(Received for publication, April 11, 1995; and in revised form, June 1, 1995)
From the
The enzymatic and cofactor subunits of human prothrombinase,
factor Xa (FXa) and factor Va (FVa), respectively, were evaluated as
modulators of Glu- and Lys-plasminogen (Pg) activation by tissue
plasminogen activator (tPA). The data revealed that both FXa and FVa
could accelerate tPA activity by as much as 60-fold for Lys-Pg and
>150-fold for Glu-Pg. This function of FVa depended on pretreatment
with plasmin (Pn), whereas the FXa fibrinolytic cofactor activity was
endogenous. In the native state, FVa was observed to inhibit the
acceleration of Pn generation by FXa. These effects were dependent on
Ca Solubilization and clearance of fibrin clot requires the
generation of plasmin (Pn)( Several proteins have been identified as
Pg receptors, including: annexin II (Cesarman etal.,
1994; Hajjar etal., 1994), a novel 45-kDa
endothelial protein (Dudani etal., 1993, 1994),
In the current study, the human blood coagulation factor Xa
(FXa), which is known from its cDNA sequence to possess the necessary
lysyl moiety (Fung etal., 1985; Messier etal., 1991), was evaluated as a Pg-binding protein and
accelerator of Pn generation by tPA. The established function of the
serine protease FXa in hemostasis is to produce the final enzyme in the
coagulation pathway, thrombin (Krishnaswamy etal.,
1994; Mann etal., 1990). To do so, FXa must
associate in the presence of Ca
Figure 1:
Demonstration of Pg binding to FXa/FVa.
96-well microtiter plates were coated with PCPS, PC, or no phospholipid
(0.3 µg) and then blocked with BSA. PanelA,
coated microtiter wells were preincubated with FXa and/or FVa (0.05
µM) in HBS/BSA for 30 min, washed four times with HBS/BSA,
then incubated for 30 min with
Figure 2:
Effect of FXa/FVa on Lys-Pg activation by
tPA. Lys-Pg (0.5 µM) was activated by tPA (10 nM)
in HBS/BSA containing PCPS (50 µM) (
When only FVa
was included in the Lys-Pg activation mixture, the amount of Pn
generated was the same as tPA alone over the first 12-17 min.
Following this period, the reaction rate was appreciably accelerated by
FVa. Since the proteolysis of FVa by Pn has been reported (Lee and
Mann, 1989; Omar and Mann, 1987), we speculated that the FVa may be
acquiring fibrinolytic cofactor activity during the experiment by a
Pn-dependent feedback mechanism. To test this hypothesis, the FXa
and/or FVa were preincubated with Pn. The effect of this treatment (Fig. 3) was to abolish the lag phase observed in the previous
experiment when FVa alone was included in the reaction mixture. The FVa
fibrinolytic activity remained the same as the system without Pn
pretreatment if aprotonin was added before the Pn (data not shown).
Figure 3:
Effect of Pn pretreatment of FXa/FVa on
Lys-Pg activation by tPA. Experimental conditions were identical to
those for Fig. 2except that before adding the Pg and tPA, the
various reaction mixtures were incubated for 25 min at 22 °C in the
presence of Pn (1.0 nanokatal/ml) and then inhibited with aprotonin
(2.5 KIU/ml). No FXa or FVa (
Pretreatment with Pn was also observed to modulate the effect of FXa
on tPA (Fig. 3). This was not as dramatic as observed for FVa
and resulted in an approximately 50% decrease of the untreated FXa
activity. To our knowledge, evidence for a Pn-sensitive cleavage site
on FXa has not been described but would account for our observed loss
in activity. We are currently investigating this possibility. As a
control, addition of aprotonin before the Pn pretreatment prevented the
loss in FXa fibrinolytic activity (data not shown). The combined
acceleratory effect of Pn-pretreated FXa and FVa on tPA activity was
approximately the sum of the individual effects. The estimated initial
velocity for Pn production was identical to that of untreated FXa,
which may indicate that the tPA is saturated under these conditions and
the kinetics are approaching V
Figure 4:
Confirmation of the effect of FXa/FVa on
Lys-Pg activation by tPA. Experimental conditions were identical to
those for Fig. 2(panelA) and Fig. 3(panelB), except that the extent of Pn
production at various times was followed by electrophoresis. No FXa or
FVa (
Figure 5:
Effect of FXa/FVa on Glu-Pg activation by
tPA. Experimental conditions were identical to those for Fig. 2(panelA) and Fig. 3(panelB) except that Glu-Pg was used as a substrate rather than
Lys-Pg. No FXa or FVa (
Figure 6:
Effect of FVa on the tPA cofactor activity
of FXa. Identical experimental conditions as Fig. 2except that
the FVa concentration was increased with constant FXa (0.1
µM). No FXa or FVa (
Figure 7:
Effect of the FXa active site,
Ca
To determine the role of
Ca
Figure 8:
Quantification of Pg binding to FXa/FVa.
Experimental conditions were identical to those for Fig. 1(panelB). Microtiter wells were coated
with 0.3 µg of PCPS or as a control for nonspecific binding with
0.3 µg of PC. Panel A, reactions were not Pn-pretreated
(as in Fig. 2); panelB, reactions were
Pn-pretreated (as in Fig. 3), except that 150 KIU/ml aprotonin
was used to inhibit the Pn.
Coagulation and fibrinolysis are opposing hemostatic
processes. To effectively coordinate the activation of procoagulant and
subsequently of profibrinolytic enzymes, a high degree of molecular
cross-talk between these pathways must exist. As an important example,
the end product of coagulation, fibrin, communicates to the
fibrinolytic pathway by functioning as an accelerator of tPA-dependent
Pn generation (Lijnen and Collen, 1994). Consequently, tPA activity is
restricted until clot has formed. In the current study, we provide
evidence that the protein components of prothrombinase, FXa and FVa,
are also capable of signaling fibrinolysis by acceleration of tPA. A
model that summarizes the molecular links between fibrinolysis and
prothrombinase is presented in Fig. 9. In previous work, Pn was
demonstrated to irreversibly block the FXa cofactor role of FVa in
thrombin production (Lee and Mann, 1989; Omar and Mann, 1987). We now
report that Pn-modulated FVa acquires tPA cofactor activity, either in
the presence or absence of FXa. Treatment of FVa with Pn also
correlates with the expression of a cryptic Pg binding site. The
apparent K
Figure 9:
Molecular links between prothrombinase and
Pg activation. The data presented here are consistent with both
prothrombinase components, FXa and FVa, being able to accelerate
tPA-dependent Pg activation after feedback modulation of FVa by Pn. See
text for details. Hatchedlines represent inhibitory
pathways.
The FXa
moiety of prothrombinase was also observed to accelerate Pn generation
by tPA. Unlike FVa, however, the tPA cofactor activity of FXa was
endogenous. Evidence that FVa directly regulates the FXa fibrinolytic
activity was obtained from experiments showing that addition of native
FVa induced a dose-dependent lag before the acceleration of Pn
generation was achieved. Following Pn pretreatment, the FVa was found
to lose this antagonistic effect. A plausible explanation is that
Pn-mediated proteolysis may break the interaction between FXa and FVa
in a manner similar to the inhibition of FVa by activated protein C
(Guinto and Esmon, 1984; Pryzdial and Mann, 1991). The general
conclusion from these data is that Pn-mediated feedback controls the
intrinsic fibrinolytic capability of FXa through an indirect route
involving FVa. Since we found that FVa did not influence the binding
affinity of Pg to FXa, this regulatory mechanism is suggested to
include an effect on tPA. The association of Pg with FXa was found to
be approximately 10-fold stronger (apparent K In this report, we have not attempted to define the kinetics of tPA
acceleration by FXa or FVa. However, to compare the efficacy of the
prothrombinase components to known tPA cofactors, we have estimated the
extent of acceleration relative to tPA alone at a single concentration
of reactants. In the presence of FXa or Pn-pretreated FVa, we observed
approximately 60-fold enhancement for the initial rate of Lys-Pg
activation. This is within the same range as the known tPA
accelerators, annexin II (Cesarman etal., 1994) and
various fibrin derivatives (Lijnen and Collen, 1994). When Glu-Pg was
used as a substrate, the generation of Pn in the presence of tPA alone
did not exceed base-line levels over the course of the experiment,
which complicates the determination of fold enhancement. Nevertheless,
we estimate the increase in observed rate due to FXa to be >150-fold
and somewhat less for Pn-pretreated FVa. These values are considerably
greater than those reported for other Pg receptors (Kelm etal., 1994; Reinartz etal., 1995) and
fibrin derivatives (Lijnen and Collen, 1994). A comprehensive
evaluation of the kinetics must be conducted to verify these
comparisons. The role of procoagulant phospholipid as an essential
prothrombinase cofactor and as a means to localize thrombin production
has been well documented (Bevers etal., 1982, 1983;
Mann etal., 1987b; Packham and Mustard, 1984).
Calcium is also required for prothrombinase assembly and functions by
facilitating the interaction between FVa subunits (Esmon, 1979;
Krishnaswamy etal., 1989), the binding of FXa to
PCPS (Nelsestuen, 1976; Nesheim etal., 1981) and the
binding of FXa to FVa (Pryzdial and Mann, 1991). In the current work,
the acceleration of tPA by FXa and FVa was also found to be dependent
on PCPS and Ca Annexin II is the only other known cellular
receptor for Pg that may also be localized to sites where procoagulant
phospholipid is expressed. This is through the preferential interaction
of annexin family proteins with procoagulant phospholipid (Andree etal., 1990; Meers etal., 1991;
Tait etal., 1989). However, unlike FXa and FVa, the
fibrinolytic cofactor activity of annexin II does not depend on the
association with procoagulant phospholipid (Cesarman etal., 1994) and consequently may not be as strictly
localized as FXa and FVa. FXa and FVa can associate with a variety
of stimulated cells and may function to generate Pn at these sites
either independently or with other Pg receptors. Activated platelets
are considered to provide the most abundant physiological source of
procoagulant phospholipid (Dahlbach and Stenflo, 1978; Miletich etal., 1978; Nesheim etal., 1981; Tracy et al., 1981) and are a significant constituent of clots. As a
result, prothrombinase is concentrated within a clot and may locally
participate in concert with fibrin to accelerate fibrinolysis. At this
time, the concentration of FXa and FVa at these sites is unknown. Since
the concentration of fibrin is high, it is probable that the largest
individual effect of prothrombinase constituents on fibrinolysis will
be on susceptible cells that are not trapped by fibrin. While other
Pg receptors may exist on platelets, annexin II is not found in
association with these cells (Eldering etal., 1993).
The fibrinolytic activity of annexin II has been identified on the
endothelial surface (Hajjar etal., 1994), which is
also an important site for prothrombinase assembly (Rodgers and Shuman,
1985). Therefore annexin II, FXa, and FVa may act together to stimulate
fibrinolysis on the endothelium. A functional difference is suggested
from the possibility that annexin II may exist on the surface of
resting cells (Hajjar etal., 1994), whereas
recruitment of FXa and FVa requires cell stimulation. At least one
other endothelial cell receptor for Pg has been identified (Dudani etal., 1993), but whether the expression of this
45-kDa protein is modulated following cell stimulation is unknown. The formation of prothrombinase has also been described in
association with monocytes (Mcgee and Li, 1991; Tracy etal., 1985) and carcinoma cells (Sakai etal., 1990). In previous studies, Pg binding to these
cells and generation of Pn on the surface of monocytes was observed to
be enhanced by Pn pretreatment (Gonzalez-Gronow etal., 1991). This is similar to our current observation
with FVa. Certain carcinoma cells are known to secrete FV (Sakai etal., 1990), but whether these reports are due to FV/FVa
Pn-mediated modulation or other Pg receptor(s) is unknown. In addition
to a fibrinolytic function, the possibility that FVa and FXa may
accelerate Pn generation at the surface of monocytes or carcinoma cells
suggests a role in tissue remodeling (Kwaan etal.,
1991). The interaction of Pg with fibrin (Lijnen and Collen, 1994),
various cells (Dudani etal., 1993; Hajjar etal., 1994; Holaerts etal., 1982; Miles etal., 1991), or collagen matrices (Kelm etal., 1994) is known to depend on the accessibility of
specific Lys residues in the receptor protein. Like many Pg receptors,
the FXa-mediated acceleration of Pg activation by tPA was observed by
us to involve a COOH-terminal Lys in FXa. This suggests that in
addition to the control of FXa fibrinolytic function by FVa, a recently
identified thrombin-activated plasma protein having carboxypeptidase B
activity (TAFI) (Bajzr et al., 1995)
may be involved. Whether the acquisition of FVa fibrinolytic activity
following Pn treatment is the result of exposing a new COOH-terminal
Lys is under investigation.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and procoagulant phospholipid. Interactions
between plasminogen and prothrombinase components were quantified. The
apparent K
for binding to FXa was 35
nM. Strikingly, the affinity between FVa and Pg was increased
by approximately 2 orders of magnitude when the FVa was Pn-pretreated (K
= 0.1 µM). These
data cumulatively suggest a mechanism by which Pn production is
coordinated with coagulation and localized to sites where procoagulant
phospholipid is exposed on a cell surface.
)
from its inactive
precursor, plasminogen (Pg) (Lijnen and Collen, 1994; Robbins, 1982).
One of the proteases that mediates Pg activation is tissue plasminogen
activator (tPA), which has relatively poor intrinsic reactivity toward
Pg. Thus, a fundamental regulatory step in fibrinolysis is the
acceleration of tPA through the assembly of a ternary complex between
tPA, Pg, and a protein cofactor such as fibrin or a cell surface
receptor. The colocalization of Pg and tPA functions to increase the
rate of catalysis by 1-2 logs through a K
effect and to restrict tPA activity to sites of fibrin
deposition. Additionally, the association of Pg with endothelial cell
receptors is known to accelerate the conversion of the native
NH
-terminal Glu-Pg to the truncated Lys-Pg species (Hajjar
and Nachman, 1988). The outcome is that Lys-Pg is activated by tPA
approximately 10 times more efficiently than Glu-Pg (Holaerts etal., 1982).
-enolase (Miles etal., 1991), osteonectin (Kelm etal., 1994), and complement component C7 (Reinartz etal., 1995). Through the use of lysine analogues
and carboxypeptidase B, these proteins have been shown to have in
common a COOH-terminal lysine that may be the only necessary structural
feature for binding to Pg (Miles etal., 1991). Most
of the Pg receptors have been shown to accelerate tPA-mediated
activation of Pg (Cesarman etal., 1994; Kelm etal., 1994; Reinartz etal., 1995) or to
bind to tPA and Pg, which is presumed to form the necessary ternary
complex for rapid Pn generation (Dudani etal., 1993,
1994).
with the cofactor Va
(FVa) and procoagulant phospholipid (e.g. phosphatidylserine),
which synergistically accelerate FXa activity by 5 orders of magnitude
(Nesheim etal., 1979). Procoagulant phospholipid is
accessible only on the surface of activated cells and therefore
restricts prothrombinase assembly to sites of vascular damage (Tracy et al., 1992; Zwaal et al., 1993). Since FVa
stabilizes the interaction of FXa with biological membranes, a role for
FVa in fibrinolysis was also evaluated. We now report that both FXa and
FVa can participate as cofactors for tPA-mediated Pg activation.
Chemicals and Reagents
HEPES, EDTA (Sigma),
Glu-Gly-Arg chloromethyl ketone (EGRck),
2-guanidinoethylmercaptosuccinic acid (GEMSA), aprotonin, porcine
carboxypeptidase B (CPB, Calbiochem),
benzoyl-Ile-Glu-(piperidyl)-Gly-Arg-p-nitroanilide (S-2337),
Glu-Pro-Arg-p-nitroanilide (S-2366, Helena Laboratories) were
obtained commercially.Proteins
Human coagulation factor X was purified
from fresh frozen plasma (Bajaj etal., 1981) or from
prothrombin complex concentrate diluted to 1 unit/ml factor X clotting
activity (obtained as generous gifts from the Canadian Red Cross
Society, Ottawa Collection Centre or from Miles Therapeutics, Inc.,
respectively). Factor Xa (approximately 1000 units/mg) was generated
from factor X (Jesty and Nemerson, 1976; Krishnaswamy etal., 1987) by treatment with the purified activator from
Russell's viper venom (Haematologic Technologies) (Jesty and
Nemerson, 1976), followed by affinity chromatography using
benzamidine-Sepharose (Pharmacia Biotech Inc.) to remove the factor X
activation peptide, Russell's viper venom, and residual
inactivated factor X (Krishnaswamy etal., 1987). The
irreversible inhibition of factor Xa was accomplished by treatment with
a 4-fold molar excess of EGRck (to produce EGR-factor Xa) (Pryzdial and
Mann, 1991). Complete inhibition of factor Xa (1.5 µM) was
confirmed by the lack of detectable conversion of the chromogenic
substrate S-2337 (200 µM) over a 15-min period using a
kinetic microplate reader (Vmax, Molecular Devices) with full scale set
to 0.03 OD. Excess EGRck was removed by Sephadex G-25 gel filtration
chromatography (Pharmacia). Human Glu-plasminogen and Lys-plasminogen
were purified as described (Nesheim etal., 1990) and
passed over benzamidine-Sepharose (Pierce) to remove any trace of
serine protease activity. Plasminogen was radioiodinated using Iodogen
(Pierce) and chromatographically desalted (Excellulose 5, Pierce) to
remove unincorporated I. Human factor Va was commercially
prepared (Haematological Technologies) according to established
protocols (Fair etal., 1975; Katzmann etal., 1981). The homogeneity of proteins was assessed by
sodium dodecyl sulfate polyacrylamide electrophoresis (Laemmli, 1970)
and where applicable by autoradiography using X-Omat AR film (Kodak)
and Quanta III intensifying screens (DuPont). Purified human plasmin
(Diagnostica Stago), single-chain tPA (American Diagnostica), and
CNBr-treated human fibrinogen (tPA activator, Kabi Vitrum) were
purchased.
Pg Binding
To follow the interaction between
prothrombinase proteins and I-Pg, an adaptation of a
method employed by Kalafatis et al.(1994) was used. In these
experiments, microtiter wells (Immulon 3) were coated with 0.3 µg
of a mixture of phosphatidylserine (PS) and phosphatidylcholine (PC)
(25%:75%), pure PC or no phospholipid in 100 µl of methanol/well by
evaporation in a vacuum desiccator. On the same day, coated wells (and
uncoated wells to control for a possible methanol effect) were blocked
for 2 h with 200 µl of 5 mg/ml bovine serum albumin (BSA) at room
temperature and rinsed once with 20 mM HEPES, 150 mM NaCl, 2 mM CaCl
, pH 7.2 (HBS) with 1 mg/ml
BSA (HBS/BSA). In 100 µl/well of HBS/BSA, FXa, FVa, or a mixture of
the two proteins was equilibrated with
I-Pg each at 50
nM for 1 h at 22 °C. In some experiments, the
prothrombinase proteins were preincubated for 1 h and then washed three
times with HBS/BSA before addition of
I-Pg. Aprotonin
(150 KIU/ml) and benzamidine (2 mM) were kept throughout to
inhibit any potential Pn activity and proteolysis by FXa, respectively.
After incubation, the wells were washed four times with HBS/BSA and the
I-Pg remaining bound was quantified. To determine the
effect of Pn on the binding of FXa or FVa to
I-Pg, the
FXa and FVa were pretreated for 30 min with Pn (1.0 nanokatal/ml) and
then inhibited by addition of a large excess of aprotonin (150 KIU/ml)
before introducing the
I-Pg into the mixture.
Chromogenic Assay for Pg Activation
The tPA (10
nM)-mediated activation of Pg (0.5 µM) was
followed at 22 °C in HBS/BSA using a chromogenic assay. At various
times, 5 µl of the reaction mixture was combined with 150 µl of
chromogenic substrate (S2366, 0.2 µM) and the rate of
color development was quantified using a kinetic multiwell plate reader
(Vmax, Molecular Devices). tPA was the final component added to
initiate the process. The effect of FXa and FVa (in most cases 0.1
µM) was evaluated by including these proteins prior to the
Pg. In some experiments the FXa and FVa (0.125 µM) were
pretreated with Pn (1.0 nanokatal/ml) for 25 min at 22 °C. The Pn
was then inhibited with a small excess of aprotonin (2.5 KIU/ml). To be
consistent, the experiments not pretreated with Pn were incubated
identically. All reactions and pretreatments were done in the presence
of synthetic unilamellar phospholipid vesicles composed of 75%
phosphatidylcholine and 25% phosphatidylserine (PCPS, 50
µM) (Krishnaswamy etal., 1994) due to
the known acceleratory effect on Pn cleavage of FVa (Omar and Mann,
1987) and requirement for PCPS in prothrombinase assembly (Lim etal., 1977; Mann etal., 1987a; Nesheim etal., 1992).Electrophoretic Assay for Pg Activation
To confirm
the observations made with the chromogenic assay, under identical
conditions I-Pg (60,000 cpm/µg) activation was
followed by electrophoresis. Samples were reduced using dithiothreitol
(20 mM) to resolve the Pg from Pn, then run on 10% acrylamide
gels (Laemmli, 1970). After drying, the gels were subjected to
autoradiography using X-Omat AR film (Kodak) and Quanta III
intensifying screens (DuPont). The Pn bands were excised from the gel
to quantify using a
-counter (Beckman). To correct for background,
a slice from each lane that did not correspond to a band on the
autoradiograph was quantified and subtracted from those of Pn.
Initial Observation of a FXa-Pg Interaction
Our
first attempt to identify a possible involvement of FXa in
fibrinolysis, was to follow the specific interaction of I-Lys-Pg to prothrombinase components that were bound to
PCPS-coated microtiter wells (Fig. 1). Selective binding of
I-Lys-Pg was observed. However, the preincubation
experiment (panelA) was dependent on the presence of
both FXa and FVa, whereas in the coincubation system FXa alone or the
FXa/FVa mixture facilitated
I-Lys-Pg binding. Since FVa
is known to stabilize the interaction of FXa with PCPS, this
discrepancy is likely the result of the additional washing steps in the
former. An additional difference between the two incubation conditions
is that considerably less
I-Lys-Pg remained bound to the
microtiter wells in panelB. This may be the result
of solution-phase inhibition by FXa. Both experiments demonstrated that
I-Lys-Pg binding is dependent on PS and
Ca
. The association of
I-Lys-Pg was
inhibitable by addition of a 100-fold molar excess of unlabeled Lys-Pg
to the level observed with BSA alone (data not shown). Cumulatively,
these observations are consistent with FXa functioning as a bridge
between Pg and PCPS that is stabilized by FVa.
I-Lys-Pg (0.05
µM). The counts/min remaining bound after another cycle of
washing are presented. PanelB, as in panelA except that the FXa/FVa was coincubated with the
I-Lys-Pg. The requirement for Ca
was
evaluated by adding 10 mM EDTA. The average of triplicates
with standard deviation is shown.
Chromogenic Measurement of FXa/FVa as tPA
Cofactors
Having demonstrated that FXa is indeed capable of
functioning as a receptor for Pg, we next determined whether FXa and/or
FVa can accelerate the activation of Lys-Pg by tPA. Although the
chromogenic substrate used in these experiments (S2366) is not as
specific for Pn as the conventional substrate (S2251), it was selected
to increase the assay sensitivity by 3-4-fold. FXa and FVa were
used at 0.1 µM, which approximates the K for Pg binding to other receptors (Ganz etal., 1991; Hajjar etal., 1994).
As shown in Fig. 2, FXa greatly enhanced the rate of Lys-Pg
activation. An initial velocity was estimated from the slope to the
first time point (2 min) and compared to the first observation of Pn
activity in the absence of FXa (8 min), which suggested a 60-fold
increase in the rate of tPA catalysis by FXa. Addition of an equimolar
amount of FVa to the FXa did not dramatically alter the kinetics
profile, although a modest lag was consistently observed. As a
comparison to a well documented fibrinolytic cofactor, the effect of
CNBr-cleaved fibrinogen at a weight equal to that of the FVa (
0.02
mg/ml) was observed to have approximately 50% of the effect of FXa. The
dependence of the reaction on tPA and Pg was confirmed by the lack of
chromogenic substrate cleavage after 40 min if either were omitted in
the presence or absence of FXa/FVa (data not shown).
). The extent of
activation was determined at various times using a chromogenic
substrate. FXa (0.1 µM, ▾), FVa (0.1
µM,
), FXa + FVa (both at 0.1 µM,
), or CNBr-cleaved fibrinogen (20 µg/ml,
) were added
before the Pg or tPA. All reactions were incubated at 22 °C for 25
min before adding the Pg or tPA. The average of triplicates with
standard deviation is shown.
), FXa (0.1 µM,
▾), FVa (0.1 µM,
), FXa + FVa (both at
0.1 µM,
), or CNBr-cleaved fibrinogen (20
µg/ml,
).
. Pn pretreatment
of the CNBr-cleaved fibrinogen appeared to reduce its tPA cofactor
activity. Since this was opposite to the effect of Pn on the FXa/Va
mixture or FVa alone, it can be inferred that contaminating fibrinogen
is not the basis for our observations.
Electrophoretic Measurement of FXa/Va as tPA
Cofactors
To confirm our chromogenic assays, we followed
conversion of I-Lys-Pg to
I-Pn by
electrophoresis. These data (Fig. 4) are consistent with the
conclusions made from Fig. 2and Fig. 3. 1) FXa and a
mixture of FXa/FVa greatly accelerate tPA activity to approximately the
same extent (panelA); 2) in the presence of FVa
alone, Pn generation undergoes a 12-min lag before the rate is
accelerated (panelA); 3) Pn pretreatment of FVa
eliminates the lag (panelB); 4) the FXa acceleratory
effect is decreased by Pn pretreatment (panelB); and
5) the mixture of Pn-pretreated FXa/FVa has the same initial rate of
Lys-Pg conversion as untreated FXa (panelB).
), FXa (0.1 µM, ▾), FVa (0.1
µM,
), FXa + FVa (both at 0.1 µM,
).
Effect of FXa/FVa on tPA-mediated Activation of Glu-Pg
Fig. 5shows that FXa and FVa also enhance the rate of
Glu-Pg activation by tPA. 1) FXa has endogenous tPA cofactor activity (panelA); 2) FVa induced a lag in the endogenous FXa
effect (panelA), although this was much more
pronounced compared to Lys-Pg (Fig. 2); 3) Pn pretreatment of
the FVa converted it into a tPA accelerator; 4) Pn pretreatment
decreased the FXa cofactor activity; and 5) Pn-pretreated FXa and FVa
combined to increase the rate of Pn generation. As reported in the
literature, Lys-Pg is converted more rapidly to Pn than Glu-Pg by tPA.
We observed this characteristic in the absence of either prothrombinase
constituent where over 30 min of Glu-Pg activation was negligible. As a
result, the feedback modulation of FVa by Pn was not observed using
Glu-Pg (panelA).
), FXa (0.1 µM, ▾), FVa
(0.1 µM,
), FXa + FVa (both at 0.1
µM,
).
Regulation of FXa Fibrinolytic Activity by
FVa
Since a lag was observed in the rate that FXa-enhanced Pn
production when equimolar FVa was present (Fig. 2), we explored
the possibility that before cleavage by Pn the FVa was a regulator.
Shown in Fig. 6is the effect of increasing the FVa
concentration on Pn generation from Lys-Pg at a constant level of FXa.
The data show that the extent of the lag phase is dependent on the
amount of FVa added and is lengthened to 22 min at the highest
concentration of FVa. Following the lag, a rapid rate of Lys-Pg
activation is resumed, which is likely contributed to by both FXa and
Pn-cleaved FVa fibrinolytic activity. This experiment suggests that
within the prothrombinase complex, the participation of FXa as a
fibrinolytic cofactor is inhibited until the FVa is feedback-modulated
by Pn.
); FXa with no FVa (▾); 1:1
FVa:FXa (
) (data from figure 2); 2:1 (
); 5:1 (
);
8:1 (
); 11:1 (
). Averaged duplicates with standard error
is shown.
Effect of Other Prothrombinase Components on tPA
Acceleration
Having established that FXa and Pn-treated FVa can
participate in the acceleration of Pn production by tPA, the
contributions of the FXa serine protease active site,
Ca, and procoagulant phospholipid were evaluated. Fig. 7(panel A and B) shows the effect of
blocking the active site of FXa with a tripeptidylchloromethyl ketone
(EGRck). In contrast to native FXa, EGR-FXa did not demonstrate
significant tPA cofactor activity alone (panelsA and B). Since Pg is believed to interact exclusively with the COOH
terminus of known receptors (Cesarman etal., 1994;
Dudani etal., 1993; Kelm etal.,
1994; Miles etal., 1991), the effect of blocking the
active site of FXa may be on tPA. Without Pn pretreatment (panelA), active site-independent synergy between EGR-FXa and
FVa was observed. This effect was not apparent after Pn pretreatment (panelB), which suggests that FXa may function to
accelerate the cleavage of FVa by Pn.
and PCPS on Pg activation by tPA in the presence
of FXa/FVa. Upperpanels (A, C, and E), reactions were not Pn-pretreated (as in Fig. 2). Lowerpanels (B, D, and F), reactions were Pn-pretreated (as in Fig. 3). PanelsA and B, EGRck-inactivated factor Xa
was used instead of active FXa; panelsC and D, following the 25-min incubation in the presence of
Ca
, 10 mM EDTA was added; panelsE and F, PCPS was omitted from the various
reaction mixtures. No FXa or FVa (
), FXa (0.1 µM,
▾), FVa (0.1 µM,
), or FXa + FVa (both at
0.1 µM,
).
in the tPA cofactor function of FXa and FVa,
excess EDTA was added to the various reaction mixtures. The data show
that without Pn treatment (panelC), the cofactor
effects of FXa and FVa were abolished in the absence of
Ca
. The data also suggest that the
Ca
-dependent association of FXa with PCPS is
necessary for tPA cofactor activity. Interestingly, when the FXa and
FVa were pretreated with Pn in the presence of Ca
(panelD), the subsequent addition of EDTA
caused a complete loss of the FXa-dependent tPA activity, but
approximately 50% of the initial FVa acceleratory effect on tPA
remained. When the entire experiment was conducted in the presence of
Ca
, but PCPS was completely omitted (panelsE and F), neither FXa nor FVa had an influence
on the rate of Lys-Pg activation regardless of whether a Pn
pretreatment step was performed.
Binding of
The data presented above establish that Pn is
capable of altering the participation of FVa and to a lesser extent of
FXa in Pg activation by tPA. To determine whether this may be an effect
on the ability of FVa and FXa to interact with Pg, the various
equilibria were quantified. The binding isotherms shown in Fig. 8, were obtained by coincubating the I-Lys-Pg to
FXa/FVa
I-Lys-Pg
and prothrombinase constituents. Specific binding was calculated by
subtracting the amount of
I-Lys-Pg bound to PC-coated
microtiter wells from the amount bound to PCPS-coated wells. The
apparent dissociation constants (K
) and
number of moles bound at saturation (B
) were
derived by fitting the data to a simple rectangular hyperbola, which
implicitly assumed a single class of sites (Table 1). Perhaps the
most striking conclusion from these data is that Pn treatment of FVa
enhances the affinity for Lys-Pg by approximately 2 orders of
magnitude. This may account for the acquisition of tPA cofactor
activity by FVa when treated with Pn. Similarly the decrease in the tPA
cofactor activity of FXa after Pn treatment may be explained by the
10-fold decrease observed in affinity for Lys-Pg. An inconsistency with
the kinetic experiments is that prior to Pn treatment, FVa did not
influence the binding of Lys-Pg to FXa. Furthermore, after Pn
treatment, the FXa and FVa were shown to have a combined effect on Pg
activation by tPA (Fig. 3), but FVa had no effect on Lys-Pg
binding to FXa. These discrepancies may be reconciled by direct effects
on tPA that have yet to be identified.
I-Pg was titrated in the
presence of the following prothrombinase components. FXa (0.05
µM,
), FVa (0.05 µM, ▾), or FXa
+ FVa (both at 0.05 µM,
). Averaged triplicates
with standard error is shown.
Involvement of FXa COOH-terminal Lysine on tPA
Acceleration
A possible function for the FXa COOH-terminal Lys
was investigated by pretreating the FXa with CPB (Table 2). The
data clearly revealed that the tPA cofactor function of FXa is
sensitive to CPB, which supports the involvement of a COOH-terminal Lys
in FXa. To control for the possibility that the CPB inhibitor GEMSA or
residual CPB activity had a direct effect on Pn generation by tPA, the
FXa was simultaneously incubated with CPB and GEMSA. A small decrease
in the initial velocity compared to no pretreatment of FXa was
observed, but was insignificant compared to the loss in FXa cofactor
activity when pretreated first with CPB. Similar observations were made
when Glu-Pg was used instead of Lys-Pg as a substrate (data not shown).
of 0.1 µM is well
below the plasma concentration of Pg (2.4 µM) and may be a
driving force for the participation of FVa in fibrinolysis. Whether a
direct effect on tPA also results after FVa is cleaved by Pn is
currently unresolved. Thus Pn induces a functional switch in FVa from a
procoagulant in the native state to a profibrinolytic.
= 35 nM) than Pg binding to other receptors.
This may make FXa a particularly effective profibrinolytic cofactor.
. This observation may be partially
explained by the known increased rate of FVa Pn-mediated proteolysis in
the presence of PCPS (Omar and Mann, 1987). However, the modest
enhancement that was reported (approximately 5-fold) cannot account for
the complete loss of tPA acceleration by Pn-pretreated FVa in the
absence of PCPS. Interestingly, when Pn proteolysis of FVa was
conducted in the presence of PCPS and Ca
, and then
EDTA added prior to assaying for tPA activity with PCPS, 50% of the FVa
cofactor activity remained. Taken together these data suggested that an
interaction between FVa and PCPS is necessary for FVa to express tPA
cofactor function and the Ca
-independent association
of the 30-kDa Pn-derived fragment of the FVa light chain with PCPS may
be directly involved. In the case of FXa, we observed that acceleration
of tPA was facilitated only when a
FXa-Ca
-phospholipid complex could form. The
interaction of FXa with Ca
(Church etal., 1989; Nelsestuen, 1976; Pryzdial and Mann, 1991;
Radcliffe and Barton, 1972) and with phospholipid (Krishnaswamy etal., 1992) is known to induce a conformational change
that may be required for tPA cofactor activity. Thus the
profibrinolytic effect of FXa and FVa is localized to sites only where
exposure of procoagulant phospholipid initially triggered the
generation of thrombin.
We thank G. Kessler and Y. Coulombe for purification
of proteins, T. Raynor for reading the manuscript, and Dr. A. Dudani
for helpful suggestions. We also thank Dr. S. Krishnaswamy and Dr. P.
Lollar for critical evaluation of the initial data.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.