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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 621-631
Amino Acid Sequence of Trocarin, a Prothrombin Activator From
Tropidechis carinatus Venom: Its Structural Similarity to
Coagulation Factor Xa
By
Jeremiah S. Joseph,
Maxey C.M. Chung,
Kandiah Jeyaseelan, and
R.
Manjunatha Kini
From the Bioscience Centre, Faculty of Science, the Department of
Biochemistry, Faculty of Medicine, and the Bioprocessing Technology
Centre, Faculty of Engineering, National University of Singapore,
Singapore; and the Department of Biochemistry and Molecular Biophysics,
Medical College of Virginia, Virginia Commonwealth University,
Richmond, VA.
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ABSTRACT |
Among snake venom procoagulant proteins, group II prothrombin
activators are functionally similar to blood coagulation factor Xa. We
have purified and partially characterized the enzymatic properties of
trocarin, the group II prothrombin activator from the venom of the
Australian elapid, Tropidechis carinatus (rough-scaled snake).
Prothrombin activation by trocarin is enhanced by Ca2+,
phospholipids, and factor Va, similar to that by factor Xa. However,
its amidolytic activity on peptide substrate S-2222 is significantly lower. We have determined the complete amino acid sequence of trocarin. It is a 46,515-Dalton glycoprotein highly homologous to factor Xa and shares the same domain architecture. The
light chain possesses an N-terminal Gla domain containing 11  -carboxyglutamic acid residues, followed by two epidermal growth
factor (EGF)-like domains; the heavy chain is a serine proteinase. Both
chains are likely glycosylated: the light chain at Ser 52 and the heavy
chain at Asn 45. Unlike other types of venom procoagulants, trocarin is
the first true structural homologue of a coagulation factor. It clots
snake plasma and thus may be similar, if not identical, to snake blood
coagulation factor Xa. Unlike blood factor Xa, it is expressed in high
quantities and in a nonhepatic tissue, making snake venom the richest
source of factor Xa-like proteins. It induces cyanosis and death in
mice at 1 mg/kg body weight. Thus, trocarin acts as a toxin in venom and a similar, if not identical, protein plays a critical role in hemostasis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE ACTIVATION OF prothrombin to thrombin
is a reaction central to the blood coagulation cascade. This is
accomplished in vivo by the prothrombinase complex consisting of a
serine proteinase factor Xa and a cofactor factor Va formed on
negatively charged phospholipid membranes in the presence of
Ca2+ ions.1 Prothrombin is also specifically
targeted by various exogenous hemostatic factors that affect blood
coagulation.2 Over the years, many such exogenous proteins
from snake venom sources have been identified.3,4 These
proteins proteolytically activate prothrombin and are grouped into four
categories based on structural characteristics, functional properties,
and cofactor requirements in prothrombin activation.5,6
Group IA activators, such as ecarin,7 are
metalloproteinases that efficiently convert prothrombin into
meizothrombin. Their activity is not enhanced by the addition of the
nonenzymatic cofactors of the prothrombinase complex: factor Va,
phospholipids, and Ca2+ ions. On the other hand, group IB
activators are metalloproteinase-C-type-lectin complexes whose
activity is Ca2+-dependent. They are exemplified by
carinactivase-1 and multactivase.6,8 The ability of group
II prothrombin activators, such as notecarin,9 to convert
prothrombin to thrombin is greatly enhanced by factor Va in the
presence of negatively charged phospholipids and Ca2+
ions. This group of proteins has been found exclusively in
Australian elapid venoms. Group III activators also activate
prothrombin to thrombin by cleaving both the necessary peptide bonds.
However, their prothrombin-converting abilities are enhanced by only
Ca2+ ions and phospholipids, not by factor Va. Such
prothrombin activators have been isolated from Oxyuranus
scutellatus (oscutarin) and Pseudonaja textilis
venoms.10,11
Functionally, group II prothrombin activators strongly resemble blood
coagulation factor Xa.2,5,9 Like factor Xa, these serine
proteinases cleave both peptide bonds (Arg 274-Thr 275 and Arg 323-Ile
324) of bovine prothrombin, meizothrombin and prethrombin-2 being
formed as intermediates.9 Their prothrombin-converting activities are stimulated to an extent comparable to that of factor Xa
by the addition of the other cofactors required for the formation of
the prothrombinase complex.9 As with factor Xa, factor Va forms a tight 1:1 complex with these prothrombin activators in the
presence of Ca2+ ions and phospholipids.9 The
molecular size and subunit compositions of these proteinases and factor
Xa are also comparable. The molecular masses of group II proteinases
range from 54 to 64.5 kD,2,12 whereas that of factor Xa is
45 kD. All of these activators, like factor Xa, have two
disulfide-linked subunits, a light chain (molecular weight [Mr],
~23 kD) and a heavy chain (Mr range, 32 to 37 kD). Furthermore, the
active site of these serine proteinases is also found in the heavy chain.
Although functional similarities between group II prothrombin
activators and factor Xa have been well documented, structural details,
including sequence information on this group, are not yet available.
Structural aspects of these proteinases should contribute significantly
towards understanding structure-function relationships and mechanisms
of factor Xa-mediated prothrombin activation. Furthermore, the
phylogenetic distance and functional similarities between these
proteinases and factor Xa could help define molecular details of
interactions among the protein components of the prothrombinase
complex. Therefore, we initiated studies on trocarin, a group II
prothrombin activator from the venom of the Australian rough-scaled
snake, Tropidechis carinatus. We describe here its
purification, characterization, and complete amino acid sequence. (The SWISS-PROT accession no. of trocarin is P81428.) The
data presented here indicate that trocarin is the first exogenous procoagulant protein produced in a nonhepatic source, namely, the venom
gland, which shares structural and functional similarities with
coagulation factor Xa.
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MATERIALS AND METHODS |
Materials.
T carinatus venom was obtained from Venom Supplies Pty Ltd
(Tanunda, South Australia). Lysyl endopeptidase, endoproteinase Glu C,
and endoproteinase Asp N were purchased from Wako Pure Chemicals
(Osaka, Japan).  -Mercaptoethanol was purchased from Nacalai Tesque
(Kyoto, Japan). Trypsin, 4-vinylpyridine, phosphatidylcholine (PC),
phosphatidylserine (PS), ovalbumin, bovine serum albumin (BSA), and
soybean trypsin inhibitor were bought from Sigma Chemical Co (St Louis,
MO). Cyanogen bromide was bought from JT Baker (Phillipsburg, NJ).
BNPS-skatole was obtained from Fluka Chemika-BioChemika (Buchs, Switzerland). S-2222 and S-2238 were purchased from Chromogenix (Mölndal, Sweden). Bovine prothrombin, bovine factor Xa, and bovine factor Va were obtained from Haematologic Technologies Inc
(Essex Junction, VT). Human plasma was donated by healthy volunteers.
Python reticulatus plasma was obtained by heart puncture of
pythons anaesthetized with sodium phenobarbital (60 mg/kg body weight).
All other chemicals and reagents were of the highest quality available.
Procoagulant activity.
The procoagulant activity of T carinatus venom and its
fractions was determined by their effect on the recalcification time of
human plasma using a BBL fibrometer (Becton-Dickinson
Microbiology Systems, Sparks, MD). Clotting of a mixture of 100 µL
human plasma and 250 µL (50 mmol/L Tris-HCl buffer, pH 7.5, plus
sample) at 37°C was initiated by adding 50 µL of 50 mmol/L
CaCl2, and the recalcification time was measured. The
procoagulant effect of the purified prothrombin activator, trocarin, on
P reticulatus plasma was also determined. Dose
responses for trocarin and human factor Xa using both human and P
reticulatus plasmas were performed.
Reconstitution of the prothrombinase complex.
The effect of the various cofactors of the prothrombinase complex
(phospholipids, factor Va, and Ca2+ ions) on prothrombin
activation by trocarin was determined by measuring the generation of
thrombin. The prothrombinase complex was reconstituted as follows.
Appropriate dilutions of trocarin (or bovine and human factor Xa, for
comparison) were preincubated for 15 minutes at 25°C in a buffer
containing 50 mmol/L Tris-HCl, 100 mmol/L NaCl, 0.5 mg/mL ovalbumin, pH
7.5, in the presence or absence of various cofactors such as
phospholipid vesicles (9:1 PC:PS; prepared according to de Kruiff et
al13; 100 µmol/L), factor Va (10 nmol/L), and
CaCl2 (5 mmol/L). When CaCl2 was not present in
the mixture, EDTA was added (final concentration, 10 mmol/L).
Activation was initiated with the addition of prothrombin (final
concentration, 5.6 µmol/L). After different time intervals, aliquots
of 1 µL were withdrawn from the reaction mixture (total volume, 50 µL) and added to 190 µL of 50 mmol/L Tris-HCl, 100 mmol/L NaCl, pH
7.5, containing 10 µg/mL soybean trypsin inhibitor, and 25 mmol/L
EDTA (to inhibit further conversion of prothrombin). The
thrombin-specific chromogenic substrate
H-D-Phe-pipecolyl-Arg-p-nitroaniline hydrochloride (S-2238) was
subsequently added (10 µL; final concentration, 100 µmol/L), and
the amidolytic activity of the thrombin formed was determined by
measuring its hydrolysis at 405 nm. Measurements were made in
triplicate using 96-well microtiter plates on a Ceres UV 900C ELISA
plate reader (Bio-Tek Instruments Inc, Winooski, VT).
Soybean trypsin inhibitor inhibits the low amidase activity of factor
Xa on S-2238 but does not affect the activity of thrombin on the
substrate.14
Determination of cleavage site(s) of prothrombin by trocarin.
The cleavage products formed during prothrombin activation by trocarin
were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Prothrombin (5 µg) was incubated with
trocarin or human factor Xa (5 ng each) in the presence of Ca2+ ions (5 mmol/L), 9:1 PC:PS phospholipid vesicles (100 µmol/L), and factor Va (10 nmol/L) in 50 mmol/L Tris-HCl, 100 mmol/L
NaCl, pH 7.5, at 37°C for 4 hours. The reaction mixtures were
electrophoresed on a nonreducing gradient gel (4% to 20%), according
to Laemmli.15 The gels were stained with Coomassie
Brilliant Blue R-250.
To determine the cleavage site(s) of prothrombin by trocarin, we
activated prothrombin in the presence of factor Va and Ca2+
ions. Bovine prothrombin (1 µmol/L), trocarin (or human factor Xa,
for comparison; 10 nmol/L), and bovine factor Va (5 nmol/L) were
dissolved in a buffer of pH 7.5, containing 50 mmol/L Tris-HCl, 100 mmol/L NaCl, 5 mmol/L CaCl2, and 1 µmol/L BSA, and
incubated at 37°C overnight. The cleavage products were separated
by reverse-phase high pressure liquid chromatography (RP-HPLC) on a
Sephasil C18 (100 × 2.1 mm) column. The amino terminal sequence
of thrombin was determined by Edman degradation, as described below.
Amidolytic activity on chromogenic substrate.
The amidolytic activity of trocarin on the factor Xa-specific
chromogenic substrate
N-benzoyl-L-Ile-L-Glu-L-Gly-L-Arg-p-nitroaniline hydrochloride
(S-2222) was studied as follows. Varying amounts of S-2222
were added to 50 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.5, containing
trocarin (1 µmol/L) or factor Xa (10 nmol/L) in disposable cuvettes
(total volume, 1 mL) at 25°C. The release of p-nitroaniline
was monitored at 405 nm using a Jasco V-560 UV/Vis spectrophotometer
(Jasco Corp, Tokyo, Japan). The Km and Vmax were estimated from Lineweaver-Burk plots.
Protein purification.
Trocarin was purified by gel filtration of T
carinatus crude venom, followed by anion exchange and RP-HPLC.
Crude venom (200 mg in 2 mL distilled water) was applied to a Superdex
75 gel filtration column (1.6 × 60 cm) equilibrated with 50 mmol/L Tris-HCl buffer, pH 7.5, using a Pharmacia FPLC system
(Pharmacia, Uppsala, Sweden). The pooled procoagulant
fraction was subfractionated on an anion exchange column UNO Q-1
(Bio-Rad, Hercules, CA; column volume, 1 mL) also using
the FPLC system. Approximately 20 mg of protein was loaded on to the
column equilibrated with 50 mmol/L Tris-HCl buffer, pH 7.5. Bound
proteins were eluted with a linear gradient of 1 mol/L NaCl in the same
buffer. In the final step, trocarin was purified by RP-HPLC on a
Nucleosil C18 (250 × 10 mm) column using a Vision Workstation
(Perkin-Elmer Biosystems, Foster City, CA). The column was
equilibrated with 0.1% trifluoroacetic acid (TFA), and a linear
gradient of 80% acetonitrile in 0.1% TFA was used for elution.
Capillary zone electrophoresis.
Homogeneity of the protein was assessed by capillary electrophoresis
essentially as described earlier.16
Electrospray ionization mass spectrometry.
Precise masses (±0.01%) of the native protein and peptides were
determined by electrospray ionization mass spectrometry using a
Perkin-Elmer Sciex API 300 LC/MS/MS System. Theoretical masses of the
peptides were calculated by a custom software written by Chiew Fook
Loong (School of Computing, National University of Singapore, Singapore).
Separation of subunits.
The protein subunits were separated by reduction and pyridylethylation.
The native protein (1 mg) was dissolved in 500 µL of 0.13 mol/L Tris
HCl, 1 mmol/L EDTA, 6 mol/L guanidine HCl, pH 8.5. After adding 20 µL
of the reducing agent, -mercaptoethanol, this solution was incubated
under N2 for 2 hours at 37°C. The alkylating reagent,
4-vinylpyridine (200 µL), was subsequently added and the mixture was
incubated under N2 for another 2 hours at room temperature.
The reaction mixture was immediately desalted and the two
pyridylethylated chains separated by RP-HPLC on a Nucleosil C18 (250 × 4.6 mm) column using a linear gradient of acetonitrile in 0.1% TFA.
Enzymatic cleavage.
Peptides of the light and heavy chains were generated by digestions
with the enzymes lysyl endopeptidase (Lys C), endoproteinase Glu C, and
endoproteinase Asp N. In each case, approximately 200 µg of protein
was dissolved in 200 µL of 50 mmol/L Tris-HCl buffer, 4 mol/L urea, 5 mmol/L EDTA, pH 7.5. The enzymes Lys C, Glu C, or Asp N (5 µg each)
were added and digestions were performed overnight at 37°C.
Chemical cleavage.
Heavy chain peptides were also obtained by chemical cleavage using
cyanogen bromide (Met-specific) and
3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole;
Trp-specific) performed essentially as described in Current
Protocols in Protein Science,17 except that, in the former procedure, 70% TFA was used, instead of 70% formic acid. For
cleavage with CNBr, the heavy chain (500 µg) was dissolved in 250 µL of 70% TFA to which 5 µL of -mercaptoethanol was added. A
200-fold molar excess (over Met residues) of CNBr in 70% TFA was added
to make a final protein concentration of 1 mg/mL. After 24 hours of
incubation in the dark at room temperature, 10 vol of Milli-Q water was
added to the mixture and lyophilized. Tryptophanyl bond cleavage was
performed as follows. The heavy chain (500 µg) was dissolved in 500 µL 0.1% TFA. Approximately 1.5 mL of 1 mg/mL BNPS-skatole solution
in glacial acetic acid was added, and the mixture was incubated
overnight in the dark at room temperature for 24 hours. An equal volume
of Milli-Q water was subsequently added and the digest was centrifuged
at 10,000g to precipitate residual reagent and reaction byproducts.
The peptides generated by enzymatic and chemical cleavage were
separated by RP-HPLC using a Sephasil C18 (100 × 2.1 mm), a Jupiter C18 (250 × 4.6 mm), or a Hypersil BDS C18 (150 × 1 mm) column, using linear gradients of acetonitrile in 0.1% TFA.
Amino terminal sequencing.
Amino terminal sequencing of the native protein, its light and heavy
chains, and the peptides generated by digesting them was performed by
automated Edman degradation using a Perkin-Elmer Applied Biosystems 494 pulsed-liquid phase protein sequencer (Procise) with an on-line 785A
PTH-amino acid analyzer.
Decarboxylation of light chain.
The light chain was decarboxylated according to Poser and
Price.18 The reduced and alkylated light chain was
lyophilized to dryness from 50 mmol/L HCl and then heated at 110°C
overnight under vacuum. Before sequencing, the sample was cleaned up by RP-HPLC using a Jupiter C18 (250 × 4.6 mm) column.
Carbohydrate detection.
The method of Dubois et al19 was used to detect the
presence of carbohydrate in individual chains of the protein.
Concentrated sulphuric acid (5 mL) was dropped directly into 1 mL of
5% phenol containing 400 µg of the light chain or 480 µg of the
heavy chain, and the absorbance at 490 nm was measured. Glucose was
used as the standard; BSA and insulin were negative controls.
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RESULTS AND DISCUSSION |
Purification of prothrombin activator from T carinatus venom.
The procoagulant effects of the venom of T carinatus are well
known.20,21 A factor Xa-like activity responsible for these procoagulant effects was partially purified by Morrison et
al.22 Recently, Marsh et al12 isolated and
partially characterized the prothrombin activator and showed that it
belongs to group II. We purified it to homogeneity from T
carinatus venom as follows. The crude venom was separated into
eight fractions on a Superdex 75 gel filtration column
(Fig 1A). Procoagulant fractions were pooled and subfractionated using anion exchange chromatography on a UNO
Q-1 column (Fig 1B). Of the eight fractions obtained, procoagulant
activity was found only in fraction 6. This fraction was homogeneous,
as judged from its RP-HPLC profile (Fig 1C). This procoagulant protein
was named trocarin in accordance with the recommendations of the
Subcommittee for the Nomenclature of Exogenous Hemostatic
Factors.23 It comprised approximately 5% of total venom
protein (wt/wt). Trocarin is not highly soluble in aqueous buffers (pH
3.0 to 7.5). When pure, it dissolves only up to approximately 1 mg/mL.
Its late elution on a Nucleosil C18 column also indicates its
hydrophobic nature.
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| Fig 1.
Purification and determination of homogeneity of
trocarin. (A) Gel chromatography of T carinatus venom on a
Superdex 75 column. Flow rate: 1.5 mL/min. Procoagulant activities of
all fractions were tested and fractions indicated by a horizontal bar
were pooled. (B) Subfractionation of pooled procoagulant fraction by
anion exchange chromatography on a UNO Q-1 column. Bound proteins were
eluted by a linear gradient of NaCl. Flow rate: 2 mL/min. Procoagulant
activities of all fractions were tested and fractions indicated by a
horizontal bar were pooled. (C) RP-HPLC of fraction 6 from UNO Q-1
column. Column: Nucleosil C18. Bound proteins were eluted by a linear
gradient of buffer B (80% ACN in 0.1% TFA). Flow rate: 2 mL/min. The
procoagulant protein peak is labeled as trocarin. (D) Capillary zone
electropherogram of trocarin. Electrophoresis runs of the protein (1 mg/mL) were performed on a BioFocus 3000 HPCE system (Bio-Rad) using a
coated capillary (24 cm × 25 µm) from positive to negative
polarities at 12 kV. Buffer: 100 mmol/L phosphate buffer, pH 2.5. Temperature: 18°C. Duration: 20 minutes. Sample injected using
pressure mode 10 psi/s. Trocarin migrates as a single peak at 4.32 min.
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The homogeneity of trocarin was determined by capillary zone
electrophoresis and electrospray ionization mass spectrometry. The
electropherogram (Fig 1D) shows a single peak, indicating a single
molecular species. The mass spectrum (data not shown) showed peaks of
mass/charge ratios ranging from 19 to 38 charges and the molecular mass
of trocarin was found to be 46,515 based on the reconstructed spectrum.
Some preparations showed a variation in molecular mass (±0.1%).
This is most likely due to heterogeneity in glycosylation or
fragmentation of carbohydrate groups during mass spectrometry, because,
during sequence determination, no heterogeneity in any amino acid
residue was observed.
Procoagulant activity and catalytic properties of trocarin.
Trocarin exhibits potent procoagulant effects. It shortened the
recalcification time of human plasma (control clotting times of 161 seconds) to 22 seconds at a concentration of 16.9 nmol/L (Fig 2). A similar procoagulant protein has
been isolated from T carinatus venom,12
which is known to clot factor X-deficient plasma, but not factor V- and
II-deficient plasmas, suggesting that it activates prothrombin and
requires factor V for optimum activity. Furthermore, like all group II
prothrombin activators and factor Xa, its blood clotting activity was
enhanced by the addition of phospholipids and Ca2+
ions.12 We determined the effect of Ca2+ ions,
negatively charged phospholipid vesicles (9:1 PC:PS), and bovine factor
Va on the rate of bovine prothrombin activation by trocarin and factor
Xa (bovine and human). The addition of Ca2+ ions,
phospholipids, and factor Va greatly stimulates the activity of
trocarin (Table 1), confirming that it is a
group II prothrombin activator. Trocarin has a higher basal activity in
the absence of all cofactors compared with bovine and human factor Xa.
The stimulation of trocarin by the cofactors is generally about an order of magnitude lower than that of bovine and human factor Xa. The
complete complex containing trocarin is twofold and fourfold less
active than prothrombinase complexes containing bovine and human Xa,
respectively (Table 1). This could be due to subtle differences in the
interaction between reptilian and mammalian enzymes with mammalian
factor Va. In addition, optimal cofactor concentrations for trocarin
may be different from those for mammalian enzymes.
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| Fig 2.
Effect of trocarin on coagulation of human and python
plasma. Procoagulant activity measured by determining recalcification
time of plasmas in the presence of various amounts of trocarin or human
factor Xa using BBL fibrometer at 37°C. Clotting of plasma was
initiated by the addition of CaCl2. Trocarin
shows very similar procoagulant effects to human factor Xa on both
human and python plasma.
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Table 1.
Effect of Ca2+ Ions, Phospholipids, and
Bovine Factor Va on Trocarin- and Factor Xa-Mediated Prothrombin
Activation
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The cleavage products obtained by trocarin-mediated prothrombin
activation were examined by SDS-PAGE on a nonreducing gel. The
electrophoretic pattern was identical to that for prothrombin cleavage
products by human Xa (Fig 3). Human factor
Xa cleaves bovine prothrombin at two sites, Arg 274-Thr 275 and Arg
323-Ile 324. We determined the cleavage site(s) of prothrombin by
trocarin in the presence of factor Va and Ca2+ ions.
Amino-terminal sequencing of the first four residues of one of the
cleavage products (Mr, 38 kD) yielded two parallel sequences,
Thr-Ser-Glu-Asp and Ile-Val-Glu-Gly. These correspond to the
amino-termini of the light and heavy chains of thrombin generated when
cleavage of prothrombin occurs at Arg 274-Thr 275 and Arg 323-Ile 324. This indicates that trocarin has identical cleavage specificities to
mammalian factor Xa, cleaving both peptide bonds of bovine prothrombin
required for conversion to physiological thrombin. This is in contrast
to group IA and IB activators, which cleave only one peptide bond,
converting prothrombin to meizothrombin.5,6
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| Fig 3.
SDS-PAGE analysis of prothrombin activation. Prothrombin
(5 µg) was activated with trocarin or human factor Xa (5 ng each) in
the presence of Ca2+ ions (5 mmol/L), 9:1 PC:PS
phospholipid vesicles (100 µmol/L), and factor Va (10 nmol/L) at
37°C for 4 hours. The reaction mixture was loaded on a nonreducing
4% to 20% gradient gel. Untreated prothrombin (lane a), cleavage
products of prothrombin by trocarin (lane b), and factor Xa (lane c)
were electrophoresed according to Laemmli.15
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We tested the amidolytic activity of trocarin on a synthetic substrate
specific for factor Xa. Trocarin did cleave S-2222, indicating
identical sequence specificity of cleavage to factor Xa. We determined
the Km and Vmax for the S-2222 hydrolysis by trocarin and human and bovine factor Xa
(Table 2). Whereas the Km's
for the three enzymes are comparable, the Vmax of trocarin is approximately 800 and 1,400 times lower than that of human and
bovine factor Xa, respectively. Similar results were reported earlier
with notecarin. It showed approximately 120-fold lower amidolytic
activity than bovine factor Xa on CBS 31.39, the best synthetic
substrate among those that were tested.9 It is interesting to note that trocarin has a lower amidolytic activity on synthetic substrate S-2222, but a higher activity on a macromolecular substrate, prothrombin, in the absence of Ca2+ ions than mammalian
factor Xa (Table 1).
Amino terminal sequencing of trocarin.
Amino terminal sequencing of the native protein yielded two parallel
sequence signals of equivalent intensity, indicating that it consisted
of two chains. These two sequences were highly homologous to the light
and heavy chains of factor Xa. Upon reduction and pyridylethylation,
the native protein yielded two chains: a light and a heavy chain 17 and
30 kD in mass, respectively. The individual chains were
sequenced separately.
The light chain.
Amino-terminal sequencing of the light chain yielded the first 40 residues. Eleven residues at positions 6, 7, 14, 16, 18, 19, 25, 26, 29, 32, and 35 could not be identified. These positions correspond to
those of Gla residues in factor Xa. Most of the sequence of the light
chain was completed with peptides generated by a Lys C digest
(Fig 4A). Seven Lys C peptides (Lc1-21,
Lc22-36, Lc37-62, Lc63-79, Lc80-83, Lc84-101, and Lc102-137) covered
the first 137 residues (Fig 5A). The
C-terminal peptide, Lc128-151, and other overlapping peptides (Lc39-77,
Lc78-82, Lc83-106, and Lc114-127) were obtained and sequenced from a
Glu C digest (data not shown). Lysine was confirmed to be the
C-terminal residue of the light chain, because it was the last residue
sequenced on peptides, Lc129-141 and Lc128-141, generated by Asp N
(data not shown) and Glu C, respectively (Fig 5A). The sequences of all
peptides were unambiguously identified and their masses corroborate the
sequence data (Table 3). In addition to the
Gla residues, residue 52 could not be identified during sequencing,
indicating that there may be some posttranslational modification at
this location (see below).
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| Fig 4.
RP-HPLC separation of peptides from Lys C digests of
trocarin subunits. (A) LC-MS profile of peptides from light chain
digest on a Hypersil BDS C18 column. Flow rate: 50 µL/min. (B) HPLC
profile of peptides from heavy chain digest on a Jupiter C18 column.
Flow rate: 1 mL/min. Peptides were eluted with a linear gradient of
buffer B (80% ACN in 0.1% TFA).
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| Fig 5.
Complete amino acid sequence of trocarin. Strategy for
determination of the sequence of light (A) and heavy chains (B).
N-term.: Amino terminal sequence of intact chain. Peptides were derived
from a digest of light or heavy chains by endoproteinase Lys C (Lys C),
endoproteinase Glu C (Glu C), endoproteinase Asp N (Asp N), cyanogen
bromide (CNBr), and BNPS-skatole (BNPS-sk). Solid lines represent
regions whose sequences were determined by Edman degradation and/or
confirmed by mass spectrometry. Broken lines represent regions of
peptides that were not sequenced.
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The heavy chain.
The first 43 residues were identified by amino-terminal sequencing of
intact, reduced, and alkylated heavy chain. The sequence was completed
using peptides generated with Lys C (Fig 4B) and Glu C (data not shown)
digests. Eleven Lys C peptides, Hc9-22, Hc23-48, Hc52-61, Hc62-66,
Hc67-95, Hc122-136, Hc137-144, Hc145-212, Hc215-220, Hc224-229, and
Hc231-235, were sequenced (Fig 5B). Long peptide Hc145-212 was
subdigested with trypsin (data not shown) and a resultant peptide,
Hc195-211, was sequenced. An additional eight peptides obtained from
the Glu C digest, Hc1-21, Hc22-52, Hc25-52, Hc53-102, Hc103-117,
Hc118-195, Hc196-207, and Hc208-235, were needed to complete the
sequence (Fig 5B). Endoproteinase Glu C also cleaved after Asp207 and
resulted in the peptide Hc208-235. To confirm the heavy chain sequence,
peptides were also generated by chemical cleavage with CNBr and
BNPS-skatole (data not shown). Cyanogen bromide peptides, Hc6-94,
Hc95-120, Hc121-157, and Hc169-232, and BNPS-skatole peptides, Hc1-14,
Hc15-205, Hc206-227, and Hc228-235, were sequenced (Fig 5B). Only the
N-termini of peptides Hc6-94 and Hc15-205 were sequenced. The
C-terminal Lys was the final residue on Glu C peptide, Hc208-235, and
BNPS-skatole peptide, Hc228-235. Verification of the sequence of the
heavy chain was obtained from the masses of peptides from various
digests (Table 3). The masses of seven peptides (Hc81-85, Hc88-111,
Hc112-122, Hc123-160, Hc161-173, Hc174-178, and Hc179-194) obtained
from an Asp N digest of the heavy chain (data not shown) provided
further verification of sequence data (Table 3). The identity of
residue 45 could not be ascertained by sequencing, indicating
posttranslational modification (see below).
Posttranslational modifications in trocarin:
-Carboxylation of glutamate residues.
As mentioned above, residues 6, 7, 14, 16, 18, 19, 25, 26, 29, 32, and
35 of the light chain were unidentifiable by sequencing and
corresponded to positions of Gla residues in factor Xa. Both mass
spectrometric and sequencing approaches were used to identify and
confirm the presence of Gla residues in these positions. The masses of
two Lys C-generated peptides, Lc1-21 and Lc22-36, were consistent with
the presence of Gla residues in the unidentified positions (Table 3).
In the mass spectra of these peptides, we could identify partially
decarboxylated peptides with masses corresponding to successive loss of
up to 3 or 4 carbon dioxide molecules (Fig 6). For further confirmation, we decarboxylated18 the light chain and sequenced its amino terminus. Glutamate residues were identified in positions 6, 7, 14, 16, 18, 19, 25, 26, 29, 32, and 35 that were blank during the sequencing of the untreated light chain.
Thus, these results confirm the presence of Gla residues at these
positions of the light chain.
View larger version (24K):
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| Fig 6.
Evidence for -carboxylation of glutamic acid residues
in the Gla domain. Reconstructed mass spectra of peptides spanning the
Gla domain: (A) Lc1-21 and (B) Lc22-36. Total masses of these peptides
are consistent with the presence of Gla residues in them. Partially
decarboxylated peptides with masses corresponding to successive loss of
up to 4 and 3 carbon dioxide molecules (~44 mass units),
respectively, can also be identified.
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The existence of Gla residues is documented in venom proteins. Tans et
al9 earlier showed the presence of Gla residues in
notecarin, the group II prothrombin activator from N scutatus scutatus. They estimated eight Gla residues per molecule, in
contrast to trocarin's 11 Gla residues. The group III prothrombin
activator, oscutarin (Oxyuranus scutellatus), has also been
shown to possess Gla residues.10
O-glycosylation.
As mentioned earlier, light chain residue 52 could not be identified
during sequencing. During preliminary characterization, we identified
the presence of carbohydrate moieties in both light and heavy chains
using the method of Dubois et al19 (data not shown). Thus,
we predicted that position 52 is probably glycosylated. Mass
spectrometric data on a peptide containing residue 52 indicates that it
is a Ser that is O-glycosylated with a disaccharide moiety (unpublished
observations). Characteristically, O-glycosylation sites
are found in Pro/Ser/Thr-rich regions. In trocarin, Ser 51 and Pro 54 are found in the immediate vicinity of Ser 52 (51-SSNP-54). Although
Ser or Thr occupies position 52 in several factor Xa proteins
(Fig 7) and these regions are similar to
that of trocarin, these residues are not glycosylated. However,
disaccharide (xylose-glucose) and trisaccharide [xylose(2)-glucose]
units have been found to be O-linked to corresponding Ser residues on
the light chains of human and bovine factors VII and IX and human
protein Z.24,25
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| Fig 7.
Alignment of amino acid sequence of trocarin with factor
Xa sequences. The sequences are taken from the following sources.
T carinatus: Tropidechis carinatus (rough-scaled snake,
this report, SWISS-PROT no. P81428); G gallus: Gallus
gallus (chicken, SWISS-PROT no. P25155); R norvegicus:
Rattus norvegicus (rat, Genbank no. 2144491); O
cuniculus: Oryctolagus cuniculus (rabbit, SWISS-PROT no.
O19045); B taurus: Bos taurus (bovine, SWISS-PROT no.
P00743); H sapiens: Homo sapiens (human, SWISS-PROT no.
P00742); and M musculus: Mus musculus (mouse, Genbank
no. 2664220). Identical and homologous residues are shaded in red and
blue, respectively. Gla, EGF-I, and EGF-II domain boundaries are
indicated above light chain sequences. Gaps ( ) are inserted for
optimal alignment.
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|
N-glycosylation.
As mentioned above, the heavy chain was also found to be glycosylated.
Because only position 45 could not be identified during sequencing of
the heavy chain, it was recognized as the glycosylation site.
Furthermore, it falls within the consensus sequence N-X-T, where N is
glycosylated. Based on mass difference, we estimate the size of
carbohydrate moiety to be approximately 3 kD. Deconvolution of the mass
spectra of Lys C peptide Hc23-48, Glu C peptides Hc22-52 and Hc25-52,
CNBr peptide Hc6-94, and BNPS-skatole peptide Hc15-205 was difficult,
probably due to either heterogeneity in glycosylation or fragmentation
of the carbohydrate during ionization. Further studies are underway to
determine the structure of the carbohydrate moiety.
Lack of -hydroxylation of Aspartate 63.
In vitamin K-dependent blood coagulation factors containing
epidermal growth factor (EGF) domains, including factor X, Asp 63 (64),
is modified to erythro--hydroxyaspartate (Hya).26,27 However, during sequencing of Lys C peptide Lc63-79 of trocarin, we
identified Asp at position 63. Mass spectrometric data on peptide Lc63-79 also indicated the lack of -hydroxylation of Asp 63 (data not shown). According to Stenflo et al,28 hydroxylation is
associated with the consensus sequence
Cys-X-Hya/Hyn-X-X-X-X-Tyr/Phe-X-C-X-C. Because trocarin fulfills these
sequence requirements, perhaps additional secondary and/or tertiary
structural features required for hydroxylation are missing.
Homology and domain structure of trocarin.
Trocarin shares high identity (53% to 60.0%) and homology (62% to
70%) with factor Xa (Fig 7). Based on homology, we propose similar
domain architecture for trocarin. The light chain is homologous to the
light chains of vitamin K-dependent coagulation factors, especially
that of factor Xa (53% to 59% identity, 60% to 67% homology with
factor Xa). It consists of an N-terminal Gla domain (residues 1-39),
followed by two EGF-like domains, EGF-I (residues 50-81) and EGF-II
(residues 89-124). Cysteine residues are completely conserved. By
homology, intrachain disulfide linkages should be between cysteines 17 and 22, 50 and 61, 55 and 70, 72 and 81, 89 and 100, 96 and 109, and
111 and 124. Cysteine 132 most probably links the light chain to Cys
108 of the heavy chain.
The amino-terminal domain of trocarin has 11 Gla residues. The first 10 lie in positions conserved in the Gla domains of vitamin K-dependent
coagulation factors. The eleventh Gla residue in position 35 of
trocarin is conserved in other factor Xas but is replaced by Asp in
human factor Xa. On the other hand, Gla 39 found in other factor Xas is
missing in trocarin. This indicates that, although the positions of the
first 10 Gla residues (6, 7, 14, 16, 19, 20, 25, 26, 29, and 32) are
critical, either one of the additional Glas in positions 35 and 39 can
be dispensed without severely compromising activity.
The organization of Gla residues into a domain homologous to the Gla
domains of vitamin K-dependent coagulation factors has been shown for
the first time in a protein isolated from venom. A number of peptide
toxins that have been sequenced from the venoms of the Conus
species of mollusks also contain Gla residues.29,30 However, the organization of these Gla residues does not resemble Gla
domains of vitamin K-dependent coagulation factors. Although Gla residues are involved in calcium binding, they play different overall functions in Conus peptide toxins and vitamin
K-dependent coagulation factors. In many Conus toxins, Gla
residues, upon binding calcium, contribute to the formation of a
functionally important amphipathic helix.31-34 On the other
hand, the Gla domains of factor Xa and other vitamin K-dependent
coagulation factors mediate Ca2+-dependent binding to
negatively charged phospholipids. Calcium and negatively charged
phospholipids have a stimulatory effect on trocarin's
prothrombin-converting ability (Table 1). Other Gla-containing proteins
from snake venom, notecarin9 and oscutarin,10 also possess the same property (ie, Ca2+- and
phospholipid-stimulated activity).
All factor Xas have a short hydrophobic (helical) stack of residues
forming a linker between the Gla and EGF-I domains and so does
trocarin. As mentioned above, it is known that the first EGF domain of
factor Xa is involved in calcium binding. This domain of trocarin
shares 56% to 75% identity and 66% to 84% homology with those of
other factor Xas, and the residues that act as calcium ligands in
factor Xa EGF-I domains are completely conserved. Hence, it is probable
that the EGF-I domain of trocarin also binds calcium. In factor Xa,
there is some evidence that the second EGF module may mediate factor Va
binding, which may be further enhanced by the presence of EGF-I
domain.35 The second EGF domain of trocarin has only a 39%
to 53% identity and a 44% to 58% homology with factor Xa EGF-II
domains, which is significantly lower than that between their EGF-I
domains. However, factor Va is still able to enhance the activity of
trocarin. Thus, it would be interesting to examine the
structure-function relationships of this domain.
The heavy chain is the catalytic subunit and is homologous to serine
proteinases. It contains all functional residues, including the triad
of the charge-relay system: His 42, Asp 88, and Ser 185. Comparison
with other factor Xas show identities and homologies ranging from 50%
to 60% and 62% to 71%, respectively. Cysteine residues are
completely conserved even in the heavy chain. By homology, intrachain
disulfide bonds are probably formed between cysteines 7 and 12, 27 and
43, 156 and 170, and 181 and 209. The residues forming the
substrate-binding pocket (Asp 179, Ala 180, Cys 181, and Gln 182) and
those involved in forming the hydrogen-bonded structure antiparallel to
the substrate (Ser 204, Trp 205, Gly 206, and Gly 216) are identical to
those found in factor Xa. We noted that the region from residues 48 to
84 shows very little homology with factor Xa. Here, identities and
homologies range from only 6% to 14% and 11% to 25%, respectively.
However, synthetic peptides based on the sequence of this region in
human factor Xa (heavy chain residues 60 to 75 and 69 to 80) were
highly effective in preventing factor Xa-factor Va
interaction.36 The role of the corresponding segments of
trocarin in its interaction with factor Va would be of interest.
Probable physiological role of trocarin.
Snake venom contains up to 11.5 mg/mL of trocarin, whereas human blood
contains only 10 µg/mL of factor X. Thus, snake venom is the richest
source of factor Xa-like proteins. It is important to note that
mammalian coagulation proteins are synthesized primarily in the liver.
Trocarin, on the other hand, is expressed in a nonhepatic tissue, the
venom gland. The only other known exogenous (ie, nonblood) factor Xa is
the virus-activating protease isolated from chick embryo allantoic and
amniotic fluid, but this is found at concentrations of only about 2 ng/mL37 and 165 ng/mL,38 respectively. Trocarin induced cyanosis and death in mice within approximately 320 and 150 minutes at 1 and 10 mg/kg (intraperitoneal), respectively. A
prothrombin activator from P textilis venom causes death in rats within several minutes of intravenous injection at doses as low as
23 µg/kg body weight.11 Preinjection of heparin
(intravenous) neutralized the lethal effects of the
procoagulant,11 indicating that lethality was due to
disseminated intravascular coagulopathy.4 Thus, trocarin
appears to play the role of a toxin in the venom of T
carinatus.
Trocarin induces clotting in snake plasma (Fig 2). Based on its
structural and functional similarities to coagulation factor Xa and the
existence of a similar extrinsic coagulation cascade in snake
blood,39 it is logical to propose that trocarin is similar,
if not identical, to snake blood coagulation factor Xa. However, blood
coagulation factor X will be in the zymogen form, unlike active
trocarin found in the venom. Furthermore, this protein would play a
critical role in hemostasis. Thus, two closely related, if not
identical, proteins play distinctly different physiological roles.
| |
ACKNOWLEDGMENT |
The authors thank Prof P. Gopalakrishnakone for providing P
reticulatus plasma and Chiew Fook Loong for writing the peptide analysis software.
| |
FOOTNOTES |
Submitted September 29, 1998; accepted March 17, 1999.
Supported by Academic Research Grants No. RP 960304 A and RP 950377 to
R.M.K.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to R. Manjunatha Kini, PhD, Bioscience Centre,
Faculty of Science, National University of Singapore, Singapore 117600;
e-mail: bsckinim{at}nus.edu.sg.
| |
REFERENCES |
1.
Suttie JW, Jackson CM:
Prothrombin structure, activation and biosynthesis.
Physiol Rev
57:1, 1977[Free Full Text]
2.
Rosing J, Tans G:
Inventory of exogenous prothrombin activators.
Thromb Haemost
65:627, 1991[Medline]
[Order article via Infotrieve]
3.
Markland FS Jr:
Snake venoms.
Drugs
54:1, 1997 (suppl 3)
4.
Hutton RA, Warrell DA:
Action of snake venom components on the haemostatic system.
Blood Rev
7:176, 1993[Medline]
[Order article via Infotrieve]
5.
Rosing J, Tans G:
Structural and functional properties of snake venom prothrombin activators.
Toxicon
30:1515, 1992[Medline]
[Order article via Infotrieve]
6.
Yamada D, Sekiya F, Morita T:
Isolation and characterization of carinactivase, a novel prothrombin activator in Echis carinatus venom with a unique catalytic mechanism.
J Biol Chem
271:5200, 1996[Abstract/Free Full Text]
7.
Morita T, Iwanaga S, Suzuki T:
The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venom and characterization of the new active intermediates.
J Biochem
79:1089, 1976[Abstract/Free Full Text]
8.
Yamada D, Morita T:
Purification and characterization of a Ca2+-dependent prothrombin activator, multactivase, from the venom of Echis multisquamatus.
J Biochem
122:991, 1997[Abstract/Free Full Text]
9.
Tans G, Govers-Riemslag JWP, van Rihn JLML, Rosing J:
Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus.
J Biol Chem
260:9366, 1985[Abstract/Free Full Text]
10.
Speijer H, Govers-Riemslag JWP, Zwaal RFA, Rosing J:
Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake).
J Biol Chem
261:13258, 1986[Abstract/Free Full Text]
11.
Masci PP, Whitaker AN, de Jersey J:
Purification and characterization of the prothrombin activator of the venom of Pseudonaja textilis, in
Gopalakrishnakone P,
Tan CK
(eds):
Progress in Venom and Toxin Research. Singapore, National University of Singapore, 1987, p 209.
12.
Marsh NA, Fyffe TL, Bennett EA:
Isolation and partial characterization of a prothrombin-activating enzyme from the venom of the Australian rough scaled snake (Tropidechis carinatus).
Toxicon
35:563, 1997[Medline]
[Order article via Infotrieve]
13.
de Kruiff B, Cullis PR, Radda GK:
Differential scanning calorimetry and 31P NMR studies on sonicated and unsonicated liposomes.
Biochim Biophys Acta
406:6, 1975[Medline]
[Order article via Infotrieve]
14.
Rosing J, Tans G, Govers-Riemslag JWP, Zwaal RFA, Hemker C:
The role of phospholipids and factor Va in the prothrombinase complex.
J Biol Chem
255:274, 1980[Abstract/Free Full Text]
15.
Laemmli UK:
Cleavage of structural proteins during the assembly of the head of the bacteriophage T4.
Nature
227:680, 1970[Medline]
[Order article via Infotrieve]
16.
Chow G, Subburaju S, Kini RM:
Purification, characterization, and amino acid sequence determination of acanthins, potent inhibitors of platelet aggregation from Acanthophis antarticus (common death adder) venom.
Arch Biochem Biophys
354:232, 1998[Medline]
[Order article via Infotrieve]
17.
Coligan JE:
Chemical analysis, in
Coligan JE,
Dunn BM,
Ploegh HL,
Speicher DW,
Wingfield PT,
Chanda VB
(eds):
Current Protocols in Protein Science, vol 1. New York, NY, Wiley, 1995, p 11.4.1.
18.
Poser JW, Price PA:
A method for decarboxylation of gamma-carboxyglutamic acid in proteins. Properties of decarboxylated gamma-carboxyglutamic acid protein from calf bone.
J Biol Chem
254:431, 1979[Free Full Text]
19.
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F:
Colorimetric method for determination of sugars and related substances.
Anal Chem
28:350, 1956
20.
Chester A, Crawford GPM:
In vitro coagulant properties of venoms from Australian snakes.
Toxicon
20:501, 1982[Medline]
[Order article via Infotrieve]
21.
Marshall LR, Herrmann RP:
Coagulant and anticoagulant actions of Australian snake venoms.
Thromb Haemost
50:707, 1983[Medline]
[Order article via Infotrieve]
22.
Morrison JJ, Masci PP, Bennett EA, Gauci M, Pearn J, Whitaker AN, Korschenko LP:
Studies of the venom and clinical features of the Australian rough-scaled snake (Tropidechis carinatus), in
Gopalakrishnakone P,
Tan CK
(eds):
Progress in Venom and Toxin Research. Singapore, National University of Singapore, 1987, p 220.
23.
Pirkle H, Marsh N:
Nomenclature of exogenous hemostatic factors.
Thromb Haemost
66:264, 1991[Medline]
[Order article via Infotrieve]
24.
Iwanaga S, Nishimura H, Kawabata S, Kisiel W, Hase S, Ikenaka T:
A new trisaccharide sugar chain linked to a serine residue in the first EGF-like domain of clotting factors VII and IX and protein Z.
Adv Exp Med Biol
281:121, 1990[Medline]
[Order article via Infotrieve]
25.
Hase S, Kawabata S, Nishimura H, Takeya H, Sueyoshi T, Miyata T, Iwanaga S, Takao T, Shimonishi Y, Ikenaka T:
A new trisaccharide sugar chain linked to a serine residue in bovine blood coagulation factors VII and IX.
J Biochem
104:867, 1988[Abstract/Free Full Text]
26.
McMullen BA, Fujikawa K, Kisiel W, Sasagawa T, Howald WN, Kwa EY, Weinstein B:
Complete amino acid sequence of the light chain of human blood coagulation factor X: Evidence for identification of residue 63 as -hydroxyaspartic acid.
Biochemistry
22:2875, 1983[Medline]
[Order article via Infotrieve]
27.
Fernlund P, Stenflo J:
-Hydroxyaspartic acid in vitamin K-dependent proteins.
J Biol Chem
258:12509, 1983[Abstract/Free Full Text]
28.
Stenflo J, Lundwall A, Dahlback B:
-Hydroxyasparagine in domains homologous to the epidermal growth factor precursor in vitamin K-dependent protein S.
Proc Natl Acad Sci USA
84:368, 1987[Abstract/Free Full Text]
29.
Haack JA, Rivier J, Parks TN, Mena EE, Cruz LJ, Olivera BM:
Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-d-aspartate antagonist activity.
J Biol Chem
265:6025, 1990[Abstract/Free Full Text]
30.
McIntosh JM, Olivera BM, Cruz LJ, Gray WR:
Gamma-carboxyglutamate in a neuroactive toxin.
J Biol Chem
259:14343, 1984[Abstract/Free Full Text]
31.
Rivier J, Galyean R, Simon L, Cruz LJ, Olivera BM, Gray WR:
Total synthesis and further characterization of the gamma-carboxyglutamate-containing "sleeper" peptide from Conus geographus venom.
Biochemistry
26:8508, 1987[Medline]
[Order article via Infotrieve]
32.
Prorok M, Warder SE, Blandl T, Castellino FJ:
Calcium binding properties of synthetic gamma-carboxyglutamic acid-containing marine cone snail "sleeper" peptides, conantokin-G and conantokin-T.
Biochemistry
35:16528, 1996[Medline]
[Order article via Infotrieve]
33.
Rigby AC, Baleja JD, Li L, Pedersen LG, Furie BC, Furie B:
Role of gamma-carboxyglutamic acid in the calcium-induced structural transition of conantokin G, a conotoxin from the marine snail Conus geographus.
Biochemistry
36:15677, 1997[Medline]
[Order article via Infotrieve]
34.
Lin CH, Chen CS, Hsu KS, King DS, Lyu PC:
Role of modified glutamic acid in the helical structure of conantokin-T.
FEBS Lett
407:243, 1997[Medline]
[Order article via Infotrieve]
35.
Hertzberg MS, Ben-Tal O, Furie B, Furie BC:
Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.
J Biol Chem
267:14759, 1992[Abstract/Free Full Text]
36.
Chattopadhyay A, James HL, Fair DS:
Molecular recognition sites on factor Xa which participate in the prothrombinase complex.
J Biol Chem
267:12323, 1992[Abstract/Free Full Text]
37.
Gotoh B, Ogasawara T, Toyoda T, Inocencio NM, Hamaguchi M, Nagai Y:
An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo.
EMBO J
9:4189, 1990[Medline]
[Order article via Infotrieve]
38.
Gotoh B, Yamauchi F, Ogasawara T, Nagai Y:
Isolation of factor Xa from chick embryo as the amniotic endoprotease responsible for paramyxovirus activation.
FEBS Lett
296:274, 1992[Medline]
[Order article via Infotrieve]
39.
Denson KWE:
The clotting of a snake (Crotalus viridis helleri) plasma and its interaction with various snake venoms.
Thromb Haemost
35:314, 1976[Medline]
[Order article via Infotrieve]
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ABSTRACT
INTRODUCTION