(Received for publication, October 14, 1994)
From the
The incorporation of factor Xa into the prothrombinase complex,
factor Xa-factor Va-phospholipid-Ca(II), results in an approximately
10-fold higher rate of substrate activation than that of
the enzyme alone. To examine the role of the prothrombin kringle
domains in the interaction with prothrombinase we have employed
site-directed mutagenesis to produce prothrombin species that lack
either the first kringle domain, PT/
K1, or the second kringle
domain, PT/
K2. Previously, we have shown that these proteins are
fully carboxylated and that they bind to phospholipid vesicles. In this
investigation we demonstrate that cleavage at
Arg
-Thr
and Arg
-Ile
peptide bonds occurs upon activation with prothrombinase to yield
normal thrombin from both PT/
K1 and PT/
K2. In the absence of
factor Va, the K
(app) for the activation
of PT/
K1, PT/
K2, or plasma-derived prothrombin by factor
Xa-phospholipid-Ca(II) are equivalent. The K
(app) for the activation of PT/
K2
by prothrombinase is approximately 4-5-fold higher than that
obtained for plasma-derived prothrombin or PT/
K1. These data
demonstrate that the prothrombin kringle domains do not contribute
significantly to the binding affinity of the substrate-enzyme
interaction. In the absence of factor Va, equivalent k
values were obtained for all of the prothrombin species when they
were activated by factor Xa-Ca(II)-phospholipid. In contrast, a 7-fold
lower k
value was obtained for the activation of
PT/
K2 by prothrombinase as compared with that obtained for plasma
prothrombin or PT/
K1. Collectively, these data suggest that
determinants within the second prothrombin kringle domain interact with
factor Va to elicit a significant acceleration in the catalytic rate of
substrate turnover. Indeed, in contrast to plasma-derived prothrombin,
no direct binding of PT/
K2 to factor Va to form the
PT/
K2factor Va complex could be demonstrated by 90° light
scattering.
The generation of thrombin concentrations suitable for blood
clot formation on a physiologically relevant time scale requires
prothrombin activation mediated by prothrombinase, a complex consisting
of the enzyme factor Xa and cofactors calcium, factor Va, and membrane
surfaces (for a review see Mann, 1987). Incorporation of factor Xa into
the prothrombinase complex confers a catalytic rate advantage resulting
in an approximately 2.8 10
-fold higher rate of
prothrombin activation than the rate of catalysis by the enzyme alone
in solution (Nesheim et al., 1979). A body of data suggests
that factor Xa associates with factor Va on the membrane surface with a
1:1 stoichiometry (Nesheim at al., 1979), independent of substrate
(Nesheim et al., 1981). Both factor Va and factor Xa bind to
phospholipid with high affinity. The binding of factor Va to
phospholipid independently increases the affinity of factor Xa for the
membrane (Krishnaswamy, 1990). The factor Va-factor Xa interaction
results from lateral diffusion of the two proteins on the membrane
surface (Krishnaswamy et al., 1988). The interaction of factor
Va and factor Xa is stabilized at the membrane surface relative to
their binding in solution (Krishnaswamy, 1990). The calcium-stabilized
conformer of prothrombin initially binds with phospholipid forming a
ternary complex, which then serves as the substrate for the
membrane-bound enzymatic complex (Nesheim et al., 1984). From
these data, the primary role of the phospholipid membrane appears to be
as a vehicle for the concentration of reactants, factor Xa, factor Va,
and prothrombin. Indeed, in the absence of factor Va, phospholipid
lowers the apparent K
of factor Xa
activation of prothrombin from 131 µM to 1 µM but causes little or no change in the V
(Rosing et al., 1980). In contrast, the cofactor appears
to function in the enhancement of the catalytic rate. In the absence of
phospholipid, factor Va caused little change in the apparent K
value for factor Xa activation of
prothrombin but promoted a 1,000-fold increase in the V
value (Rosing et al., 1980; van Rijn et al., 1984). It is currently unclear how factor Va effects
this dramatic increase in the catalytic rate.
We are interested in
understanding the nature of the interaction between prothrombin and the
prothrombinase complex. Various structural domains of prothrombin are
thought to play important functional roles in binding to the different
components of the prothrombinase complex, thereby regulating the
generation of thrombin activity. Our previous data have suggested that
membrane binding is mediated by the -carboxyglutamic acid-rich
domain and/or aromatic amino acid stack domain (Kotkow et al.,
1993). Indeed, we have recently demonstrated that the homologous Gla
domain and aromatic amino acid stack domain of Factor IX express
phospholipid binding properties (Jacobs et al., 1994). The
precise role of the kringle domains of prothrombin in the activation
process is unknown. However, these structural motifs are found in many
other proteins involved in hemostasis and fibrinolysis and have been
implicated in protein-protein interaction. For example, studies
examining the mechanism of plasmin generation by tissue type
plasminogen activator have determined that the fibrin specificity
displayed by tissue type plasminogen activator is caused by its ability
to assemble with plasminogen bound on a fibrin surface (Hoylaerts et al., 1982). Fibrin recognition and enhanced activity of
tissue type plasminogen activator appear to be localized to
determinants within the second kringle domain (van Zonneveld et
al., 1986; Larsen et al., 1988; Urano et al.,
1989). Previous studies examining prothrombin activation in solution by
factor Xa-factor Va-Ca(II) using fragments of prothrombin have
suggested that the second kringle domain interacts with factor Va
(Esmon and Jackson, 1974; Bajaj et al., 1975). We have
examined the interaction of recombinant prothrombins lacking the first
kringle (PT/
K1) or the second kringle (PT/
K2) as substrates
for the complete prothrombinase complex. We now suggest that
determinants within the second kringle domain of prothrombin are
important for the factor Va-dependent rate enhancement observed upon
activation of prothrombin by the prothrombinase complex. Also, kinetic
studies performed in the absence of factor Va indicate that the kringle
domains of prothrombin do not play any direct role in the interaction
of prothrombin with factor Xa.
Previously we have shown that recombinant prothrombins
lacking the first kringle (PT/K1) or the second kringle
(PT/
K2) have the mature amino-terminal sequence of prothrombin and
are fully
-carboxylated (Kotkow et al., 1993). These
proteins are able to assume both calcium-induced conformational
transitions and to bind to phospholipid vesicles. Despite these
properties, PT/
K2 has only 10% of the coagulant activity of
plasma-derived prothrombin, whereas PT/
K1 has 50% of the coagulant
activity of plasma-derived prothrombin (Kotkow et al., 1993).
Figure 1:
SDS-PAGE analysis of the activation of
plasma-derived prothrombin, PT/K1, and PT/
K2 by
prothrombinase. For each reaction 2 µM prothrombin
substrate was activated with 0.3 nM factor Xa in the presence
of 5 nM factor Va, 50 µM phospholipid, 5 mM CaCl
and 20 µM I-2581. A,
plasmaderived prothrombin; B, PT/
K1; C, PT/
K2. Pre 1, prethrombin 1; F1
2,
fragment 1
2; II
, thrombin; F1,
fragment 1.
Amino-terminal sequencing of the thrombins derived
from limited proteolysis of PT/K1 and PT/
K2 by the
prothrombinase complex showed that the only observable cleavages
occurred at Arg
-Thr
and
Arg
-Ile
, thereby producing
-thrombin.
These were also the cleavages observed upon activation of the
plasma-derived prothrombin sample under the same conditions. These data
demonstrate that the deletion of a kringle domain does not alter the
specificity of the factor Xa cleavage site in the substrate.
Figure 2:
Comparison of the rate of activation of
plasma-derived prothrombin, PT/K1, and PT/
K2 as a function of
factor Va concentration. Prothrombin substrates (10 nM) were
activated with 0.10 nM factor Xa, 35 µM phospholipid vesicles, 2.5 mM CaCl
, and
indicated concentrations of factor Va in TBS containing 0.1% BSA at 37
°C. Aliquots were removed at 2-min intervals.
,
plasma-derived prothrombin;
, PT/
K1;
,
PT/
K2.
Figure 3:
Activation of plasma-derived prothrombin,
PT/K1, and PT/
K2 by prothrombinase. Prothrombin substrates
(0.03-3.12 µM) were activated with 0.01 nM factor Xa, 10.0 nM factor Va, 35 µM phospholipid vesicles, and 2.5 mM CaCl
in TBS
containing 0.1% BSA at 37 °C. Aliquots were removed at 2-min
intervals and assayed for thrombin activity with the chromogenic
substrate CBS.34.47 (375 µM). A, Plasma-derived
prothrombin (
); B, PT/
K1 (
); C,
PT/
K2 (
).
Figure 4:
Activation of plasma-derived prothrombin,
PT/K1, and PT/
K2 by factor Xa, Ca(II), and phospholipid.
Prothrombin substrates (0.63-4.0 µM) were activated
with 5 nM factor Xa, 35 µM phospholipid vesicles,
and 2.5 mM CaCl
in TBS containing 0.1% BSA at 37
°C. Aliquots were removed at 0, 15, 25, and 40 min after initiation
of the reaction and assayed for thrombin activity with the chromogenic
substrate S-2238 (375 µM). A, plasma-derived
prothrombin (
); B, PT/
K1 (
); C,
PT/
K2 (
).
Figure 5:
Binding of plasma-derived prothrombin and
PT/K2 to factor Va. The interaction of the prothrombin species
with factor Va was monitored by 90° light scattering.
Plasma-derived prothrombin (52.8 µM) or PT/
K2 (32.8
µM) were added to a cuvette containing Factor Va (2.7
µM, 100 µl) to achieve the concentrations shown. The
change in relative molecular weight is expressed as M2/M1(molecular weight of the protein complex/molecular weight
of factor Va).
, plasma-derived prothrombin;
,
PT/
K2.
Our approach toward understanding the functional role of the
prothrombin kringle domains in the interaction with the prothrombinase
complex was to study mutant prothrombin proteins that lack either the
first kringle (PT/K1) or the second kringle domain (PT/
K2).
Previously, we have provided evidence that posttranslational
-carboxylation of these mutants is complete and that the
-carboxyglutamic acid-rich domains of our purified PT/
K1 and
PT/
K2 proteins are properly folded (Kotkow et al., 1993).
In this study we have shown that activation of PT/
K1 by
prothrombinase results in the generation of prethrombin 1 and fragment
1, whereas these fragments are not detected upon activation of
PT/
K2. This pattern of activation fragments is consistent with
those predicted from the primary sequence of the mutated proteins.
Cleavage of both mutant zymogens by factor Xa occurred at the expected
sites, resulting in the production of
-thrombin. Equivalent
apparent K
and k
values
were obtained when the mutant and plasma-derived substrates were
activated by factor Xa, Ca(II), and phospholipid. These data
demonstrate that in the absence of factor Va the mutant substrates are
recognized and processed as efficiently as the plasma-derived
substrate, making it unlikely that the observed differences in thrombin
generation were actually caused by differences in the specific activity
of thrombin species derived from these mutants. These observations
indicate that functional differences between the mutant prothrombin
species and plasma-derived prothrombin are caused by the absence of
amino acid residues on the surface of the deleted kringle domain rather
than incomplete processing events or global distortion of the structure
of the recombinant proteins.
The first kringle domain of prothrombin
does not appear to interact with phospholipid, factor Va, or factor Xa.
Kinetic analyses of PT/K1 activation by prothrombinase or factor
Xa-Ca(II)-phospholipid yielded equivalent apparent K
and k
values as plasma-derived
prothrombin. Despite these results, the coagulant activity of this
prothrombin species was 50% when compared with that of plasma-derived
prothrombin. This inconsistency may reflect inherent differences
between the two assays. The coagulant activity assays, which are
performed under conditions that may not follow Michaelis-Menten
kinetics, may enhance slight differences between the prothrombin
species.
Data from these kinetic studies have established that the
kringle domains of prothrombin do not contribute significantly to the
overall binding affinity of the substrate-enzyme interaction.
Equivalent apparent K values were obtained when
PT/
K1, PT/
K2, or plasma-derived prothrombin was activated by
factor Xa-Ca(II)-phospholipid or when PT/
K2 was activated by
prothrombinase (Table 1). These apparent K
values were approximately 4-5-fold higher than those
obtained for the activation of PT/
K1 or plasma-derived prothrombin
by prothrombinase (Table 1). These results indicate that the
binding affinity of the PT/
K2 substrate for prothrombinase is
weaker than that of plasma-derived prothrombin and that this result is
caused solely by the loss of binding affinity resulting from the
interaction of the second kringle domain with factor Va. Indeed, we
were unable to demonstrate direct binding between PT/
K2 and factor
Va, in contrast to the binding of prothrombin to factor Va. These
results are consistent with previous studies, which have shown that the
phospholipid component, not the protein cofactor, contributes
significantly to the overall binding affinity of the substrate-enzyme
complex (Rosing et al., 1980).
This investigation provides
new insight into the mechanism by which factor Va effects an
enhancement on the catalytic rate of substrate turnover when it is
assembled in the prothrombinase complex (Rosing et al. 1980,
Nesheim et al., 1979). Previous studies have suggested that
this rate enhancement could be attributed to a direct influence on the
conformation of the enzyme active site by the cofactor, factor Va. A
perturbation of the active site of factor Xa has been observed upon
incorporation of the enzyme into the prothrombinase complex by
perturbation of the active site bound fluorophore dansyl-Glu-Gly-Arg,
suggesting that factor Va induces an allosteric conformational
transition in the active site of factor Xa (Krishnaswamy et
al., 1988; Husten et al., 1987) Recent studies have shown
that this conformational transition results in subtle changes in the
accessibility of the factor Xa active site rather than in a general
increase in the reactivity of the active site histidine residue (Walker
and Krishnaswamy, 1992). In addition to influencing the conformation of
the enzyme, other observations have suggested that the interaction of
factor Va with the substrate may be critical for the enhancement of the
catalytic rate. Nesheim et al.(1981) have observed that, in
contrast to activation of prothrombin, the V for
activation of small peptide substrates by factor Xa is unaffected by
the presence of factor Va. We have observed that the k
value determined for PT/
K2 is approximately 7-fold lower
than that determined for plasma-derived prothrombin, thus implying that
one aspect of the factor Va-dependent catalytic rate enhancement is
dependent upon the association of the cofactor and the substrate.
Previous studies examining the ability of various prothrombin activation fragments to serve as substrates for the prothrombinase complex for the solution phase factor Xa-Ca(II)-factor Va complex have suggested that prothrombin fragment 2, comprising the second kringle domain, contains a site for factor Va interaction, which affects the factor Va-dependent catalytic rate acceleration (Esmon and Jackson, 1974; Bajaj et al., 1975). Our results confirm these data. However, using prothrombin species that bind phospholipid membranes the concentrations of reactants and the time required to monitor thrombin formation approach physiologically relevant conditions. By including the contributions of the phospholipid component to assembly of the prothrombin-prothrombinase complex, we may have also ensured that the conclusions reached as a result of these studies reflect the interactions that occur in the physiological complex.
The second
kringle domain of prothrombin is not the only domain that is necessary
for factor Va interaction, since we observed that the factor
Va-dependent rate enhancement, approximately 200-fold for
plasma-derived prothrombin, was not completely ablated in PT/K2.
Indeed, our data indicate that PT/
K2 displays an approximately
35-fold higher k
value in the presence of
saturating concentrations of factor Va than that determined for
activation of the mutant by factor Xa-Ca(II)-phospholipid (Table 1). This may be because the first kringle domain is able
to functionally substitute for the second kringle domain, although it
is more likely that an additional site for factor Va interaction is
present. This additional site may be localized to some portion of the
serine protease domain. Support for these conclusions are derived from
previous studies of Esmon and Jackson(1974), which have shown no effect
of fragment 1 on the factor Va-dependent rate enhancement, thus
indicating that the first kringle domain probably can not substitute
for the second kringle domain. Also, Esmon and Jackson(1974) have shown
that the rate of prethrombin 2 activation by factor Xa and Ca(II) is
stimulated 5-fold by the addition of factor Va, suggesting that
portions of the serine protease domain are involved in the factor
Va-dependent rate enhancement. In addition, recent studies employing a
chimeric factor Xa have determined that the binding of factor Va in the
prothrombinase complex is mediated by the second EGF-like domain and
the serine protease domain of factor Xa (Hertzberg et al.,
1992) Thus, it seems likely that optimal alignment between the active
site of factor Xa and the activation sites of the prothrombin serine
protease domain is also mediated by factor Va.
In summary, we have determined that the first kringle domain of prothrombin is not involved in phospholipid, factor Xa, or factor Va binding. The second kringle domain of prothrombin appears to contribute only modestly to the overall binding affinity of the substrate-enzyme complex. However, our data suggest that this domain interacts directly with factor Va to effect an acceleration in the catalytic rate of thrombin formation.