(Received for publication, October 3, 1994)
From the
The ability of intact peripheral blood monocytes to modulate
factor V procoagulant activity was studied using electrophoretic and
autoradiographic techniques coupled to functional assessment of
cofactor activity. Incubation of plasma concentrations of factor V with
monocytes (5 10
/ml) resulted in the time-dependent
cleavage of the 330-kDa protein. Activation occurred via several high
molecular mass intermediates (
200 kDa) to yield peptides of 150,
140, 120, 94, 91, 82, and 80 kDa, which paralleled the expression of
cofactor activity. The cleavage pattern observed differed from that
obtained with either thrombin or factor Xa as an activator. The
incubation time required to achieve full cofactor activity was
dependent on the monocyte donor and ranged from 10 min to 1 h and was
consistently slightly lower than that obtained with thrombin-activated
factor Va. Cofactor activity was not diminished by additional
incubation. The cofactor activity generated bound to the monocyte such
that a competent prothrombinase complex was formed at the monocyte
membrane surface. Furthermore, within 5 min of factor V addition to
monocytes, near maximal cofactor activity (
70%) was bound and
expressed on the monocyte membrane. The proteolytic activity toward
factor V was associated primarily with the monocyte membrane, as little
proteolytic activity was released into the cell-free supernatant.
Proteolytic activity was inhibited by diisopropyl fluorophosphate and
phenylmethanesulfonyl fluoride. However, the inhibitor profile obtained
with
-antiproteinase inhibitor,
-antichymotrypsin, and
-macroglobulin
suggested membrane-bound forms of elastase and cathepsin G were
mediating, in large part, the proteolysis observed. These data were
confirmed using purified preparations of both proteases and a specific
anti-human leukocyte elastase antibody. Thus, expression of these
proteases at the monocyte surface may contribute to thrombin generation
at extravascular tissue sites by catalyzing the activation of the
essential cofactor, factor Va, which binds to the monocyte surface and
supports the factor Xa-catalyzed activation of prothrombin.
Monocytes and macrophages play key roles in the physiological and pathophysiological processes of wound repair, acute/chronic inflammation, and atherosclerosis. These processes, which in several instances proceed at extravascular tissue sites, are characterized by extensive fibrin deposition and an active fibroproliferative response (1, 2, 3, 4) . Since thrombin serves as a potential effector of fibrin deposition, leukocyte chemoattraction(5) , and mesenchymal cell growth (6) , it seems likely that the production of thrombin at the monocyte/macrophage membrane surface provides an important bioregulatory effector molecule at these extravascular sites. Thus, we and others have begun to explore the hypothesis that the ability of these cells to participate in the molecular events leading to thrombin formation is an important mechanism by which they function.
Monocytes/macrophages can provide the appropriate membrane surface
for the assembly and function of virtually all the coagulation
complexes involved in thrombin production. Monocytes can be stimulated
to express tissue factor at their membrane surface, which binds factor
VIIa, and catalyzes the activation of factor X to factor Xa, thereby
initiating the extrinsic pathway of coagulation (for review, see (7) ). The tissue factor-factor VIIa complex also activates
factor IX to factor IXa(8) , which in complex with the
cofactor factor VIIIa bound to the monocyte/macrophage membrane can
provide additional factor Xa(9) . Propagation of the coagulant
response is accomplished by the assembly and function of
prothrombinase, a stoichiometric complex of the nonenzymatic cofactor
factor Va and the enzyme factor Xa bound to the monocyte surface in the
presence of calcium ions and which effects the proteolytic conversion
of prothrombin to thrombin(10, 11) .
While it is
well established that both factor IXa and factor Xa formation are
accomplished at the monocyte/macrophage membrane surface, little is
known concerning how these cells may participate in the events
resulting in the proteolytic activation of the plasma procofactors,
factors V and VIII. The provision of these cofactors is essential to
the proper assembly and function of the coagulant enzyme complexes in
which they participate(12) . For example, factor Va is
responsible for mediating the majority of the protein-protein and
protein-membrane interactions required for prothrombinase assembly,
such that the deletion of factor Va from the complex reduces the rate
of thrombin generation by 4 orders of magnitude(13) . The
central role that factor Va assumes in complex assembly, coupled with
its profound influence on the rate of thrombin formation, provides
strong evidence that the activation of factor V to factor Va is a key
regulatory event. This notion is underscored by the inability of the
procofactor factor V to participate, to any significant degree, in
thrombin generation(13, 14) .
Several proteases have been identified that cleave human factor V to yield different levels of cofactor activity. Thrombin (12) and factor Xa (15) are believed to be the most potent physiological activators of human factor V and by far the most widely recognized with respect to coagulant and hemostatic responses. In contrast to thrombin activation of factor V, the factor Xa-catalyzed activation of factor V is absolutely dependent on the presence of a membrane surface(14, 15) . Whether the membrane surface is required for substrate or enzyme binding, or both, has not yet been determined. Factor V activators that can be released from platelets(16, 17) , endothelial cells(18) , and neutrophils (19) have also been described. In contrast to the other cell-derived activators, leukocyte elastase released from neutrophils first activates factor V and then rapidly inactivates the formed factor Va(19) . The extent to which these cell-associated proteases contribute to factor V activation/inactivation in normal or pathophysiological events has not been elucidated.
In attempts to determine if factor Xa bound to the monocyte membrane (20, 21) could activate factor V, the observation was made that freshly isolated peripheral blood monocytes alone effected the rapid cleavage and activation of plasma concentrations of factor V. Cofactor activity was expressed at the monocyte membrane surface, which supported the factor Xa-catalyzed activation of prothrombin. As detailed in this report, membrane-bound forms of elastase and cathepsin G appear to be responsible, at least in part, for the monocyte-mediated activation of factor V.
Factor V was radioiodinated using the IODO-GEN
(Pierce) transfer technique purified and characterized as previously
detailed(15) . I-Factor V was >95%
precipitable with 10% trichloroacetic acid, expressed specific
radioactivities of 1000-5000 cpm/ng (0.1-0.5 mol of
iodine/mol of protein), and was stored at -20 °C in 50%
glycerol.
I-Factor V retained full clotting activity.
Certain experimental protocols required the analysis of I-factor V either free in solution or bound to the cell
surface. Accordingly, free and bound
I-factor V/Va were
obtained by centrifugation (12,000
g, 30 s) of an
aliquot (100 µl) of the reaction mixtures over an oil mixture
containing 1 part Apiezon A oil (Apiezon Products, Ltd., London) and 9
parts n-butyl phthalate. The supernatant fractions were made
10% in acetic acid, and the cell pellets were lysed by addition of 10%
acetic acid. Both fractions were subsequently frozen and lyophilized
and then processed for SDS-PAGE and autoradiography as outlined above.
In experimental protocols where the effect of a protease inhibitor or a specific anti-protease antibody on factor V proteolysis was being assessed, the inhibitor or antibody was incubated at 37 °C with the monocytes for 2 or 15 min, respectively, prior to the addition of factor V to the reaction mixture.
Figure 1:
Cleavage and activation of human factor
V by a monocyte-associated protease(s). Monocytes (5
10
/ml) were incubated with human factor V (67 nM)
plus trace
I-factor V (1000 cpm/µl) in 20 mM HEPES, 0.15 M NaCl, 5 mM CaCl
, pH
7.4, at 37 °C. At the time points indicated, aliquots were removed
and centrifuged at 12,000
g for 10 s. The supernatant
was removed, and the aliquots were assayed. A depicts SDS-PAGE
(5-10% gradient) followed by autoradiography. Equal amounts of
isotope were applied to each lane. The symbols at the bottom of each lane correspond to the factor Va
cofactor activity assays made during the course of the incubation as
shown in B. In B, factor Va cofactor activity (
1
nM) was assessed by its ability to support the factor
Xa-catalyzed (5 nM) activation of prothrombin (1.39
µM) in the presence of defined phospholipid vesicles (20
µM). -
-, activity observed with factor
Va
. Inset depicts initial rates of thrombin
generation for each assay.
The time-dependent cleavages observed paralleled the expression of factor Va cofactor activity (Fig. 1B), as indicated by the shortening of the lag period preceding steady state thrombin formation in the assay and an increase in the steady state rate of thrombin generation. Both observations were dependent on the length of time monocytes were incubated with the factor V. Increases in cofactor activity were apparent with loss of the 330-kDa parent molecule but could not be ascribed with any certainty to a particular combination of the peptides observed.
Based on the five replicate experiments
performed, the length of time required to achieve full cofactor
activity was donor-dependent and in some cases required less than a
15-min incubation of monocytes with factor V, in marked contrast to the
1-h incubation shown in Fig. 1A. As can be seen clearly
in Fig. 1B, total cofactor activity generated was
consistently slightly lower than that observed with thrombin as
activator (--), since addition of thrombin following a 60-min
incubation of factor V with monocytes (-
-) did not result in rates
of prothrombin activation achieved with thrombin-activated factor V,
factor Va
(-
-). Even though lymphocytes represented
as much as 20% of the cells present in the reaction mixtures, purified
populations of T-lymphocytes (
98%) neither activated nor cleaved
factor V when identical experimental protocols to those described in Fig. 1were used (data not shown). These combined observations
indicate that intact, freshly isolated peripheral blood monocytes
express a protease(s) capable of activating factor V, that the factor
Va generated is a slightly less effective cofactor than factor
Va
, and that the cofactor is comprised of novel peptides.
Figure 2:
Monocyte-mediated proteolysis of factor V
requires the continued presence of cells. Monocytes (5
10
/ml) were incubated with factor V (20 nM plus
trace label) as described in Fig. 1. After 5 min, the reaction
mixture was divided, and both aliquots were centrifuged at 200
g for 10 min. One aliquot was resuspended and assayed for
proteolytic activity toward factor V (panelI). The
cell-free supernatant was removed from the second and assayed similarly (panelII). In a third reaction mixture (panelIII), factor V was added to a cell-free supernatant
obtained subsequent to the centrifugation step. A, SDS-PAGE
and autoradiographic visualization of the factor V peptides. B, densitometric analysis of the data shown in A monitoring the appearance of the 94/91 peptide doublet (indicated
by the arrow).
Figure 3:
Assessment of factor Va cofactor activity
associated with peptides bound to the monocyte membrane as visualized
by autoradiography. Monocytes (5 10
/ml) were
incubated with 20 nM factor V (A) or factor
Va
(B) each containing trace label as previously
described. At the specified time points, aliquots were removed and
centrifuged at 12,000
g (10 s). The supernatant (I) or pellet (II) fractions were then processed for
SDS-PAGE and autoradiography. In parallel reaction mixtures, the
pelleted cells with bound factor Va peptides were resuspended in 20
mM HEPES, 0.15 M NaCl, 5 mM CaCl
0.35% bovine serum albumin, pH 7.4, and assayed for their ability
to support the factor Xa-catalyzed (5 nM) activation of
prothrombin (1.4 µM) in the absence of additional factor
Va or lipid. Initial rates are indicated above the corresponding lanes in panelII.
Comparison of the prothrombin
activation rate data listed in panelsA and B indicated that at any time during the course of the reaction, the
factor V(a) peptides associated with the monocyte membrane due to
cell-mediated proteolysis (panelA) expressed
60% of the cofactor activity provided by bound factor Va
(panelB). The time-dependent increase in
expression of cofactor activity observed for both species of
monocyte-bound peptides most likely reflects their increased binding
over time. It is interesting to note that within 5 min of factor V
addition, 60-70% of the total cofactor activity, which can
associate with the monocyte membrane surface, is expressed. Thus, at
the earliest time point measured (5 min subsequent to factor V addition
to a monocyte suspension), the factor Va cofactor activity expressed at
the monocyte membrane surface was capable of promoting the generation
of 50 nmol/liter/min (5 NIH units/ml/min).
Comparison of the
peptides bound to the monocyte surface versus those remaining
in the supernatant in panelA allows for several
observations: 1) only one peptide resulting from factor V cleavage by a
monocyte-associated protease(s) did not bind to the monocyte membrane (arrow); 2) factor V bound poorly or alternatively was rapidly
cleaved upon binding; and 3) peptides of 94 kDa selectively bound,
suggesting that they comprise the active cofactor. In contrast, the
data shown in panelB indicated that only the heavy
(105 kDa) and light chain (74 kDa) subunits comprising factor
Va
(12, 15) bound to the monocyte to
any appreciable degree. The 150-kDa activation peptide did not bind nor
did a peptide migrating similarly to the 280-kDa factor V activation
intermediate as indicated by the arrows. Bound to a very small
degree were the 220- and 150-kDa activation intermediates, which
contain the 74- and 105-kDa factor Va subunits, respectively. It is
interesting to note that the bound factor Va
was further
cleaved to products not observed in the supernatant fraction,
suggesting that factor Va
is also a substrate for the
monocyte-associated protease(s). However, this additional proteolysis
did not appear to affect cofactor activity. Although it is not yet
clear which peptides are expressing cofactor activity, these data
clearly demonstrate that incubation of plasma concentrations of factor
V with monocytes results in the production of a cofactor bound to the
monocyte surface that expresses significant cofactor activity and
facilitates the assembly of a functional prothrombinase complex at the
monocyte surface.
Figure 4:
Effect of protease inhibitors on the
cleavage of factor V by a monocyte-associated protease(s). Monocytes (5
10
/ml) were incubated with the protease inhibitors
indicated for 2 min at 37 °C prior to the addition of factor V (20
nM) plus trace label. Following a 30-min incubation, the
reaction mixtures were processed for SDS-PAGE and autoradiography as
previously described. Densitometric analysis was used to calculate the
amount of single chain factor V remaining, as its loss represented the
presence of proteolytic activity. PMSF, phenylmethanesulfonyl
fluoride; TLCK, N
-p-tosyl-L-lysine
chloromethyl ketone.
In five similar experiments using different donors for monocyte
isolation, similar inhibition trends were observed. Whereas
-proteinase inhibitor consistently elicited the most
pronounced inhibition (
90%), the inhibition effected by
-macroglobulin, the elastase-specific peptide, and
-antichymotrypsin was substantially more variable.
These data were interpreted to indicate that monocytes exhibit
significant heterogeneity with respect to the levels of the proteases
expressed.
Based on these observations, experiments with purified
human leukocyte elastase (HLE) and cathepsin G were performed to
determine their ability to cleave and activate factor V. Preliminary
experiments were done to determine concentrations of both proteases,
which produced factor V cleavages over a similar time frame as 5
10
monocytes/ml to accurately compare the cleavage
patterns obtained. Low concentrations of both proteases were required
as detailed in Fig. 5. Both proteases cleaved factor V (Fig. 5), concomitant with an increase in cofactor activity
(data not shown). The cleavage patterns were remarkably similar to each
other and to that obtained with monocytes alone. The co-migration
studies shown allowed for the unequivocal identification of two
peptides unique to elastase-mediated cleavage as indicated by the arrows. These observations were supported by data shown in Fig. 6in which an
-HLE antibody, at a concentration that
substantially inhibited purified HLE, was only partially inhibitory
toward total monocyte proteolytic activity yet completely inhibited the
formation of peptides unique to elastase cleavage, as indicated by the arrows. The inhibitory activity could not be increased by
higher concentrations of antibody in subsequent experiments (data not
shown). These combined data strongly suggest that both elastase and
cathepsin G may be active on the monocyte surface to effect the
cleavage and activation of factor V.
Figure 5:
Monocyte-, cathepsin G-, and HLE-mediated
proteolysis of factor V. Factor V (20 nM) plus trace label was
incubated with monocytes (5 10
/ml), cathepsin G
(0.1 nM), or HLE (0.3 nM) for 5 min at 37 °C and
then prepared for autoradiography (middle, left, and rightlanes, respectively). The lanes to the left and right of middle represent equal
mixtures of the monocyte and cathepsin G or monocyte and HLE gel
samples, respectively. Right arrows represent cleavages
specific to HLE.
Figure 6:
Partial inhibition of monocyte-associated
proteolytic activity with an -HLE antibody. Monocytes (5
10
/ml) or purified HLE (0.3 nM) were incubated
± sheep
-HLE antibody (10 µM) for 15 min at 37
°C prior to the addition of factor V (20 nM) plus trace
label. At the times indicated, aliquots were removed and processed for
autoradiography. A, -
-HLE; B,
+
-HLE; C, control sheep IgG plus monocytes or
HLE.
Since a membrane-bound form of
HLE appeared to be responsible for the proteolytic activity observed,
experiments were done to ensure that neutrophils present at extremely
low levels (0.05%) in our monocyte preparations were not
responsible for the activity observed. The factor V cleavage pattern
obtained with neutrophils was identical to that obtained with purified
HLE and thus almost identical to that obtained with monocytes. Even
though on a per cell basis neutrophils expressed 25-40 times the
activity observed with monocytes (data not shown), they did not
contribute significantly to factor V cleavage and activation in our
system.
The assembly and function of a competent prothrombinase
complex at the monocyte/macrophage surface requires the participation
of the nonenzymatic cofactor factor
Va(10, 11, 21) , which is produced by limited
proteolysis of the procofactor factor V(12) . In this report,
we demonstrate that addition of factor V to a suspension of freshly
isolated whole blood monocytes leads to factor V cleavage and the
generation of factor Va cofactor activity. Proteolysis occurs through a
novel cleavage pattern when compared with those that result from
activation by the well described activators-thrombin and factor
Xa (15) , and may explain the observation that the cofactor
activity generated by the monocyte-bound proteases is just slightly
less than that obtained with factor Va. However, the
novel cofactor generated remains associated with the monocyte membrane
such that in the presence of added factor Xa, a functional
prothrombinase complex is assembled at its surface. Based on the data
depicted in Fig. 3, pathophysiologically relevant concentrations
of thrombin may be produced quite rapidly; for example, 1 NIH unit/ml
(
l0 nM) can be produced within 1 min of the addition of a
plasma concentration of factor V to a monocyte suspension (5
10
cells/ml). Furthermore, within 5 min, 70% of the
cofactor binding sites on the monocyte membrane surface are occupied
and competent to support the factor Xa-catalyzed activation of
prothrombin. However, the activation of factor V catalyzed by the
monocyte-associated proteases proceeds to completion and is thus
capable of generating a substantial concentration of active cofactor in
the surrounding milieu.
The proteolytic activity observed appears to
be due in large part, if not completely, to membrane-bound forms of
elastase and cathepsin G. Immunohistochemical evidence indicates that
human peripheral blood monocytes contain enzymes antigenically similar
to leukocyte and cathepsin G(36) . While monocytes appear to be
heterogeneous with respect to enzyme content, the antigens are present
predominantly in peroxidase-positive cytoplasmic granules(36) ,
although membrane-bound forms of elastase have been
reported(35) . Other studies have demonstrated that human
leukocyte elastase binds to monocytes and remains in an active form at
the membrane surface for as long as 24 h (37) . Since these
proteases are susceptible to inhibition by the plasma protease
inhibitors anti-proteinase inhibitor,
-macroglobulin, or
-antichymotrypsin,
we suspect that the proteases would not be active in plasma under
normal conditions. This notion was confirmed by demonstrating that
resuspension of proteolytically active monocytes in autologous, normal
plasma completely abrogated their ability to activate factor V (data
not shown). Therefore, we propose that the monocyte-associated
proteolytic activity may play a significant role in thrombin generation
primarily at extravascular tissue sites, as well as in clinical
settings where extensive depletion of plasma protease inhibitors
occurs. Examples might include
-proteinase inhibitor
deficiency, disseminated intravascular coagulation, and thrombolytic
therapy. Finally, since our initial studies of plasma inhibition by
autologous plasma were done using cells in suspension, those studies
must now be done with an adherent cell population since enzyme
expression may be different, possibly modifying the ability of plasma
inhibitors to regulate monocyte-associated proteolysis. In fact,
several studies have been reported indicating that cell- or
matrix-bound proteases expressed by, or released from, adherent
monocytes/macrophages appear to be protected from their respective
plasma inhibitors(38, 39, 40) .
The functional significance of monocyte/macrophage-derived thrombin is underscored by the fibrin deposition and fibroproliferative responses that accompany wound repair, chronic inflammation, and atherosclerosis, processes in which these cells are known to participate. As cells that support procoagulant enzymatic reactions, monocytes/macrophages are unique in that they provide the appropriate membrane surface required for the assembly and function of all the coagulation enzyme complexes required for thrombin generation in vivo. Through the provision of the membrane-bound enzymes, elastase and cathepsin G, which can effect the generation of an essential cofactor in this process, these cells appear to enhance their procoagulant phenotype substantially.
Continued maintenance of this procoagulant phenotype may be accomplished through additional elastase- and cathepsin G-mediated events. For example, elastase can proteolytically inactivate proteins C (41) and S(42) , which would effectively abrogate activated protein C-catalyzed inactivation of factor Va(43) . In addition, while platelets are not activated by elastase directly, elastase enhances the platelet activation-dependent responses induced by low concentrations of cathepsin G, indicating that both enzymes may function synergistically to activate platelets(44, 45) . Further, elastase can proteolytically inactivate tissue factor pathway inhibitor(46, 47) , which would ordinarily limit thrombin production through inhibition of factor Xa and a factor Xa-factor VIIa-tissue factor complex.
Elastase and in some cases
cathepsin G have been demonstrated to proteolytically inactivate
several inhibitors of coagulant proteases including antithrombin
III(48, 49) , heparin cofactor II(50) ,
chlorine inactivator(51, 52) , and
-antiplasmin(51, 53) . The decreased
concentration of active inhibitor would slow the protease inhibition,
thereby increasing the local steady state level of the target protease.
In such circumstances, the inactivation of potential inhibitors would
contribute to the local progression of coagulation at inflammatory
sites. In the case of tissue factor pathway inhibitor, elastase
cleavage leads to regeneration of factor Xa and tissue factor activity
from previously formed inhibitory complexes. These collective
observations suggest that monocyte-associated elastase and cathepsin G
would play a stimulatory role in coagulant events and would begin to
more firmly establish the monocyte procoagulant phenotype and its
relationship to both intra- and extravascular thrombin generation and
fibrin deposition.
In stark contrast, elastase and cathepsin G, have
been demonstrated to proteolytically inactivate coagulant
zymogens(54, 55, 56, 57, 58) ,
proteases(59) , and cofactors (19, 60) and
thus may produce an anticoagulant effect. However, these studies have
all been done with ``free'' proteolytic inactivators, while
our studies explored the effect of membrane-bound proteases. For
example, whereas the ability of cathepsin G to activate factor V is a
new finding, other investigators have shown that purified neutrophil
elastase, in solution, will first activate factor V then rapidly
inactivate the formed cofactor(19) . However, as shown in these
studies, elastase associated with the monocyte membrane surface rapidly
activated factor V, but neither inactivated the formed cofactor nor
exogenously added factor Va. The factor Va
bound to the monocyte surface was further cleaved, but these
additional cleavages were without effect on its cofactor activity.
These combined data would suggest that the monocyte membrane surface is
in some way modulating the inactivation event. The three-dimensional
conformation of the enzyme, the substrate, or both may be different
when associated with monocyte membrane. In our studies with purified
neutrophils, a similar observation was made. However, when studies were
performed with purified human leukocyte elastase, factor V, and
synthetic phospholipid vesicles, we found that the presence of a
synthetic lipid membrane did not prevent the inactivation event; it
merely slowed the rate of both factor V activation and factor Va
inactivation (data not shown). Thus, it appears that the presence of a cell membrane, with possible attendant carbohydrate or protein
receptors, may be required for full biological regulation.