(Received for publication, October 20, 1995)
From the
The venom of the viper Echis carinatus contains a
metalloprotease, ecarin, that is a potent prothrombin activator. We
here show that the venom is also rich in another prothrombin activator,
which does not belong to any known category of prothrombin activators.
The novel enzyme, designated carinactivase-1 (CA-1), consists of two
subunits held together non-covalently but very tightly. One subunit is
a 62-kDa polypeptide that has metalloprotease activity and is
homologous to the single-chain enzyme ecarin; the other subunit of 25
kDa consists of two disulfide-linked polypeptides of 17 and 14 kDa, and
this subunit resembles the anticoagulant in the habu snake venom,
IX/X-bp, that specifically binds the Gla domains of coagulation factors
IX and X in a Ca-dependent fashion. The activation of
prothrombin by CA-1 requires Ca
ions at millimolar
concentrations and in the absence of Ca
ions this
enzyme is virtually inactive. By contrast, activation by ecarin is
completely independent of Ca
ions. CA-1, unlike
ecarin, does not activate prothrombin derivatives, in which binding of
Ca
ions has been perturbed, namely prethrombin-1 and
acarboxyprothrombin. Furthermore, the isolated catalytic subunit,
although its activity is greatly reduced as compared to that of the
holoenzyme, no longer requires Ca
ions for the
activation of prothrombin. Reconstitution with the non-catalytic 25-kDa
subunit restores high level activity and the dependence on
Ca
ions. Finally, prothrombin activation by CA-1 is
inhibited by prothrombin fragment 1, and the isolated non-catalytic
subunit is capable of binding fragment 1 in the presence of
Ca
ions. From these observations, we postulate the
following unique mechanism for the activation of prothrombin by CA-1.
The enzyme primarily recognizes the Ca
-bound
conformation of the Gla domain in prothrombin via the 25-kDa regulatory
subunit, and the subsequent conversion of prothrombin to active
thrombin is catalyzed by the 62-kDa catalytic subunit.
Compounds that affect the mammalian blood coagulation system, in
particular those that cause acute thrombosis, are often the major
active principals of the lethal toxins in viper
venoms(1, 2) . Thrombogenic components in these venoms
exhibit considerable heterogeneity in terms of function as well as of
structure. Many types of protease that convert quiescent clotting
proenzymes to their active forms (or inactive procofactors to active
cofactors) are known, and various prothrombin activators have been
reported(2) . To date, three types of prothrombin activator
have been identified in venoms(3) : group 1 enzymes, which are
metalloproteases whose actions on prothrombin are independent of any
plasma or exogenous cofactors; group 2 enzymes, which are
Gla-containing, factor Xa-like serine proteases that require factor Va,
anionic phospholipids and Ca ions, resembling in this
respect the physiological activator factor Xa; and group 3 enzymes,
which are hybrid proteins that consist of factor Xa-like catalytic
subunits and factor Va-like regulatory subunits and require
phospholipids and Ca
ions for their action. The group
1 enzymes are widely distributed in venoms of many kinds of viper, e.g. genera Echis and Bothrops, and they are
presumably the most toxic since they are resistant to the natural
coagulation inhibitors (serpins) present in mammalian plasma, such as
antithrombin-III. Another difference between metalloprotease-type
prothrombin activators and the physiological activator factor Xa or the
venom serine proteases involves the cleavage sites in the prothrombin
molecule. The metallo-enzymes cleave only the bond between the A chain
and the B chain (Arg
-Ile
in human
prothrombin; Arg
-Ile
in the bovine protein)
with the resultant production of meizothrombin, which is ultimately
converted to
-thrombin by autolysis(4) . The serine-type
enzymes cleave one additional site (the junction between fragment 2 and
the A chain; Arg
-Thr
in the human protein)
to produce
-thrombin directly(5) .
The venom of Echis carinatus contains a high level of a metallo-type prothrombin activator and has been widely used in laboratory studies as a convenient tool for the production of thrombin from prothrombin. The enzyme in E. carinatus venom, named ecarin, is a single-chain protein of 55 kDa that exhibits very strict substrate specificity. Prothrombin is the only protein that is cleaved by ecarin in plasma, and other structurally related coagulation factors, e.g. factors IX and X, are scarcely affected(6) . The primary structure of ecarin was recently determined by molecular cloning(7) . The mature protein consists of three independent motifs. From N to C terminus, there is a metalloprotease catalytic domain of approximately 200 amino acid residues, a disintegrin-like domain of approximately 90 residues, and a Cys-rich domain of approximately 120 residues. At present, however, the roles of non-catalytic regions remain unclear as to the factors that determine the strict specificity of this enzyme, and further information, in particular those about the three-dimensional structure, is necessary to clarify these issues. A significant number of proteins with the same domain organization and with unique respective functions has recently been identified in mammalian tissues (see (8) , and references therein). Thus, this venom protein should serve as a good model in efforts aimed at an understanding of the biochemistry of these mammalian proteins as well as of details of the evolution of these proteins.
During the purification of ecarin, we found a novel
prothrombin activator in the same venom preparation, which could not be
assigned to any of the above mentioned categories. This enzyme,
designated carinactivase-1 (CA-1),()(
)is
strongly dependent on Ca
ions for the activation of
prothrombin, in sharp contrast to ecarin, whose action is unaffected by
exogenous Ca
ions. In the present report, we describe
the purification of CA-1 and discuss the relationship between its
structure and function and the unique mechanism by which it activates
prothrombin.
The effects of various
protease inhibitors were determined directly by this method. Each
inhibitor to be tested (e.g. EDTA, Mn,
Co
, or other heavy metal ions at 10 mM) was
incubated with the enzyme for 10 min at the ambient temperature and
then assayed as described above.
The venom of E. carinatus was first fractionated by
gel filtration and assayed for activation of prothrombin (Fig. 1A). Ecarin activity, which could be detected in
the absence of Ca ions, was found in the first
protein peak. When the assay was conducted in the presence of a
millimolar concentration of Ca
ions, the extent of
activation of prothrombin was considerably enhanced. Since the activity
of purified ecarin did not show any Ca
dependence
(see below), it was clear that this fraction also contained another,
hitherto unidentified prothrombin activator(s) whose activity was
dependent on Ca
ions. We isolated this activity, as
described below.
Figure 1:
Isolation
of CA-1. A (step 1), gel filtration. The venom of E.
carinatus (100 mg) was subjected to a column of Superdex 200pg.
The pooled fractions are indicated by a bar. B (step 2), Blue
Sepharose. Active fractions from A were subjected to a column
of Blue Sepharose CL-6B (1.0 20 cm) and eluted with a linear
gradient of NaCl (dotted line). Two-ml fractions were
collected. The fractions indicated by bars were pooled as
CA-1, CA-2, and ecarin, respectively. C (step 3),
anion-exchange chromatography. The CA-1 fraction from B was
subjected to a column of Q-Sepharose high performance (1.6
20
cm) and eluted with a linear gradient of NaCl (dotted line).
Two-ml fractions were collected. The pooled fraction (bar)
contained apparently homogeneous CA-1. Fractions at each purification
step were assayed for activation of prothrombin as described under
``Experimental Procedures,'' either in the presence (closed circles) or absence (open circles) of 3
mM Ca
ions.
This fraction was applied to a column of Blue
Sepharose. As is shown in Fig. 1B, two peaks of the
activity of a Ca-dependent prothrombin activator were
identified, and we designated these activities CA-1 and CA-2,
respectively. Ecarin was eluted at higher concentrations of NaCl and
was clearly separated from CA-1 and CA-2 at this step. Subsequent
purification and characterization revealed that CA-2 was almost
identical to CA-1 in terms of the molecular structure and enzymological
features, and the main focus of this report is on CA-1. Isolation of
CA-1 was accomplished by a third chromatography on Q-Sepharose (Fig. 1C). In a typical purification, we obtained 2 mg
of CA-1 and 0.1 mg of ecarin from 100 mg of crude venom. The activity
of CA-1 was irreversibly abolished by the incubation with EDTA or with
heavy metals such as Co
and Mn
, but
it was resistant to inhibitors of serine, thiol, or carboxyl proteases.
Thus, CA-1 appeared to be a metalloenzyme, as is ecarin.
SDS-PAGE of CA-1 is shown in Fig. 2A (lane 1). Two bands (60/62-kDa doublet plus 25 kDa) were obtained under non-reducing conditions, and three bands (62/64, 17, and 14 kDa) were obtained under reducing conditions. The doublet appearance of the larger polypeptide was probably due to microheterogeneity, as discussed below. We were unable to separate these polypeptides by any subsequent chromatographic procedures under nondenaturing conditions. The polypeptides also comigrated as a single band in native-PAGE. Dissociation of the polypeptides required rather extreme conditions. For example, guanidine hydrochloride (>4 M) or SDS (>0.1%) was effective, but urea was ineffective up to 8 M. Fig. 2B depicts the chromatograms after gel filtration in either the absence or the presence of 4 M guanidine hydrochloride. Without the denaturant, a single, symmetrical peak that contained all three polypeptides was obtained. By contrast, two peaks were obtained in the presence of the denaturant; the first peak contained the 60/62-kDa polypeptide, and the second peak contained the 25-kDa component (see Fig. 2A). Thus, it appeared that CA-1 was a protein that consisted of two heterogeneous subunits held together non-covalently but very tightly, and that the 25-kDa subunit consisted of two different disulfide-linked polypeptides. The stoichiometry of the two subunits was 1 to 1. When the holoenzyme was subjected directly to protein sequence analysis, almost equimolar amounts (after correction for recovery of each amino acid) of three amino acids, corresponding to residues in the sequences of each of the three polypeptides (see below), were found after each sequencing cycle.
Figure 2: Subunit composition of CA-1. A, SDS-PAGE of CA-1 before and after separation of the subunits. The fractions indicated in B were subjected to electrophoresis under non-reducing (NR) and reducing (R) conditions. Positions of molecular mass markers are shown on the left, and the subunits (polypeptide chains) consisting of CA-1 are identified on the right. B, gel filtration chromatograms of purified CA-1 in the absence (upper panel) and presence (lower panel) of 4 M guanidine HCl. Note that the second peak in the lower panel has a shoulder, but the peptides in this peak were homogeneous at least as far as could be judged by SDS-PAGE.
The N-terminal amino acid sequence of each polypeptide in CA-1 was
analyzed (Fig. 3A). The 60/62-kDa chain had a sequence
highly homologous to that of ecarin(7) , and this subunit did,
indeed, have metalloprotease activity, as described below. The 60-kDa
polypeptide had an identical sequence to that of the 62-kDa polypeptide
but lacked two N-terminal residues (Ser-Arg), suggesting the existence
of a microheterogeneity (hereafter, we refer the 60/62-kDa polypeptide
simply as the 62-kDa polypeptide). The two chains of the 25-kDa subunit
resembled one another; furthermore, these sequences were rather similar
to those of the snake venom protein IX/X-bp(20) . IX/X-bp is a
protein isolated in this laboratory from the venom of the habu snake Trimeresurus flavoviridis. It has strong anticoagulant
activity and acts by neutralizing factors IX/IXa and X/Xa through
binding to the Gla domains of these factors in a
Ca-dependent
fashion(21, 22, 23) . As is shown in Fig. 3B, the apparent molecular masses of the
respective polypeptide chains of CA-1 also resemble those of ecarin and
IX/X-bp. The structural similarity of CA-1 to these venom proteins was
confirmed by an immunochemical method. In immunoblotting analysis, the
62-kDa polypeptide of CA-1 cross-reacted with antiserum raised against
ecarin and the 25-kDa subunit cross-reacted with antiserum against
IX/X-bp (data not shown).
Figure 3: Structure of CA-1. A, N-terminal amino acid sequence of CA-1. The determined sequence of each polypeptide in CA-1 is aligned with those of ecarin (7) and IX/X-bp (20) . Identical residues are shaded. Two N-terminal residues of the 62-kDa polypeptide, indicated by dots, are absent from the 60-kDa polypeptide. B, schematic representation of the structures of CA-1, ecarin, and IX/X-bp. The apparent molecular mass of each polypeptide is shown.
We examined the enzymological features of CA-1. First, we evaluated the specificity of CA-1. Although it very efficiently activated prothrombin of bovine or human origin, it had no effect on any other vitamin K-dependent coagulation proteins. We incubated factors VII, IX, and X and protein C (all of bovine origin) with CA-1 for a long period and then analyzed by SDS-PAGE. Little if any cleavage was detected (data not shown). Thus, the specificity of CA-1 appears to be very strict, as is that of ecarin. Furthermore, the activation of prothrombin (0.1 µM) was unaffected by high concentrations of factor IX or factor X (up to 10 µM).
The most prominent enzymological difference between CA-1 and ecarin
was the former enzyme's requirement for Ca ions
for the activation of prothrombin. As shown in Fig. 4A,
Ca
ions at millimolar concentrations were necessary
for CA-1 (half-maximal and maximal activation occurred at 0.2 and 2
mM Ca
, respectively), while purified ecarin
had no such requirement. In the absence of Ca
ions,
the generation of active thrombin by CA-1 was extremely slow (less than
1/100 of that in the presence). The Ca
-dependent
thrombin generation was confirmed by SDS-PAGE; when the incubation
mixture of bovine prothrombin with CA-1 was subjected to SDS-PAGE under
reducing condition, the band corresponding to the B chain of thrombin
appeared only when Ca
ions had been present (Fig. 4B). In a non-reducing gel, the band
corresponding to meizothrombin but not
-thrombin was observed
(data not shown). It appears that CA-1 cleaves only the bond between
the A chain and the B chain, as does ecarin. By contrast to the
reaction with the natural substrate, hydrolysis by CA-1 (and also by
ecarin) of a peptidyl fluorogenic substrate, which was synthesized to
resemble the scissile site in prothrombin, was unaffected by exogenous
Ca
ions (Table 1). Thus, it seemed likely that
Ca
ions were required for the recognition of
prothrombin by CA-1; prothrombin is capable of binding Ca
ions via the N-terminal Gla domain and undergoes a dramatic
change in conformation at Ca
-concentrations around 1
mM. It appeared possible that CA-1 might recognize the Gla
domain of prothrombin with bound Ca
ions, primarily
via the IX/X-bp-like 25-kDa subunit, which is not present in ecarin.
Figure 4:
Requirement for Ca ions
of the activation of prothrombin by CA-1. A, Ca
titration curve. Bovine prothrombin (1 µM) was
incubated with 4 pM CA-1 (closed circles) or 25
pM ecarin (closed triangles) for 10 min at 37 °C
in the presence of Ca
ions at the indicated
concentrations. The thrombin generated was then quantified as described
under ``Experimental Procedures.'' B, analysis of
incubation products of prothrombin with CA-1 or ecarin. Prothrombin (5
µM) was incubated with 20 nM CA-1 or 20 nM ecarin for 20 min at 37 °C in the presence and in the absence
of 1 mM Ca
ions, and then subjected to
SDS-PAGE under reducing condition. Benzamidine HCl (10 mM) and p-amidinophenylmethanesulfonyl fluoride (1 mM) were
included in the incubation mixture to prevent the secondary cleavage of
prothrombin by the thrombin generated and thus to isolate the direct
actions of venom activators on prothrombin. Positions of molecular mass
markers are shown on the left, and derivatives of prothrombin
are identified on the right: PT, prothrombin; B, the B chain of thrombin; F1
2A, the residual
fragment consisting of fragment 1, fragment 2, and the A chain of
thrombin.
To examine this possibility, we investigated the activation of
prothrombin in detail using prothrombin derivatives and isolated CA-1
subunits. First, we tested prethrombin-1, which lacks prothrombin
fragment 1, the N-terminal portion of prothrombin that includes the Gla
domain. As is shown in Table 1, removal of the
Ca-binding site from the prothrombin molecule had a
striking negative effect on the action of CA-1. Moreover, the
activation of this derivative no longer required Ca
ions, even though the rate of activation was greatly diminished,
and this rate was close to that for intact prothrombin without
Ca
ions. The steady-state kinetic parameters for the
activation of prothrombin were obtained. The apparent K
and V
values of CA-1 for the activation of
bovine prothrombin in the presence of 5 mM Ca
ions were 1.1 µM and 33 mol of prothrombin/min/mol
of CA-1, respectively, while those of ecarin were 0.44 µM and 17 mol/min/mol. In the absence of Ca
ions,
the K
of CA-1 was greatly increased (to >100
µM) but that of ecarin was essentially unchanged. Human
prothrombin was also a good substrate, and results similar to those
with bovine protein were obtained. Apparently, the action of CA-1 was
strongly dependent on both of Ca
ions and the Gla
domain of prothrombin, while that of ecarin was influenced by neither
of these factors.
Next, we evaluated the role of the subunits of
CA-1 (Table 2). When we examined the amidolysis of the
fluorogenic substrate, we found that the isolated 62-kDa subunit had
enzymatic activity almost equivalent to that of the intact CA-1, but
the rate of activation of prothrombin, the natural substrate, was much
reduced. The 25-kDa subunit had no enzymatic activity (data not shown).
Removal of the 25-kDa subunit also led to the loss of the dependence on
Ca ions. It appeared, therefore, that the 62-kDa
component is the metalloprotease catalytic subunit, while the 25-kDa
non-catalytic component is an accessory, regulatory subunit.
Reconstitution with the 25-kDa subunit restored both the high potency
and the dependence on Ca
ions. By contrast, the rate
of activation of prethrombin-1 was unaffected by reconstitution, and
the rate of the reaction was very close to that for prothrombin
activation in the absence of the 25-kDa subunit and/or Ca
ions. These results strongly suggested that CA-1 recognized the
conformation of the Gla domain of prothrombin with bound Ca
ions via its 25-kDa subunit.
In order to obtain further evidence, we conducted three additional experiments. In the first one, we examined the effect of prothrombin fragment 1 on the activation of prothrombin. As is shown in Fig. 5, the activation by CA-1, but not by ecarin, was effectively inhibited by fragment 1. This result indicated that the low reactivity of prethrombin-1 with CA-1 was due to the absence of fragment 1 and not due to a secondary change in conformation of the protein upon liberation of the N-terminal portion.
Figure 5:
Inhibition of the CA-1-induced activation
of prothrombin by fragment 1. Prothrombin (50 nM) was
incubated with 10 pM CA-1 (closed circles) or 50
pM ecarin (closed triangles) in the presence of 10
mM Ca ions, and the effect of fragment 1 was
examined.
In the second experiment, we used plasma from individuals who had
taken a vitamin K antagonist, in which abnormal prothrombin with
incompletely carboxylated Gla residues was present concomitant with the
decreased level of normal prothrombin. A batch of plasma with a
clotting activity of 20% of that of normal controls, which had been
determined by a standard assay of prothrombin time, was utilized. The
plasma was mixed with prothrombin-deficient plasma, the activator, and
Ca ions, and the time required for clot formation was
measured. The prothrombin content of the tested plasma was estimated
with serially diluted normal control plasma as the reference. When CA-1
was used as the activator, the results were similar to those obtained
with the physiological activator factor Xa (Table 3). By
contrast, with ecarin, higher values were obtained, probably
representing the sum of normal and abnormal prothrombins since it is
known that ecarin can activate abnormal prothrombin as well as normal
prothrombin(24) . It was apparent that CA-1 selectively
recognized normal prothrombin with all the Gla residues intact, even in
the presence of excess acarboxyprothrombin. Ecarin did not recognize
such differences, and it seemed to recognize only the scissile site.
In the third experiment, we investigated the association between
fragment 1 and the isolated 25-kDa subunit directly. We employed a
cross-linking technique. The I-labeled 25-kDa subunit was
incubated with fragment 1 in the presence of Ca
ions,
and the complex formed was stapled by the bifunctional reagent
BS
. The resultant stable complex was subjected to SDS-PAGE
followed by analysis with a radioimaging analyzer (Fig. 6). A
small amount of the 25-kDa subunit was self-associated, and a
radioactive band corresponding to the apparent molecular mass of 43 kDa
(presumably a dimer) was found even in the absence of the cross-linker (lane 1). This species was presumably generated during the
radiolabeling procedure, which included an oxidizing reaction. In the
presence of Ca
ions, fragment 1 per se associated with one another as reported previously(25) ,
and a ladder-like pattern was visible after Coomassie Blue staining.
The radiolabeled 25-kDa subunit was indeed incorporated into these
fragment 1 polymers in the presence of Ca
ions, but
in the absence of Ca
ions no incorporation occurred (lanes 3 and 4). The association with fragment 1 was
effectively blocked by the addition of an excess of the cold 25-kDa
subunit (lane 5), indicating that this interaction was
specific.
Figure 6:
The 25-kDa subunit of CA-1 recognizes
fragment 1 in a Ca-dependent fashion. The
I-labeled 25-kDa subunit was incubated with fragment 1 (F1; 2 µM) in the presence and absence of 10
mM Ca
ions at 37 °C for 60 min, and then
it was cross-linked with BS
. The resultant SDS-stable
complex was subjected to SDS-PAGE followed by the analysis with a
BAS-2000 Bioimaging Analyzer. In the lane 5, an excess of the
cold 25-kDa subunit (6 µM) was included to confirm the
specificity of the interaction. Positions of molecular mass markers are
shown on the left. On the right, the arrowhead indicates the 25-kDa subunit, the double arrowhead shows
the subunit dimer, and arrows show complexes of fragment 1
(and fragment 1 polymers) and the subunit (and/or the subunit dimer).
Detailed methods are given under ``Experimental
Procedures.''
These results together demonstrate that the unique
structure of CA-1 explains the unique mechanism of its activation of
prothrombin; the 25-kDa subunit first recognizes the N-terminal Gla
domain of prothrombin in a Ca-dependent fashion, and
then the 62-kDa subunit cleaves the distal scissile site, the bond
between the A chain and the B chain (Fig. 7).
Figure 7:
The proposed mechanism for the recognition
and activation of prothrombin by CA-1. The 25-kDa regulatory subunit
first recognizes the Ca-bound conformation of the Gla
domain of prothrombin, and then the 62-kDa catalytic subunit cleaves
the bond between the A and B chains, generating meizothrombin.
Participation of Ca
ions in the exposure of the Gla
domain recognition site on the regulatory subunit has not been proven
and is hypothetical at present. For further details, see under
``Discussion.''
The results described herein clearly show that the venom of E. carinatus contains a hitherto novel type of prothrombin
activator. This finding necessitates reconsideration of the
classification of exogenous prothrombin activators. We propose that the
previously defined group 1 enzymes (3) be divided into two
subgroups, i.e. the ecarin-like
(Ca-independent) metalloproteases (perhaps termed
group 1A) and the carinactivase-like (Ca
-dependent)
enzymes (group 1B).
We screened the venoms of various Viperidae
snakes for carinactivase-like activity (detailed screening data will be
published elsewhere). ()All the venom preparations from Echis snakes contained both ecarin-like and carinactivase-like
activators, although the total activities as well as the relative
abundance of two enzymes varied depending upon the source. However, we
failed to detect carinactivase-like activity in venoms of Viperidae
snakes in genera other than Echis, although ecarin-like
activities were found in some of them.
CA-1 appears to be a hybrid
protein that is well adapted for the efficient and selective activation
of prothrombin in the plasma of target animals after envenomation. The
25-kDa subunit of CA-1 exhibits striking structural similarity to the
anticoagulant IX/X-bp in the T. flavoviridis venom. IX/X-bp is
a heterodimeric protein that consists of two homologous polypeptide
chains(20) , and it recognizes Ca-bound
conformations of the Gla domains in factors IX and
X(22, 23) . The function of the 25-kDa subunit is also
similar to that of IX/X-bp, i.e. the
Ca
-dependent recognition of the Gla domain of
prothrombin. Thus, the non-catalytic component is the regulatory
subunit (Fig. 7). The specificity of IX/X-bp is very strict; it
never binds other vitamin K-dependent coagulation factors such as
prothrombin(21) . This point is of great interest, since the
Gla domains in these proteins are very similar to one another in terms
of primary structure and, in view of their common function
(Ca
-dependent binding to anionic phospholipids),
their tertiary structures should be also similar. It appears that
IX/X-bp can discriminate slight difference in the tertiary structures
of Gla domains. A similar statement can be made about the binding
specificity of the regulatory subunit of CA-1. Strong substrate
specificity and the absence of inhibitory effect of factors IX and X on
the activation of prothrombin indicate that the subunit selectively
recognizes the Gla domain of prothrombin. In the presence of
Ca
ions, prothrombin should interact with the
regulatory subunit of CA-1 via its Gla domain. Then its scissile site
is presented in the proper orientation to the active center of the
62-kDa metalloprotease catalytic subunit (Fig. 7). The
regulatory subunit acts as an effective condenser of the substrate and
greatly reduces the apparent K
. This function is
analogous to that of phospholipids in the physiological prothrombin
activator, the prothrombinase complex (factor Xa plus factor Va,
anionic phospholipids, and Ca
ions). Therefore,
studies with CA-1 should provide insight into the structure and
function of the prothrombinase complex at a molecular level from a
novel perspective.
We showed previously that IX/X-bp is also capable
of binding Ca ions (2 ions/molecule) and that
occupation of the Ca
-binding sites is a prerequisite
for subsequent binding to coagulation factors(23) . It is
possible that the regulatory subunit of CA-1 might also bind
Ca
ions and, upon binding of Ca
ions, the recognition site for the Gla domain of prothrombin
would be exposed (Fig. 7).
The results indicate that IX/X-bp
and the regulatory subunit of CA-1 are highly analogous not only in
terms of structure but also in terms of function, even though they have
opposite toxicological effects. IX/X-bp is an anticoagulant, and it
should support the action of hemorrhagic factors that are present in
the same venom(26) , while CA-1 is an enzyme that causes
thrombosis via the generation of thrombin. In addition to IX/X-bp from
the habu snake venom, proteins structurally related to the regulatory
subunit of CA-1 are widely distributed in viper venoms. We have
identified homologues of IX/X-bp in venoms of Bothrops jararaca(27) and Deinagkistrodon acutus(28) .
Moreover, numerous proteins with structures very similar to IX/X-bp but
with totally different pharmacological actions have been found in
venoms of various Viperidae snakes, and appear to constitute a unique
protein family. Ligands for these proteins are very heterogeneous. For
example, botrocetin from B. jararaca binds von Willebrand
factor(29) , bothrojaracin from the same venom binds the
anion-binding exosite in -thrombin(30) , and alboaggregin
from T. albolabris binds platelet glycoprotein
Ib(31) . Each of these venom proteins has two homologous
polypeptide chains, and each chain constitutes the domain structure,
known as CRD. The name CRD originally referred to the
``carbohydrate-recognition domain'' because this structure
was first identified as the minimum functional motif of
Ca
-dependent animal lectins such as
asialoglycoprotein receptor. It is now known that the CRD is widely
distributed in the animal kingdom, from invertebrates such as sea
urchins to mammals, and it seems to be a fundamental motif that acts as
an important domain in the construction of
proteins(32, 33) .
Another component in CA-1, the catalytic subunit, also has numerous relatives in the venoms of Viperidae. These relatives include ecarin in the same venom and hemorrhagic factors in T. flavoviridis and Crotalus atrox venoms(26, 34) . In addition, many proteins that resemble to these venom metalloproteases have recently been identified in mammalian tissue, in particular in reproductive organs, e.g. the sperm protein fertilin (PH-30)(35) . Together, all these proteins constitute a superfamily for which the name ADAM has been proposed(8) .
It is noteworthy that the factor X
activator RVV-X in Vipera russelli venom has a structure very
similar to that of CA-1. This protein also has three polypeptide
chains, i.e. 57.6-, 19.4-, and 16.4-kDa chains, with a
stoichiometry of 1:1:1, and these chains are held together by disulfide
bonds, as recently proven unequivocally by Gowda et
al.(36) . In an earlier report, Takeya et al.(37) determined the complete amino acid sequences of the
57.6- and 16.4-kDa chains, and showed that the 57.6-kDa chain has a
structure very similar to that of ecarin while the 16.4-kDa chain has a
sequence homologous to that of IX/X-bp. Furthermore, the 19.4-kDa chain
also has an N-terminal sequence homologous to IX/X-bp. ()The
action of RVV-X is also dependent on both Ca
ions and
the Gla domain in factor X(38, 39) . Therefore, it
seems likely that the catalytic mechanism of RVV-X might be similar to
that of CA-1. However, isolation of the intact catalytic chain of RVV-X
appears to be impossible and thus unequivocal biochemical evidence
cannot be obtained, because the chain is covalently linked to
IX/X-bp-like chains. Both CA-1 and RVV-X are composed of two different
components, which have a totally different genetic origin, and both
proteins are likely to have originated from a single ancestral hybrid
protein. It is still unclear how they are synthesized and correctly
folded. The topology of the polypeptides in these enzymes is also
unknown. These issues require further investigations.
In conclusion, we have shown that a newly isolated novel prothrombin activator, CA-1, has a unique machinery for the recognition and subsequent processing of its substrate. CA-1 should be useful as a convenient probe for biochemical studies in vitro of prothrombin and as a good diagnostic reagent for monitoring normal prothrombin levels in plasma (cf.Table 3). Furthermore, this enzyme should be a good model for attempts to elucidate details of the evolution and the biosynthesis of multi-subunit proteins.