venom of all elapids by immunoblotting with a number of different primary antibodies. For all western blot analysis, a total of 10 μg of lyophilized venom resuspended in 50% glycerol from O. scutellatus, O. microlepidotus, P. textilis, N. scutatus, T. carinatus, H. stephensii, P. porphyriacus, and P. australis were run on a sodium dodecyl sulfate (SDS)–polyacrylamide gel in that order, with a prestained molecular weight marker (Fermentas, Hanover, Md.). All gels were also run with an appropriate quantity of purified P. textilis factor Xa–like protease.
Venom proteins separated by electrophoresis on a 12% SDS–polyacrylamide gel under either reducing or nonreducing conditions were then transferred to nitrocellulose membranes at 100 V for 1 h at 4°C in transfer buffer (100 mM Tris, 40 mM glycine, 0.036% SDS, and 20% methanol). Blots were then probed with a range of primary antibodies including an antibody raised in sheep against a recombinant form of the heavy chain from P. textilis (residues D302 L467), an antibody against the purified prothrombin activator complex from P. textilis raised in rabbits, and finally a commercial monoclonal antibody raised against human -carboxyglutamyl (GLA) residues in mice (American Diagnostica Inc., Stamford, Conn.). Membranes were then probed with appropriate secondary antibodies conjugated to horseradish peroxidase and the protein signal was detected with enhanced chemiluminescence reagents according to manufacturer's instructions (PerkinElmer).
Results
Identification of Factor Xa–like Proteases from Australian Elapids
Full-length cDNA clones corresponding to the coding sequence of the factor Xa–like prothrombin activator were isolated from the venom glands of Australian elapids based on the P. textilis protease gene sequence (Filippovich et al., unpublished data) which has also recently been described by Rao, Swarup, and Kini (2004). Two primers designed from the propeptide and heavy chain gene sequences were used to amplify an approximately 1.4-kb fragment. The results in figure 1 demonstrate the presence of a product in P. textilis, O. scutellatus, P. porphyriacus, N. scutatus, T. carinatus, and O. microlepidotus. A similar product was also amplified from the venom gland cDNA of H. stephensii (results not shown); however, protease mRNA was not detected in the mulga (P. australis) even when additional primer pairs, based on the internal gene sequence of P. textilis, were employed. This is not surprising given that the venom of this snake does not demonstrate procoagulant activity (Tan and Ponnudurai 1990; Dambisya, Lee, and Gopalakrishnakone 1995).
FIG. 1.— PCR amplification of the factor X–like protease cDNA. A complete 1.4-kb PCR product was amplified from venom gland cDNA isolated from (1) Pseudonaja textilis, (2) Oxyuranus scutellatus, (3) Pseudechis porphyriacus, (4) Notechis scutatus, (5) Tropidechis carinatus, (6) Pseudechis australis, (7) Oxyuranus microlepidotus, (8) no template control, and (9) molecular weight marker. A similar product was also amplified from the venom gland cDNA of Hoplocephalus stephensii at a later date (not shown).
Multiple clones were generated to identify the protease cDNA sequence in each snake for nucleotide comparisons. The overall identity at the nucleotide level was 82.5% when an alignment was performed between all seven snake species. A protein alignment of the predicted amino acid sequence from these cDNAs is depicted in figure 2. The overall identity between the predicted snake proteins is 71.5%. Comparison of the two taipan species, O. scutellatus and O. microlepidotus, reveals an identity of 95% (table 1). These proteases were most closely related to that of P. textilis with a three-way identity of 91%. While the degree of homology between all snake species was high (80%–97%), the protease from P. porphyriacus was least related to the others.
FIG. 2.— Alignment of the factor X–like proteases. An alignment was performed of the deduced amino acid sequence obtained from multiple cDNA clones of the factor X–like protease isolated from the venom glands of seven Australian snakes, including Oxyuranus scutellatus (AY940204), Oxyuranus microlepidotus (AY940205), Pseudonaja textilis (AY631238), Pseudechis porphyriacus (AY940207), Hoplocephalus stephensii (AY940208), Notechis scutatus (AY940206), and Tropidechis carinatus (AY769963). GenBank accession numbers are given in parentheses. Sequence identity is shown in black, gaps are inserted for optimal alignment, and the cleavage sites for the propeptide, light chain, activation peptide, and heavy chain sequences are also marked. The sites for -carboxylation of GLA residues are marked by and active site residues with an *. Conserved cysteine residues involved in putative disulfide bond formation are shaded gray.
Table 1 Identity of the Factor X–like Proteases from the Venom Glands of Australian Elapids Compared to that of the Human Homolog
Phylogenetic analysis of the deduced amino acid sequences resolved the snake factor X–like prothrombin activators into two distinct clades, each with high bootstrap support (fig. 3). It is interesting to note that this clustering, based on sequence relatedness, separates the elapids into two distinct groups which have previously been defined on the basis of their requirement for mammalian factor Va as a cofactor for activity. Group C prothrombin activators, including proteases isolated from members of the Pseudonaja and Oxyuranus genera, do not require mammalian coagulation factor Va for activity as they have their own factor Va–like molecule complexed to the factor Xa–like protease. On the other hand, the group D prothrombin activators do have a requirement for mammalian factor Va. The classification of this group described here on the basis of sequence relatedness corresponds exactly with previous subgrouping based on factor Va requirement. Although the phylogenetic analyses strongly support a close relationship between the venom group D sequences, relationships within the group C clade are not well supported, which explains the implied paraphyly of the genus Oxyuranus. The diverged nature of the P. porphyriacus sequence is also clear in the tree shown in figure 3, although the analyses strongly support its basal position within the venom group D clade.
FIG. 3.— Phylogenetic relationships of the factor X–like proteins. The maximum likelihood method was employed to determine the phylogenetic relationship of the deduced amino acid sequences of factor X–like prothrombin activators from multiple Australian elapids. Numbers above branches indicate the percentage of 1,000 bootstrap replicates supporting the topology shown. Clades corresponding to group C and group D factor X–like proteases are clearly resolved with high bootstrap support.
Comparison of the snake protease sequence with that of human factor X reveals that it possesses a similar overall domain arrangement, including a propeptide, light chain, activation peptide, and heavy chain in that order (MacGillivray and Fung 1989). In all cases, the 40–amino acid propeptide sequence was highly conserved, differing by only a single residue in P. porphyriacus and H. stephensii (fig. 2). This sequence contains a high content (60%) of hydrophobic residues consistent with its role as secretion signal/signal peptide, and the only two substitutions observed also led to conservation of hydrophobic residues. It is evident from the domain arrangement that this precursor protein requires processing to produce the mature heavy and light chain cross-linked protease, similar to that of its mammalian counterpart (Di Scipio et al. 1977). The cleavage site between the propeptide and light chain is highly conserved between all snake species being R ANS in group C proteases and R SNS in group D prothrombin activators. The cleavage sites at either end of the activation peptide in the precursor protease are absolutely conserved in all snake species, with the exception of a hydrophobic amino acid substitution in the case of P. porphyriacus (V211I).
While the protease sequences are highly conserved overall, the three most closely related snakes among the group D prothrombin activators (H. stephensii, T. carinatus, and N. scutatus) have a premature stop codon that deletes the final 11 C-terminal amino acids compared to the group C proteases and P. porphyriacus. In addition, deletions of a variable number of amino acids were observed between residues 292 and 304 of precursor protein in the group D proteases, plus another area of low homology in the heavy chain within residues 353–360 (fig. 2).
In human factor Xa, 12 disulfide bonds have been identified in total, seven in the light chain, four in the heavy chain, and one interchain disulfide linkage (Davie, Fujikawa, and Kisiel 1991). All these cysteine residues are conserved in all seven snake sequences identified in this study, with the exception of a C216S substitution in the H. stephensii precursor protease, consistent with the previously published amino acid sequence of hopsarin D (P83370 [GenBank] ) (Rao, Joseph, and Kini 2003). Similarly, vitamin K–dependent posttranslational carboxylation of glutamic acid (GLA) residues in the light chain has been described for human factor Xa and the group D prothrombin activators, including 11 GLA residues identified in the N-terminal domain of trocarin D (Joseph et al. 1999). Results in figure 2 demonstrate that these 11 GLA residues are also conserved in all Australian elapids with the exception of an E72K substitution in the P. porphyriacus precursor protein.
A further posttranslational modification reported in the prothrombin activator of pseutarin C from P. textilis is the O-linked glycosylation of a serine at position 92 of the precursor protein which is also present in the other six elapid sequences (fig. 2) (Joseph et al. 2003). Similarly, an N-linked glycosylation of a heavy chain asparagine at position 254 of pseutarin C precursor protein is also conserved among the other snakes with P. porphyriacus again being the exception. The three active site residues of human factor Xa are conserved within all snakes in this study, corresponding to positions H251, D309, and S406 of the O. scutellatus precursor. An activation peptide located between the light and heavy chain sequences in the precursor human factor X plays a key role in the folding and processing of the protease (Baugh and Krishnaswamy 1996). A 27–amino acid sequence (approximately half that of the human equivalent), similarly positioned between the light and heavy chains, is also present and highly conserved in all snakes.
Comparative Analysis of Factor V–like Proteins
Based on the nucleotide sequence of the factor V–like nonenzymatic subunit of pseutarin C from P. textilis (AY168281 [GenBank] ), amplification of similar full-length cDNA sequences for elapids involved in this study was attempted. Because only group C prothrombin activators contain their own factor V–like sequence, it was only possible to obtain an amplified product in O. scutellatus, O. microlepidotus, as well as P. textilis. Alignment of the deduced protein sequence from multiple cDNA clones demonstrated that there was 95.7% identity between the three snakes (fig. 4).
FIG. 4.— Alignment of the factor V–like proteins. An alignment of the deduced amino acid sequence of the factor V–like nonenzymatic unit of the prothrombinase complex amplified from cDNA from venom glands of the group C Australian snakes Oxyuranus scutellatus (AY940209 [GenBank] ), Oxyuranus microlepidotus (AY940210 [GenBank] ), and the previously identified Pseudonaja textilis (AY168281 [GenBank] ) is shown. Arrows mark the boundaries of the different domains, and conserved cysteines involved in putative disulfide linkages are shaded gray.
The overall domain arrangement of the factor V–like protein observed in the Oxyuranus species was the same as that of mammalian coagulation factor V and the nonenzymatic subunit of pseutarin C (A1-A2-B-A3-C1-C2) (Rao, Swarup, and Kini 2003). A 30–amino acid signal peptide is also present with 100% identity in all three elapids. The two thrombin cleavage sites reported in P. textilis (R772 and R817 in the precursor molecule) are again conserved between the three snakes. Rao, Swarup, and Kini (2003) have recently demonstrated that the pseutarin C nonenzymatic subunit contains only a single conserved activated protein C cleavage site, whereas the mammalian equivalent has three sites required for the inactivation of factor Va (Kalafatis, Rand, and Mann 1994). Both P. textilis and O. microlepidotus contain a lysine residue at this site, whereas a conserved amino acid (arginine) is present in the O. scutellatus protein.
The association of factors Xa and Va on a phospholipid bilayer, forming the prothrombinase complex, is a key step in the blood coagulation process (Steen 2002). Kalafatis and Beck (2002) identified a binding site for factor Xa within the heavy chain of human factor Va between amino acid residues 323 and 331. The corresponding residues in the elapid sequences (amino acids E354 I362 in the precursor molecule) are completely conserved with one another, and the first eight of these residues are identical to the corresponding human sequence, with the final amino acid being a hydrophobic residue in both instances (V I). Another key component of the prothrombinase complex are phospholipids, which have been predicted from three-dimensional structural analysis to bind to the surface-exposed loop KKSWW in mammalian coagulation factor Va. Mutagenesis of these residues identified both tryptophans, which are absolutely conserved in all three elapid sequences, as important in interactions with phospholipids (Nicolaes, Villoutreix, and Dahlback 2000). Of the seven putative disulfide linkages reported for human factor Va, six are conserved in the group C sequences (fig. 4).
mRNA Expression of Factor X– and Factor V–like Components from Australian Elapids
As outlined above, the Australian elapids under investigation fall into two categories, group C which includes complete prothrombin activators and group D that require phospholipids, calcium ions, and mammalian coagulation factor Va for activity (Chester and Crawford 1982). The results in figure 5 demonstrate that all three members of group C express the factor V–like subunit mRNA. It is of interest that the highest level of expression of factor V–like protein is observed in P. textilis (approximately six times greater than that of the Oxyuranus species). This reflects the proportion of prothrombinase activator in these species' venom (Masci, Whitaker, and de Jersey 1988). As expected, it was not possible to detect expression of this factor at the mRNA level in any of the four snakes with group D prothrombin activators.
FIG. 5.— mRNA expression of the factor X– and V–like prothrombinase components. Expression levels of components of the prothrombinase complex were determined by qPCR analysis in Australian elapid snakes relative to that of the coastal taipan (Oxyuranus scutellatus). A liver RNA sample from O. scutellatus is included as a control.
Transcription of the factor X–like mRNA was detected in both group C and group D prothrombin activators with some degree of variability (fig. 5). The venom of P. australis has been shown to be largely anticoagulant in clinical pathology and laboratory studies (Tan and Ponnudurai 1990; Dambisya, Lee, and Gopalakrishnakone 1995). Thus, it was not surprising that mRNAs for either factor X– or factor V–like proteins were not expressed in the venom gland of this snake. It is also evident that neither of these factors are expressed in the liver of O. scutellatus.
Relative Expression of Factor Xa–like Proteases in Australian Snake Venoms
To further investigate the prothrombin activator complex in Australian elapids, the expression of the factor Xa catalytic and factor Va–like noncatalytic components was examined in the venom of these snakes. Under nonreducing conditions, the total mass of the factor Xa–like protease (light plus heavy chain linked by a single disulfide bond) in the venom of elapids with group C prothrombin activators (O. scutellatus, O. microlepidotus, and P. textilis) corresponded in size to the purified P. textilis protein and was somewhat greater than that of the group D proteases (fig. 6A, lanes 1–3 vs. 4–7). This antibody (which had been raised against a recombinant form of the heavy chain from P. textilis) failed to detect protein in P. australis as expected (lane 8). To gain an accurate estimate of molecular size, the venom was run under reducing conditions and protein detected with the same antibody (fig. 6B). The heavy chain of the factor Xa–like protease (Xa-HC) was approximately the same size in all three group C venoms (lanes 1–3) and again corresponded in size to the purified P. textilis protein (lane 9). The molecular mass of this protein was lower in the other group D proteases (lanes 4–7), consistent with deletions observed in the heavy chain as described in figure 2. The greater molecular mass change evident in the heavy chain of P. porphyriacus under reducing conditions (lane 7) does not appear to be solely due to changes in the number of amino acids but might be explained by a reduced degree of posttranslational modifications or a different pattern of cleavage.
FIG. 6.— Detection of components of the prothrombin activator complex in the venom of Australian elapids. In all immunoblots, each lane was loaded with 10 μg of venom protein in the following order: (1) Oxyuranus scutellatus, (2) Oxyuranus microlepidotus, (3) Pseudonaja textilis, (4) Notechis scutatus, (5) Tropidechis carinatus, (6) Hoplocephalus stephensii, (7) Pseudechis porphyriacus, (8) Pseudechis australis, and (9) purified P. textilis factor Xa–like protease. (A) Detection with an antiheavy chain factor X protease antibody run under nonreducing gel conditions. (B) Detection with an antiheavy chain antibody under reducing conditions. (C) Detection with antisera raised against the complete snake prothrombin activator from P. textilis under reducing conditions. (D) Detection with a commercial antibody raised against GLA residues under reducing conditions.
Among the group C prothrombin activators, P. textilis had the highest level of expression of the factor Xa–like protease, being approximately fivefold greater than either O. scutellatus or O. microlepidotus per microgram of total venom (fig. 6B, compare lane 3 with lanes 1 and 2). This approximates the expression observed at the mRNA level (fig. 5). The amount of the factor Xa–like protease was variable in all group D snakes, with the greatest amount of protein being observed in T. carinatus, again agreeing with observations at the level of the mRNA.
The results in figure 6C describe the presence of heavy and light chains of the factor Xa–like protease in both groups of snakes using antisera raised against the total prothrombin activator complex of P. textilis. For P. porphyriacus, only a single band was detected, which was equivalent in size to that of the heavy chain evident in figure 6B. A product corresponding in size to this band was also detected using an antibody specific for -carboxylated sites which are known to be present close to the N-terminus of the light chain (fig. 6D) (Persson, Hogg, and Stenflo 1993). This indicates that this band contains cross-reacting sequences for both light and heavy chains, suggesting some alternate or abnormal form of processing that involved different cleavage sites in the precursor protease of this elapid. Immunoblotting with antisera against recombinant forms of the heavy and light chains confirmed these results (data not shown). The antibody used in figure 6C also detected proteins of approximately 65 and 100 kDa, which correspond in size to the light and heavy chains of the nonenzymatic factor V–like subunit and were only present in the group C snakes as expected.
Discussion
The mammalian prothrombinase complex, composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va, plays a central role in the process of blood coagulation (Tracy, Eide, and Mann 1985; Davie, Fujikawa, and Kisiel 1991). An activity related to that of the prothrombinase complex has been well documented in the venom of the Australian elapid snakes (Braud, Bon, and Wisner 2000; Matsui, Fujimura, and Titani 2000). The activity of this prothrombin activator complex has been characterized functionally in both the group C proteases, which require calcium and phospholipids to clot mammalian blood and the group D proteases, which have an additional requirement for factor Va for activity (Kini, Rao, and Joseph 2001). In this study, we performed a comparative molecular analysis of the factor X– and factor V–like components of the prothrombin activators from both subgroups of elapids. The results demonstrate that there is a high degree of sequence relatedness in the factor X–like proteins in seven snake species across the two subgroups. This is evident throughout the entire length of the precursor molecule, with the exception of two short regions within the heavy chain that differentiate the proteases from each group (fig. 2, amino acids 292 304 and 353 360 of the precursor protein). The deduced amino acid sequence of the proteases described here display 78%–96% identity to the precursor catalytic subunit of pseutarin C from P. textilis, recently reported by Rao, Swarup, and Kini (2004). In that study, the cDNA sequence encoded a precursor protein of 449 amino acids compared to the 467–amino acid protein for P. textilis in our studies (Filippovich et al., unpublished data). This difference in size can be attributed to the presence of a termination codon (TAA) in the Rao, Swarup, and Kini (2004) sequence compared to a lysine (AAA) at position 450 of the precursor P. textilis sequence (fig. 2). Given that this lysine is conserved across all seven species of snake studied here, it is likely that the stop codon described by Rao, Swarup, and Kini (2004) is formed due to an error in sequencing or an aberrant clone.
The amino acid sequence for the factor Xa–like catalytic subunit from T. carinatus (trocarin D) and H. stephensii (hopsarin D) determined by protein sequencing has also been reported (Joseph et al. 1999; Rao, Joseph, and Kini 2003). Comparison to the deduced amino acid sequences from multiple cDNA clones identified in the present study reveals that the propeptide and activation peptide sequences are absent from these protein sequences, consistent with their cleavage posttranslationally during the formation of an active molecule. The propeptide in both snakes was 40 amino acids in length and highly conserved, as was the activation peptide (27 residues). The deduced amino acid sequences for T. carinatus and H. stephensii described in figure 2 contain a 11-residue segment of the heavy chain (S260 S270) not identified in either trocarin D or hopsarin D protein sequences. Given that the cDNA data described here demonstrate the presence of this sequence in all seven snakes, it would indicate that this fragment is an inherent part of the mature heavy chain. Except for this deletion, the published protein sequence for trocarin D is 100% identical to the transcript identified in this study (Joseph et al. 1999). Only three amino acid differences were observed when comparisons were made between hopsarin D and its cDNA counterpart (Rao, Joseph, and Kini 2003). In both these sequences (actual and deduced protein), a serine is present at position 216 of the precursor instead of a cysteine conserved in all other snakes. This cysteine is also conserved in mammalian coagulation factor Xa and is involved in a heavy chain intradisulfide linkage (MacGillivray and Fung 1989).
Mammalian factor X is produced in an inactive precursor form containing a propeptide. In order to generate an active molecule (factor Xa), cleavage of the propeptide is required initially, followed by removal of an activation peptide sequence between the light and heavy chains, giving rise to a correctly folded mature protease (Chattopadhyay, James, and Fair 1992). Because the corresponding precursor protein identified in snake venom contains a similar domain arrangement, it is likely that these molecules are processed in a similar fashion. The purification and identification by amino acid sequencing of the heavy and light chains of the factor Xa–like protease from T. carinatus support a similar mechanism of processing (Joseph et al. 1999). They identified the N-terminal sequence of the light chain as SNS, which is consistent with cleavage on the C-terminal side of an arginine residue (R SNS), deduced from results described by Rao, Swarup, and Kini (2004) and the data described in this report using cDNA cloning of the factor X–like protease precursor. It is of interest that this propeptide cleavage site (R ANS) is identical between human factor X and the group C prothrombin activator sequences described here, whereas a single amino acid difference is present in the group D proteases (R SNS). Both cleavage sites surrounding the activation peptide, which were observed to be highly conserved in all seven snake species, are also closely related to the corresponding sites in the human factor X precursor. In addition, the H-D-S catalytic triad, the 11 analogous sites known to be -carboxylated in the mammalian factor Xa protease, and the cysteine residues involved in disulfide linkages are also highly conserved in all species, supporting the importance of these amino acids/modifications in the function of the protein.
Mammalian factor Va enhances the prothrombin-converting activity of the group D proteases but is not required by the group C activators as they contain their own factor V–like protein. The group C prothrombin activators have previously been isolated from O. scutellatus and P. textilis and are high–molecular mass complexes made up of multiple subunits (Masci, Whitaker, and de Jersey 1988). The nonenzymatic subunit of the group C prothrombin activators enhances the catalytic activity of the snake factor Xa–like protease in the conversion of prothrombin to thrombin. Rao and Kini (2002) purified the nonenzymatic subunit of pseutarin C from P. textilis and revealed that it had a domain arrangement similar to that of mammalian coagulation Va with a homology of approximately 55%. We have described the cloning of the factor V–like gene from two other snakes containing group C prothrombin activators (O. scutellatus and O. microlepidotus) and demonstrated that it had 96% identity with the P. textilis protein sequence also cloned as part of this study. The P. textilis clone was observed to be 100% homologous to that reported by Rao, Swarup, and Kini (2003). It was not possible to identify a similar transcript by PCR in any of the snakes as predicted with group D prothrombin activators or in P. australis, given that a factor V–like protein has never been detected in the venom of these snakes. The overall domain arrangement is conserved between all group C proteins (A1-A2-B-A3-C1-C2) identified in this study and is similar to that observed in the mammalian protein. A 30–amino acid signal peptide is 100% identical between the three snakes, which is of considerable interest as this high degree of conservation is also observed within the propeptide for the factor X–like component of the complex in the same snakes. While the signal peptide bears little relatedness to the corresponding human factor V sequence, the cleavage site between this peptide and the heavy chain (A AQLR) is conserved between human and snake species. The cleavage sites at either end of the B domain, however, while conserved among the snakes, display little similarity to the human homolog. Furthermore, the B domain is conserved in size between the snakes (126–127 amino acids), but is significantly smaller than that of the human precursor protein (882 amino acids).
Previous data suggest that the prothrombin activator represents as much as 40% of the total venom protein from P. textilis, which is several-fold greater than that observed for the corresponding complex in O. scutellatus (Masci, Whitaker, and de Jersey 1988). This difference in the amount of prothrombin activator was confirmed in the present study at both the mRNA and protein levels for O. scutellatus, and in addition for another member of the same genus, O. microlepidotus. This was the case for both the factor X– and factor V–like proteins, which were present in approximately equivalent proportions in both Oxyuranus species.
As expected, we did not detect the presence of the noncatalytic factor V–like protein in any of the group D venoms; however, the heavy chain of the factor Xa–like protease was present in all four snakes. It is evident that the molecular size of the heavy chain under reducing conditions is less than that observed in the group C protein (fig. 6B and C). This is particularly evident in the protein from P. porphyriacus which migrates at an apparent molecular mass of 29 kDa. In the case of N. scutatus, T. carinatus, and H. stephensii, this molecular mass difference can be explained by a short deletion in the heavy chain gene sequence and the introduction of a premature stop codon 33 nt shorter than the open reading frame for the group C transcripts (fig. 2). In the case of the P. porphyriacus gene, a 39-nt deletion in the heavy chain sequence could account for at least some of the molecular size difference. An additional contribution to the size change may be a N254T substitution within the precursor protein, which would be predicted to result in the loss of an N-linked glycosylation site in this species. The apparent discrepancy between the migration of the factor Xa–like protein under reducing and nonreducing conditions in P. porphyriacus relative to the other group D snakes (compare fig. 6A with B) is difficult to explain but may be due to differences in posttranslational modifications affecting running conditions or an alternate pattern of cleavage. Although the light chain for the factor Xa–like protease is evident in all of the other six snakes using antisera raised against the total prothrombin activator from P. textilis, this band did not appear to be detected in P. porphyriacus. However, a -carboxylated band was identified at a position of higher molecular mass than that expected of the light chain, which corresponds in size to that of the heavy chain. Given that this modification is specific to the light chain, and because 10 of the 11 -carboxylation sites are conserved in the light chain of P. porphyriacus, it suggests that the pattern of cleavage during processing of the factor X–like protein in this elapid differs from those of the others, even though this is not evident in the primary amino acid sequence. This was further confirmed using antibodies raised against recombinant forms of the light and heavy chains. Neither component of the prothrombin activator was detected in the venom of P. australis (a member of the same genus as P. porphyriacus), confirming the inability to detect mRNA in this species and hence the absence of procoagulant activity in this snake.
The family Elapidae contains almost 300 individual species of which approximately 91 are found in Australia (Keogh, Shine, and Donnellan 1998; Shea 1999). Indeed, almost all known venomous Australian snakes are members of the Elapidae family and are among the most toxic in the world (Broad, Sutherland, and Coulter 1979). There are three major toxic components characterized from Australian elapid venoms, including prothrombin-activating enzymes, phospholipases, and potent peptidic neurotoxins (Fry 1999). The prothrombin activators are responsible solely for the procoagulant activities of these venoms, which contain no thrombin-like enzymes, and prior classification of these venoms into groups C and D has been on the basis of cofactor requirement alone (Chester and Crawford 1982). Clearly, it is desirable to have an additional means for the classification of these species in order to understand their divergence and evolutionary development. The cloning and sequence analysis of genes implicated in the coagulation process described here represent a useful approach for molecular classification. Phylogenetic analysis of the primary factor X–like protein sequence from a select representation of Australian elapids confirms their previous classification on the basis of their cofactor requirement (fig. 3). Using the sequence analysis approach described here, the elapids with group C proteases cluster with a significantly high degree of identity (91%) and differ from the group D proteases with a number of subtle variations including alternate cleavage recognition sites between the propeptide and the light chain as well as other significant changes within the heavy chain. When bootstrap analysis is applied, the relationship within the group C sequences is less well supported with an apparent paraphyly of the Oxyuranus species. However, it should be noted that the bootstrap values in establishing this relationship are low, and direct sequence comparison supports a closer relationship between the two taipans than that suggested by the phylogenetic tree (table 1). Using immunoblotting alone, it is possible to distinguish the two groups on the basis of the molecular size of the factor Xa heavy chain. Interestingly, the overall domain arrangement of the protease is retained among both groups of snakes and indeed with that of the human sequence. Conservation of this structure between mammals and a nonhepatic, nonmammalian source (i.e., venom) indicates its importance for the production of functionally active molecules in the coagulation cascade. This also holds true of the active site residues and other sections of the protein implicated in substrate/cofactor binding and posttranslational modification.
The functional differences observed between the venom of elapid group C and D prothrombin activators can be attributed to the absence of a factor V–like cofactor in the group D complexes, as further confirmed by this study. Sequence results of this protein show a high degree of identity within the group C proteins, as well as a significant level of conservation with its mammalian homolog, again confirming the importance of this structure in molecular interactions with activated factor X. In short, we describe a new means of classification of the group C and group D prothrombin activators from elapid snakes using different molecular markers including DNA sequence analysis, presence or absence of the factor V–like protein, and the molecular size of the factor Xa–like heavy chain.
In conclusion, we have detailed the cloning and characterization of both the factor X– and factor V–like cDNA components of the group C and group D prothrombin activators from Australian elapids. These data reveal a high degree of preservation in domain arrangement with the corresponding mammalian proteins, which is in keeping with their conserved function. The molecular classifications described here provide additional important information on the evolutionary relationship of these subclasses of elapid snake.
Acknowledgements
We wish to acknowledge QRxPharma and the Australian Research Council for financial support and thank Joe Sambono for the provision of venom glands.
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E-mail: martinL@qimr.edu.au.
Abstract
A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X– and factor V–like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X–like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%–97%). The factor V–like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%–98%). Expression of both the factor X– and factor V–like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.
Key Words: prothrombin activator ? factor X ? factor V ? snake venom ? gene cloning ? Oxyuranus scutellatus
Introduction
Snake venoms are a complex mixture of pharmacologically important protein and polypeptide toxins, many of which are known to affect the mammalian blood hemostatic mechanism via coagulant, anticoagulant, and/or fibrinolytic actions. These venom components are related to each other within several families of venomous snakes, including the Elapidae, the Colubridae, the Viperidae, and the Atractaspididae. Australian venomous snakes, belonging exclusively to the Elapidae family, are among the most toxic in the world (Broad, Sutherland, and Coulter 1979). A unique feature of the procoagulant properties of venoms from Australian elapid snakes is that they are limited to prothrombin activation and contain no thrombin-like enzymes. This is due to the presence of prothrombin activators that contain a component that is structurally and functionally similar to that of mammalian coagulation factor Xa (Chester and Crawford 1982).
One of the key reactions in the mammalian blood coagulation and hemostatic process is the cleavage of the zymogen prothrombin to thrombin which in turn activates fibrinogen to fibrin, eventually resulting in clot formation. The proteolytic cleavage of prothrombin is mediated by the prothrombinase complex, a multimeric complex composed of factor Xa in association with its nonenzymatic cofactor factor Va in the presence of calcium on a negatively charged phospholipid bilayer (Jackson and Nemerson 1980; Davie, Fujikawa, and Kisiel 1991). Human activated factor X is a two-chained serine protease linked via a single disulfide bond, with a 52–amino acid glycopolypeptide that is released upon activation. The presence of a vitamin K–dependent, -carboxyglutamic acid residue–rich region at the N-terminus of the light chain plays a critical role in the binding of phospholipids and calcium ions, while the heavy chain contains the catalytic site residues (Jackson 1984; Persson, Hogg, and Stenflo 1993).
Prothrombin activators from snake venoms are currently classified on the basis of their cofactor requirement (Kini, Morita, and Rosing 2001). Metalloproteinases that convert prothrombin to meizothrombin, such as ecarin A isolated from Echis carinatus, are classified as group A (requiring no cofactors for activity) or group B (requiring calcium only for activity) prothrombin activators (Morita and Iwanaga 1978). Group C prothrombin activators are serine proteases that do not require mammalian coagulation factor Va for the cleavage of prothrombin as they contain their own noncatalytic factor Va–like molecule complexed with a factor Xa–like protein (Rao and Kini 2002). As in the case of the mammalian prothrombinase complex, the factor V–like subunit of the snake prothrombin activator serves to increase proteolytic activity. Examples of group C prothrombin activators include pseutarin C (isolated from the venom of the common brown snake, Pseudonaja textilis) and oscutarin C (present in the venom of the coastal taipan, Oxyuranus scutellatus) (Walker, Owen, and Esmon 1980; Masci, Whitaker, and de Jersey 1988). Similarly, group D prothrombin activators are also serine proteases; however, they require calcium, phospholipids, and mammalian coagulation factor Va for maximal activity and include the proteases trocarin D (from the rough-scaled snake, Tropidechis carinatus), hopsarin D (from the Stephen's banded snake, Hoplocephalus stephensii), and notecarin D (identified in the venom of the mainland tiger snake, Notechis scutatus) (Tans et al. 1985; Marsh, Fyffe, and Bennett 1997; Weinstein, Williams, and White 2001). Interestingly, the prothrombin activators identified from Australian elapid snakes appear to be limited to groups C and D only.
Studies on the prothrombin-activating properties of Australian snake venoms have to date focussed primarily upon purified fractions of the native protein, with emphasis on the fact that they are the only nonmammalian and nonhepatic source of molecules capable of activating prothrombin (Joseph et al. 1999). Recently, however, both the factor X–like and much larger factor V–like nonenzymatic subunits of pseutarin C, the prothrombin activator present in the venom of P. textilis, have been isolated and the genes cloned (Rao, Swarup, and Kini 2003, 2004). Pseutarin C comprises approximately 40% of the total dry weight of the venom of P. textilis and its injection results in massive disseminated intravascular coagulation within the body of its prey, ultimately resulting in death (Masci, Whitaker, and de Jersey 1988). Its factor X–like component demonstrates a similar structure to that of mammalian coagulation factor X with a propeptide region, and light and heavy chains bound by a single disulfide linkage and separated via an activation peptide that is cleaved posttranslationally. Overall, there is 48% amino acid identity with human factor Xa. It has been demonstrated that cleavage of this activation peptide is critical for correct folding of the protease and that activity is dependent on posttranslational glycosylation and -carboxylation (Baugh and Krishnaswamy 1996; Joseph et al. 2003). The nonenzymatic subunit of pseutarin C also contains a domain structure similar to its mammalian counterpart with a typical A1-A2-B-A3-C1-C2 domain architecture, accompanied by a number of unique posttranslational modifications (Rao, Swarup, and Kini 2003).
In this study we describe the cloning, characterization, and comparative analysis of components of both group C and group D prothrombin activators isolated from the venom glands of a number of Australian elapids. Results of the study provide significant information not only on the molecular interactions between factors Va and Xa in the formation of the prothrombinase complex but also on the evolutionary relationships between members of the Australian Elapidae family.
Materials and Methods
RNA Isolation and cDNA Synthesis
Venom glands were excised from Australian elapids, including the coastal taipan (O. scutellatus), inland taipan (Oxyuranus microlepidotus), common brown snake (P. textilis), mainland tiger snake (N. scutatus), rough-scaled snake (T. carinatus), Stephen's banded snake (H. stephensii), red-bellied black snake (Pseudechis porphyriacus), and mulga (Pseudechis australis), collected under National Parks and Wildlife's permit number W4\00261\01\SAA. Snap-frozen glands were homogenized with a polytron, RNA isolated using the Tri Reagent method (Sigma-Aldrich, St. Louis, Mo.), precipitated in isopropanol, washed with 70% ethanol, and resuspended in diethylopyrocarbonate treated water. First-strand cDNA was synthesized from 1 μg of total RNA with an oligo(dT)12–18 primer via reverse transcription with 200 units of Superscript II RNase H– Reverse Transcriptase (Invitrogen, Mt. Waverly, Australia). The final reaction was ethanol precipitated, and cDNA samples were resuspended in sterile water and stored at –20°C.
Factor X–like Protease Precursor Amplification and Cloning
Polymerase chain reaction (PCR) amplification of the full-length coding sequence of the factor X–like protease was performed from all cDNAs isolated from Australian elapids. An approximately 1.4-kb product was amplified with 50 pmol of each of the forward (5'-ATG GCT CCT CAA CTA CTC CTC TG-3') and reverse (5'-TTA GAG CCG ACC AGT GCT TGA CTC-3') primers designed from the recently published P. textilis sequence (AY631238 [GenBank] ). The PCR reaction mix was made to a final volume of 25 μl containing approximately 200 ng of cDNA template, 1 unit of AmpliTaq gold (Applied Biosystems, Foster City, Calif.) in 1 x buffer, 2 mM MgCl2, and 200 μM deoxyribonucleoside triphosphates. All reactions were run with appropriate no template controls, with an initial denaturation at 95°C for 8 min, followed by 30 cycles of 95°C for 30 s, 52°C for 45 s, and 72°C for 2 min, and a final extension of 72°C for 7 min.
The resulting PCR products were analyzed on a 1% tris-acetate EDTA agarose gel, and bands of interest were excised and purified with a QIAEX II Gel Extraction kit according to the manufacturer's protocol (Qiagen, Hilden, Germany). Purified PCR products were cloned via the pGEM?-T vector system (Promega, Madison, Wis.). The ligation mix was ethanol precipitated prior to transformation into electrocompetent dH5 Escherichia coli cells, with recombinant clones selected on LB-ampicillin plates (50 μg/ml) supplemented with isopropyl-beta-D-thiogalactoside and 5-bromo-4-chloro-3-indolyl-bD-galactoside. Multiple clones were then sequenced from each snake with an ABI Big Dye Terminator cycle sequence ready reaction kit (PerkinElmer, Norwalk, Conn.). Sequencing was performed in both the forward and reverse directions, as well as with internal primers where appropriate, and results were assembled and analyzed with BioEdit software (Isis Pharmaceuticals Inc., Carlsbad, Calif.). For phylogenetic analyses, the predicted protein sequences were aligned using ClustalW, subjected to maximum likelikood analysis using MolPhy version 2.3 via the Dayhoff model and in local rearrangement of Neighbor-Joining trees mode (1,000 bootstrap replicates) (Adachi and Hasegawa 1996). In these analyses, platypus (Ornithorhynchus anatinus) (AAG00453 [GenBank] and human (Homo sapiens) (CAI41386 [GenBank] factor X sequences were defined as out-groups.
Factor V–like Protein Amplification and Cloning
PCR amplification of the full-length cDNA transcript for the nonenzymatic factor V–like unit of the snake prothrombin-activating complex was performed in elapids containing group C complexes using primer sequences designed from the previously published P. textilis sequence (AY168281 [GenBank] ). The complete mRNA transcript of 4.4 kb was amplified as two overlapping PCR fragments of 2,271 bp (fragment 1, including start codon) and 2,202 bp (fragment 2, including stop codon). PCR amplification was performed in a fashion similar to that of the factor X–like protease with fragment 1 amplified with forward (5'-ATG GAA GAT ACA GTG TGA GCC C-3') and reverse (5'-CTC TGT CTG TTC CCT GCG TGG C-3') primers, while fragment 2 was amplified with forward (5'-CCA GAC CCA GAA TCG GAT GCG C-3') and reverse (5'-GCC TTA AAA AAC TTC ACA ACC-3') primers. Reaction mixtures were thermocycled at 95°C for 10 min, followed by 37 cycles of 95°C for 30 s, 64°C for 45 s (fragment 1) or 59°C for 45 s (fragment 2), and 72°C for 2 min 30 s, followed by a final extension of 72°C for 7 min. PCR products were then visualized on a 1.25% TAE agarose gel, and the appropriate band was excised, purified, cloned, and sequenced as previously described from P. textilis, O. scutellatus, and O. microlepidotus.
Quantitative PCR Analysis
Transcription of the factor X– and factor V–like prothrombin activator genes were analyzed via quantitative PCR (qPCR). cDNA was synthesized from 1.5 μg of venom gland RNA for each snake as well as liver RNA purified from O. scutellatus. Prior to cDNA synthesis, total RNA was digested with two units of RQ1 DNase-polymerase to remove any contaminating genomic DNA that may affect expression results (Promega). DNase-treated RNA was ethanol precipitated prior to cDNA synthesis, and 1:50 dilution of the cDNA was then used in subsequent qPCR reactions as template. In all cases 7.5 μl of a 2 x Sybr Green PCR master mix was added to 5 pmol of a forward and reverse primer mix and 3 μl of diluted cDNA template in a total volume of 15 μl (Applied Biosystems). A 190-bp fragment of the factor X–like protease was amplified with a forward (5'-GAA GAC CCC TAT CCA GTT CTC-3') and reverse (5'-CAT GCA GGT GTG CCT GTC CAC A-3') primer pair, while a 201-bp section of the factor V–like protein was amplified with forward (5'-CTC AAG GAG CCA CAT CAA TGA C-3') and reverse (5'-GGA TGA TAC GAA TGA ACC TGG AC-3') primers, based on a sequence 100% homologous to all snakes. Each template was run in triplicate, and every run included the amplification of the 60S ribosomal protein housekeeping gene, which was amplified as a 195-bp product with forward (5'-GCA AGC GTA TGA ACA CCA ACC C-3') and reverse (5'-AGA GCA GCT GGG ACG ACC ATT C-3') primers.
Reaction mixtures were thermocycled at 95°C for 10 min, followed by 35 cycles of 95°C for 15 s, 64°C for 15 s, and 72°C for 30 s on a Rotor-Gene 3000 (Corbett Research, Mortlake, Australia). A standard curve of cycle time using a range of known quantities of plasmid as template was also obtained. PCR products were run on a 1% TAE agarose gel to confirm amplification, and the results including cycle time for product formation and calculated transcript concentration as determined by the Rotor-Gene software were exported into Microsoft Excel. Averages of the triplicate samples in duplicate runs were then taken, and the results normalized to that of the housekeeping gene.
Prothrombin Activator Detection in Venom
The presence and quantity of the factor X– and factor V–like components of the snake prothrombinase complex were examined in the venom of all elapids by immunoblotting with a number of different primary antibodies. For all western blot analysis, a total of 10 μg of lyophilized venom resuspended in 50% glycerol from O. scutellatus, O. microlepidotus, P. textilis, N. scutatus, T. carinatus, H. stephensii, P. porphyriacus, and P. australis were run on a sodium dodecyl sulfate (SDS)–polyacrylamide gel in that order, with a prestained molecular weight marker (Fermentas, Hanover, Md.). All gels were also run with an appropriate quantity of purified P. textilis factor Xa–like protease.
Venom proteins separated by electrophoresis on a 12% SDS–polyacrylamide gel under either reducing or nonreducing conditions were then transferred to nitrocellulose membranes at 100 V for 1 h at 4°C in transfer buffer (100 mM Tris, 40 mM glycine, 0.036% SDS, and 20% methanol). Blots were then probed with a range of primary antibodies including an antibody raised in sheep against a recombinant form of the heavy chain from P. textilis (residues D302 L467), an antibody against the purified prothrombin activator complex from P. textilis raised in rabbits, and finally a commercial monoclonal antibody raised against human -carboxyglutamyl (GLA) residues in mice (American Diagnostica Inc., Stamford, Conn.). Membranes were then probed with appropriate secondary antibodies conjugated to horseradish peroxidase and the protein signal was detected with enhanced chemiluminescence reagents according to manufacturer's instructions (PerkinElmer).
Results
Identification of Factor Xa–like Proteases from Australian Elapids
Full-length cDNA clones corresponding to the coding sequence of the factor Xa–like prothrombin activator were isolated from the venom glands of Australian elapids based on the P. textilis protease gene sequence (Filippovich et al., unpublished data) which has also recently been described by Rao, Swarup, and Kini (2004). Two primers designed from the propeptide and heavy chain gene sequences were used to amplify an approximately 1.4-kb fragment. The results in figure 1 demonstrate the presence of a product in P. textilis, O. scutellatus, P. porphyriacus, N. scutatus, T. carinatus, and O. microlepidotus. A similar product was also amplified from the venom gland cDNA of H. stephensii (results not shown); however, protease mRNA was not detected in the mulga (P. australis) even when additional primer pairs, based on the internal gene sequence of P. textilis, were employed. This is not surprising given that the venom of this snake does not demonstrate procoagulant activity (Tan and Ponnudurai 1990; Dambisya, Lee, and Gopalakrishnakone 1995).
FIG. 1.— PCR amplification of the factor X–like protease cDNA. A complete 1.4-kb PCR product was amplified from venom gland cDNA isolated from (1) Pseudonaja textilis, (2) Oxyuranus scutellatus, (3) Pseudechis porphyriacus, (4) Notechis scutatus, (5) Tropidechis carinatus, (6) Pseudechis australis, (7) Oxyuranus microlepidotus, (8) no template control, and (9) molecular weight marker. A similar product was also amplified from the venom gland cDNA of Hoplocephalus stephensii at a later date (not shown).
Multiple clones were generated to identify the protease cDNA sequence in each snake for nucleotide comparisons. The overall identity at the nucleotide level was 82.5% when an alignment was performed between all seven snake species. A protein alignment of the predicted amino acid sequence from these cDNAs is depicted in figure 2. The overall identity between the predicted snake proteins is 71.5%. Comparison of the two taipan species, O. scutellatus and O. microlepidotus, reveals an identity of 95% (table 1). These proteases were most closely related to that of P. textilis with a three-way identity of 91%. While the degree of homology between all snake species was high (80%–97%), the protease from P. porphyriacus was least related to the others.
FIG. 2.— Alignment of the factor X–like proteases. An alignment was performed of the deduced amino acid sequence obtained from multiple cDNA clones of the factor X–like protease isolated from the venom glands of seven Australian snakes, including Oxyuranus scutellatus (AY940204), Oxyuranus microlepidotus (AY940205), Pseudonaja textilis (AY631238), Pseudechis porphyriacus (AY940207), Hoplocephalus stephensii (AY940208), Notechis scutatus (AY940206), and Tropidechis carinatus (AY769963). GenBank accession numbers are given in parentheses. Sequence identity is shown in black, gaps are inserted for optimal alignment, and the cleavage sites for the propeptide, light chain, activation peptide, and heavy chain sequences are also marked. The sites for -carboxylation of GLA residues are marked by and active site residues with an *. Conserved cysteine residues involved in putative disulfide bond formation are shaded gray.
Table 1 Identity of the Factor X–like Proteases from the Venom Glands of Australian Elapids Compared to that of the Human Homolog
Phylogenetic analysis of the deduced amino acid sequences resolved the snake factor X–like prothrombin activators into two distinct clades, each with high bootstrap support (fig. 3). It is interesting to note that this clustering, based on sequence relatedness, separates the elapids into two distinct groups which have previously been defined on the basis of their requirement for mammalian factor Va as a cofactor for activity. Group C prothrombin activators, including proteases isolated from members of the Pseudonaja and Oxyuranus genera, do not require mammalian coagulation factor Va for activity as they have their own factor Va–like molecule complexed to the factor Xa–like protease. On the other hand, the group D prothrombin activators do have a requirement for mammalian factor Va. The classification of this group described here on the basis of sequence relatedness corresponds exactly with previous subgrouping based on factor Va requirement. Although the phylogenetic analyses strongly support a close relationship between the venom group D sequences, relationships within the group C clade are not well supported, which explains the implied paraphyly of the genus Oxyuranus. The diverged nature of the P. porphyriacus sequence is also clear in the tree shown in figure 3, although the analyses strongly support its basal position within the venom group D clade.
FIG. 3.— Phylogenetic relationships of the factor X–like proteins. The maximum likelihood method was employed to determine the phylogenetic relationship of the deduced amino acid sequences of factor X–like prothrombin activators from multiple Australian elapids. Numbers above branches indicate the percentage of 1,000 bootstrap replicates supporting the topology shown. Clades corresponding to group C and group D factor X–like proteases are clearly resolved with high bootstrap support.
Comparison of the snake protease sequence with that of human factor X reveals that it possesses a similar overall domain arrangement, including a propeptide, light chain, activation peptide, and heavy chain in that order (MacGillivray and Fung 1989). In all cases, the 40–amino acid propeptide sequence was highly conserved, differing by only a single residue in P. porphyriacus and H. stephensii (fig. 2). This sequence contains a high content (60%) of hydrophobic residues consistent with its role as secretion signal/signal peptide, and the only two substitutions observed also led to conservation of hydrophobic residues. It is evident from the domain arrangement that this precursor protein requires processing to produce the mature heavy and light chain cross-linked protease, similar to that of its mammalian counterpart (Di Scipio et al. 1977). The cleavage site between the propeptide and light chain is highly conserved between all snake species being R ANS in group C proteases and R SNS in group D prothrombin activators. The cleavage sites at either end of the activation peptide in the precursor protease are absolutely conserved in all snake species, with the exception of a hydrophobic amino acid substitution in the case of P. porphyriacus (V211I).
While the protease sequences are highly conserved overall, the three most closely related snakes among the group D prothrombin activators (H. stephensii, T. carinatus, and N. scutatus) have a premature stop codon that deletes the final 11 C-terminal amino acids compared to the group C proteases and P. porphyriacus. In addition, deletions of a variable number of amino acids were observed between residues 292 and 304 of precursor protein in the group D proteases, plus another area of low homology in the heavy chain within residues 353–360 (fig. 2).
In human factor Xa, 12 disulfide bonds have been identified in total, seven in the light chain, four in the heavy chain, and one interchain disulfide linkage (Davie, Fujikawa, and Kisiel 1991). All these cysteine residues are conserved in all seven snake sequences identified in this study, with the exception of a C216S substitution in the H. stephensii precursor protease, consistent with the previously published amino acid sequence of hopsarin D (P83370 [GenBank] ) (Rao, Joseph, and Kini 2003). Similarly, vitamin K–dependent posttranslational carboxylation of glutamic acid (GLA) residues in the light chain has been described for human factor Xa and the group D prothrombin activators, including 11 GLA residues identified in the N-terminal domain of trocarin D (Joseph et al. 1999). Results in figure 2 demonstrate that these 11 GLA residues are also conserved in all Australian elapids with the exception of an E72K substitution in the P. porphyriacus precursor protein.
A further posttranslational modification reported in the prothrombin activator of pseutarin C from P. textilis is the O-linked glycosylation of a serine at position 92 of the precursor protein which is also present in the other six elapid sequences (fig. 2) (Joseph et al. 2003). Similarly, an N-linked glycosylation of a heavy chain asparagine at position 254 of pseutarin C precursor protein is also conserved among the other snakes with P. porphyriacus again being the exception. The three active site residues of human factor Xa are conserved within all snakes in this study, corresponding to positions H251, D309, and S406 of the O. scutellatus precursor. An activation peptide located between the light and heavy chain sequences in the precursor human factor X plays a key role in the folding and processing of the protease (Baugh and Krishnaswamy 1996). A 27–amino acid sequence (approximately half that of the human equivalent), similarly positioned between the light and heavy chains, is also present and highly conserved in all snakes.
Comparative Analysis of Factor V–like Proteins
Based on the nucleotide sequence of the factor V–like nonenzymatic subunit of pseutarin C from P. textilis (AY168281 [GenBank] ), amplification of similar full-length cDNA sequences for elapids involved in this study was attempted. Because only group C prothrombin activators contain their own factor V–like sequence, it was only possible to obtain an amplified product in O. scutellatus, O. microlepidotus, as well as P. textilis. Alignment of the deduced protein sequence from multiple cDNA clones demonstrated that there was 95.7% identity between the three snakes (fig. 4).
FIG. 4.— Alignment of the factor V–like proteins. An alignment of the deduced amino acid sequence of the factor V–like nonenzymatic unit of the prothrombinase complex amplified from cDNA from venom glands of the group C Australian snakes Oxyuranus scutellatus (AY940209 [GenBank] ), Oxyuranus microlepidotus (AY940210 [GenBank] ), and the previously identified Pseudonaja textilis (AY168281 [GenBank] ) is shown. Arrows mark the boundaries of the different domains, and conserved cysteines involved in putative disulfide linkages are shaded gray.
The overall domain arrangement of the factor V–like protein observed in the Oxyuranus species was the same as that of mammalian coagulation factor V and the nonenzymatic subunit of pseutarin C (A1-A2-B-A3-C1-C2) (Rao, Swarup, and Kini 2003). A 30–amino acid signal peptide is also present with 100% identity in all three elapids. The two thrombin cleavage sites reported in P. textilis (R772 and R817 in the precursor molecule) are again conserved between the three snakes. Rao, Swarup, and Kini (2003) have recently demonstrated that the pseutarin C nonenzymatic subunit contains only a single conserved activated protein C cleavage site, whereas the mammalian equivalent has three sites required for the inactivation of factor Va (Kalafatis, Rand, and Mann 1994). Both P. textilis and O. microlepidotus contain a lysine residue at this site, whereas a conserved amino acid (arginine) is present in the O. scutellatus protein.
The association of factors Xa and Va on a phospholipid bilayer, forming the prothrombinase complex, is a key step in the blood coagulation process (Steen 2002). Kalafatis and Beck (2002) identified a binding site for factor Xa within the heavy chain of human factor Va between amino acid residues 323 and 331. The corresponding residues in the elapid sequences (amino acids E354 I362 in the precursor molecule) are completely conserved with one another, and the first eight of these residues are identical to the corresponding human sequence, with the final amino acid being a hydrophobic residue in both instances (V I). Another key component of the prothrombinase complex are phospholipids, which have been predicted from three-dimensional structural analysis to bind to the surface-exposed loop KKSWW in mammalian coagulation factor Va. Mutagenesis of these residues identified both tryptophans, which are absolutely conserved in all three elapid sequences, as important in interactions with phospholipids (Nicolaes, Villoutreix, and Dahlback 2000). Of the seven putative disulfide linkages reported for human factor Va, six are conserved in the group C sequences (fig. 4).
mRNA Expression of Factor X– and Factor V–like Components from Australian Elapids
As outlined above, the Australian elapids under investigation fall into two categories, group C which includes complete prothrombin activators and group D that require phospholipids, calcium ions, and mammalian coagulation factor Va for activity (Chester and Crawford 1982). The results in figure 5 demonstrate that all three members of group C express the factor V–like subunit mRNA. It is of interest that the highest level of expression of factor V–like protein is observed in P. textilis (approximately six times greater than that of the Oxyuranus species). This reflects the proportion of prothrombinase activator in these species' venom (Masci, Whitaker, and de Jersey 1988). As expected, it was not possible to detect expression of this factor at the mRNA level in any of the four snakes with group D prothrombin activators.
FIG. 5.— mRNA expression of the factor X– and V–like prothrombinase components. Expression levels of components of the prothrombinase complex were determined by qPCR analysis in Australian elapid snakes relative to that of the coastal taipan (Oxyuranus scutellatus). A liver RNA sample from O. scutellatus is included as a control.
Transcription of the factor X–like mRNA was detected in both group C and group D prothrombin activators with some degree of variability (fig. 5). The venom of P. australis has been shown to be largely anticoagulant in clinical pathology and laboratory studies (Tan and Ponnudurai 1990; Dambisya, Lee, and Gopalakrishnakone 1995). Thus, it was not surprising that mRNAs for either factor X– or factor V–like proteins were not expressed in the venom gland of this snake. It is also evident that neither of these factors are expressed in the liver of O. scutellatus.
Relative Expression of Factor Xa–like Proteases in Australian Snake Venoms
To further investigate the prothrombin activator complex in Australian elapids, the expression of the factor Xa catalytic and factor Va–like noncatalytic components was examined in the venom of these snakes. Under nonreducing conditions, the total mass of the factor Xa–like protease (light plus heavy chain linked by a single disulfide bond) in the venom of elapids with group C prothrombin activators (O. scutellatus, O. microlepidotus, and P. textilis) corresponded in size to the purified P. textilis protein and was somewhat greater than that of the group D proteases (fig. 6A, lanes 1–3 vs. 4–7). This antibody (which had been raised against a recombinant form of the heavy chain from P. textilis) failed to detect protein in P. australis as expected (lane 8). To gain an accurate estimate of molecular size, the venom was run under reducing conditions and protein detected with the same antibody (fig. 6B). The heavy chain of the factor Xa–like protease (Xa-HC) was approximately the same size in all three group C venoms (lanes 1–3) and again corresponded in size to the purified P. textilis protein (lane 9). The molecular mass of this protein was lower in the other group D proteases (lanes 4–7), consistent with deletions observed in the heavy chain as described in figure 2. The greater molecular mass change evident in the heavy chain of P. porphyriacus under reducing conditions (lane 7) does not appear to be solely due to changes in the number of amino acids but might be explained by a reduced degree of posttranslational modifications or a different pattern of cleavage.
FIG. 6.— Detection of components of the prothrombin activator complex in the venom of Australian elapids. In all immunoblots, each lane was loaded with 10 μg of venom protein in the following order: (1) Oxyuranus scutellatus, (2) Oxyuranus microlepidotus, (3) Pseudonaja textilis, (4) Notechis scutatus, (5) Tropidechis carinatus, (6) Hoplocephalus stephensii, (7) Pseudechis porphyriacus, (8) Pseudechis australis, and (9) purified P. textilis factor Xa–like protease. (A) Detection with an antiheavy chain factor X protease antibody run under nonreducing gel conditions. (B) Detection with an antiheavy chain antibody under reducing conditions. (C) Detection with antisera raised against the complete snake prothrombin activator from P. textilis under reducing conditions. (D) Detection with a commercial antibody raised against GLA residues under reducing conditions.
Among the group C prothrombin activators, P. textilis had the highest level of expression of the factor Xa–like protease, being approximately fivefold greater than either O. scutellatus or O. microlepidotus per microgram of total venom (fig. 6B, compare lane 3 with lanes 1 and 2). This approximates the expression observed at the mRNA level (fig. 5). The amount of the factor Xa–like protease was variable in all group D snakes, with the greatest amount of protein being observed in T. carinatus, again agreeing with observations at the level of the mRNA.
The results in figure 6C describe the presence of heavy and light chains of the factor Xa–like protease in both groups of snakes using antisera raised against the total prothrombin activator complex of P. textilis. For P. porphyriacus, only a single band was detected, which was equivalent in size to that of the heavy chain evident in figure 6B. A product corresponding in size to this band was also detected using an antibody specific for -carboxylated sites which are known to be present close to the N-terminus of the light chain (fig. 6D) (Persson, Hogg, and Stenflo 1993). This indicates that this band contains cross-reacting sequences for both light and heavy chains, suggesting some alternate or abnormal form of processing that involved different cleavage sites in the precursor protease of this elapid. Immunoblotting with antisera against recombinant forms of the heavy and light chains confirmed these results (data not shown). The antibody used in figure 6C also detected proteins of approximately 65 and 100 kDa, which correspond in size to the light and heavy chains of the nonenzymatic factor V–like subunit and were only present in the group C snakes as expected.
Discussion
The mammalian prothrombinase complex, composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va, plays a central role in the process of blood coagulation (Tracy, Eide, and Mann 1985; Davie, Fujikawa, and Kisiel 1991). An activity related to that of the prothrombinase complex has been well documented in the venom of the Australian elapid snakes (Braud, Bon, and Wisner 2000; Matsui, Fujimura, and Titani 2000). The activity of this prothrombin activator complex has been characterized functionally in both the group C proteases, which require calcium and phospholipids to clot mammalian blood and the group D proteases, which have an additional requirement for factor Va for activity (Kini, Rao, and Joseph 2001). In this study, we performed a comparative molecular analysis of the factor X– and factor V–like components of the prothrombin activators from both subgroups of elapids. The results demonstrate that there is a high degree of sequence relatedness in the factor X–like proteins in seven snake species across the two subgroups. This is evident throughout the entire length of the precursor molecule, with the exception of two short regions within the heavy chain that differentiate the proteases from each group (fig. 2, amino acids 292 304 and 353 360 of the precursor protein). The deduced amino acid sequence of the proteases described here display 78%–96% identity to the precursor catalytic subunit of pseutarin C from P. textilis, recently reported by Rao, Swarup, and Kini (2004). In that study, the cDNA sequence encoded a precursor protein of 449 amino acids compared to the 467–amino acid protein for P. textilis in our studies (Filippovich et al., unpublished data). This difference in size can be attributed to the presence of a termination codon (TAA) in the Rao, Swarup, and Kini (2004) sequence compared to a lysine (AAA) at position 450 of the precursor P. textilis sequence (fig. 2). Given that this lysine is conserved across all seven species of snake studied here, it is likely that the stop codon described by Rao, Swarup, and Kini (2004) is formed due to an error in sequencing or an aberrant clone.
The amino acid sequence for the factor Xa–like catalytic subunit from T. carinatus (trocarin D) and H. stephensii (hopsarin D) determined by protein sequencing has also been reported (Joseph et al. 1999; Rao, Joseph, and Kini 2003). Comparison to the deduced amino acid sequences from multiple cDNA clones identified in the present study reveals that the propeptide and activation peptide sequences are absent from these protein sequences, consistent with their cleavage posttranslationally during the formation of an active molecule. The propeptide in both snakes was 40 amino acids in length and highly conserved, as was the activation peptide (27 residues). The deduced amino acid sequences for T. carinatus and H. stephensii described in figure 2 contain a 11-residue segment of the heavy chain (S260 S270) not identified in either trocarin D or hopsarin D protein sequences. Given that the cDNA data described here demonstrate the presence of this sequence in all seven snakes, it would indicate that this fragment is an inherent part of the mature heavy chain. Except for this deletion, the published protein sequence for trocarin D is 100% identical to the transcript identified in this study (Joseph et al. 1999). Only three amino acid differences were observed when comparisons were made between hopsarin D and its cDNA counterpart (Rao, Joseph, and Kini 2003). In both these sequences (actual and deduced protein), a serine is present at position 216 of the precursor instead of a cysteine conserved in all other snakes. This cysteine is also conserved in mammalian coagulation factor Xa and is involved in a heavy chain intradisulfide linkage (MacGillivray and Fung 1989).
Mammalian factor X is produced in an inactive precursor form containing a propeptide. In order to generate an active molecule (factor Xa), cleavage of the propeptide is required initially, followed by removal of an activation peptide sequence between the light and heavy chains, giving rise to a correctly folded mature protease (Chattopadhyay, James, and Fair 1992). Because the corresponding precursor protein identified in snake venom contains a similar domain arrangement, it is likely that these molecules are processed in a similar fashion. The purification and identification by amino acid sequencing of the heavy and light chains of the factor Xa–like protease from T. carinatus support a similar mechanism of processing (Joseph et al. 1999). They identified the N-terminal sequence of the light chain as SNS, which is consistent with cleavage on the C-terminal side of an arginine residue (R SNS), deduced from results described by Rao, Swarup, and Kini (2004) and the data described in this report using cDNA cloning of the factor X–like protease precursor. It is of interest that this propeptide cleavage site (R ANS) is identical between human factor X and the group C prothrombin activator sequences described here, whereas a single amino acid difference is present in the group D proteases (R SNS). Both cleavage sites surrounding the activation peptide, which were observed to be highly conserved in all seven snake species, are also closely related to the corresponding sites in the human factor X precursor. In addition, the H-D-S catalytic triad, the 11 analogous sites known to be -carboxylated in the mammalian factor Xa protease, and the cysteine residues involved in disulfide linkages are also highly conserved in all species, supporting the importance of these amino acids/modifications in the function of the protein.
Mammalian factor Va enhances the prothrombin-converting activity of the group D proteases but is not required by the group C activators as they contain their own factor V–like protein. The group C prothrombin activators have previously been isolated from O. scutellatus and P. textilis and are high–molecular mass complexes made up of multiple subunits (Masci, Whitaker, and de Jersey 1988). The nonenzymatic subunit of the group C prothrombin activators enhances the catalytic activity of the snake factor Xa–like protease in the conversion of prothrombin to thrombin. Rao and Kini (2002) purified the nonenzymatic subunit of pseutarin C from P. textilis and revealed that it had a domain arrangement similar to that of mammalian coagulation Va with a homology of approximately 55%. We have described the cloning of the factor V–like gene from two other snakes containing group C prothrombin activators (O. scutellatus and O. microlepidotus) and demonstrated that it had 96% identity with the P. textilis protein sequence also cloned as part of this study. The P. textilis clone was observed to be 100% homologous to that reported by Rao, Swarup, and Kini (2003). It was not possible to identify a similar transcript by PCR in any of the snakes as predicted with group D prothrombin activators or in P. australis, given that a factor V–like protein has never been detected in the venom of these snakes. The overall domain arrangement is conserved between all group C proteins (A1-A2-B-A3-C1-C2) identified in this study and is similar to that observed in the mammalian protein. A 30–amino acid signal peptide is 100% identical between the three snakes, which is of considerable interest as this high degree of conservation is also observed within the propeptide for the factor X–like component of the complex in the same snakes. While the signal peptide bears little relatedness to the corresponding human factor V sequence, the cleavage site between this peptide and the heavy chain (A AQLR) is conserved between human and snake species. The cleavage sites at either end of the B domain, however, while conserved among the snakes, display little similarity to the human homolog. Furthermore, the B domain is conserved in size between the snakes (126–127 amino acids), but is significantly smaller than that of the human precursor protein (882 amino acids).
Previous data suggest that the prothrombin activator represents as much as 40% of the total venom protein from P. textilis, which is several-fold greater than that observed for the corresponding complex in O. scutellatus (Masci, Whitaker, and de Jersey 1988). This difference in the amount of prothrombin activator was confirmed in the present study at both the mRNA and protein levels for O. scutellatus, and in addition for another member of the same genus, O. microlepidotus. This was the case for both the factor X– and factor V–like proteins, which were present in approximately equivalent proportions in both Oxyuranus species.
As expected, we did not detect the presence of the noncatalytic factor V–like protein in any of the group D venoms; however, the heavy chain of the factor Xa–like protease was present in all four snakes. It is evident that the molecular size of the heavy chain under reducing conditions is less than that observed in the group C protein (fig. 6B and C). This is particularly evident in the protein from P. porphyriacus which migrates at an apparent molecular mass of 29 kDa. In the case of N. scutatus, T. carinatus, and H. stephensii, this molecular mass difference can be explained by a short deletion in the heavy chain gene sequence and the introduction of a premature stop codon 33 nt shorter than the open reading frame for the group C transcripts (fig. 2). In the case of the P. porphyriacus gene, a 39-nt deletion in the heavy chain sequence could account for at least some of the molecular size difference. An additional contribution to the size change may be a N254T substitution within the precursor protein, which would be predicted to result in the loss of an N-linked glycosylation site in this species. The apparent discrepancy between the migration of the factor Xa–like protein under reducing and nonreducing conditions in P. porphyriacus relative to the other group D snakes (compare fig. 6A with B) is difficult to explain but may be due to differences in posttranslational modifications affecting running conditions or an alternate pattern of cleavage. Although the light chain for the factor Xa–like protease is evident in all of the other six snakes using antisera raised against the total prothrombin activator from P. textilis, this band did not appear to be detected in P. porphyriacus. However, a -carboxylated band was identified at a position of higher molecular mass than that expected of the light chain, which corresponds in size to that of the heavy chain. Given that this modification is specific to the light chain, and because 10 of the 11 -carboxylation sites are conserved in the light chain of P. porphyriacus, it suggests that the pattern of cleavage during processing of the factor X–like protein in this elapid differs from those of the others, even though this is not evident in the primary amino acid sequence. This was further confirmed using antibodies raised against recombinant forms of the light and heavy chains. Neither component of the prothrombin activator was detected in the venom of P. australis (a member of the same genus as P. porphyriacus), confirming the inability to detect mRNA in this species and hence the absence of procoagulant activity in this snake.
The family Elapidae contains almost 300 individual species of which approximately 91 are found in Australia (Keogh, Shine, and Donnellan 1998; Shea 1999). Indeed, almost all known venomous Australian snakes are members of the Elapidae family and are among the most toxic in the world (Broad, Sutherland, and Coulter 1979). There are three major toxic components characterized from Australian elapid venoms, including prothrombin-activating enzymes, phospholipases, and potent peptidic neurotoxins (Fry 1999). The prothrombin activators are responsible solely for the procoagulant activities of these venoms, which contain no thrombin-like enzymes, and prior classification of these venoms into groups C and D has been on the basis of cofactor requirement alone (Chester and Crawford 1982). Clearly, it is desirable to have an additional means for the classification of these species in order to understand their divergence and evolutionary development. The cloning and sequence analysis of genes implicated in the coagulation process described here represent a useful approach for molecular classification. Phylogenetic analysis of the primary factor X–like protein sequence from a select representation of Australian elapids confirms their previous classification on the basis of their cofactor requirement (fig. 3). Using the sequence analysis approach described here, the elapids with group C proteases cluster with a significantly high degree of identity (91%) and differ from the group D proteases with a number of subtle variations including alternate cleavage recognition sites between the propeptide and the light chain as well as other significant changes within the heavy chain. When bootstrap analysis is applied, the relationship within the group C sequences is less well supported with an apparent paraphyly of the Oxyuranus species. However, it should be noted that the bootstrap values in establishing this relationship are low, and direct sequence comparison supports a closer relationship between the two taipans than that suggested by the phylogenetic tree (table 1). Using immunoblotting alone, it is possible to distinguish the two groups on the basis of the molecular size of the factor Xa heavy chain. Interestingly, the overall domain arrangement of the protease is retained among both groups of snakes and indeed with that of the human sequence. Conservation of this structure between mammals and a nonhepatic, nonmammalian source (i.e., venom) indicates its importance for the production of functionally active molecules in the coagulation cascade. This also holds true of the active site residues and other sections of the protein implicated in substrate/cofactor binding and posttranslational modification.
The functional differences observed between the venom of elapid group C and D prothrombin activators can be attributed to the absence of a factor V–like cofactor in the group D complexes, as further confirmed by this study. Sequence results of this protein show a high degree of identity within the group C proteins, as well as a significant level of conservation with its mammalian homolog, again confirming the importance of this structure in molecular interactions with activated factor X. In short, we describe a new means of classification of the group C and group D prothrombin activators from elapid snakes using different molecular markers including DNA sequence analysis, presence or absence of the factor V–like protein, and the molecular size of the factor Xa–like heavy chain.
In conclusion, we have detailed the cloning and characterization of both the factor X– and factor V–like cDNA components of the group C and group D prothrombin activators from Australian elapids. These data reveal a high degree of preservation in domain arrangement with the corresponding mammalian proteins, which is in keeping with their conserved function. The molecular classifications described here provide additional important information on the evolutionary relationship of these subclasses of elapid snake.
Acknowledgements
We wish to acknowledge QRxPharma and the Australian Research Council for financial support and thank Joe Sambono for the provision of venom glands.
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