Factor XI–Dependent Reciprocal Thrombin Generation
Consolidates Blood Coagulation when Tissue Factor Is
Not Available
Simone J.H. Wielders, Suzette Béguin, H. Coenraad Hemker, Theo Lindhout
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Objective—Feedback activation of factor XI by thrombin is a likely alternative for tissue factor-dependent propagation of
thrombus formation. However, the hypothesis that thrombin can initiate and propagate its formation in a factor
XI-dependent and platelet-dependent manner has not been tested in a plasma milieu.
Methods and Results—We investigated thrombin generation in recalcified platelet-rich plasma activated with varying
amounts of thrombin or factor VIIa. Thrombin initiates and propagates dose-dependently thrombin generation only
when platelets and plasma factor XI are present. Incubation of thrombin-activated platelets with a tissue factor
neutralizing antibody had no effect on thrombin formation, indicating that platelet-associated tissue factor, if present at
all, is not involved. In the absence of factor VIII, thrombin could not initiate its own formation, whereas factor
VIIa-induced thrombin generation was reduced. Collagen strongly stimulated both thrombin-initiated and factor
VIIa-initiated thrombin generation.
Conclusions—These findings support the notion that platelet-localized feedback activation of factor XI by thrombin plays
an important role in maintaining normal hemostasis as well as in sustaining thrombus formation when the TF pathway
is inhibited by tissue factor pathway inhibitor. (Arterioscler Thromb Vasc Biol. 2004;24:1138-1142.)
Key Words: factor XI 䡲 thrombin 䡲 clot formation 䡲 activated platelets 䡲 factor VIIa 䡲 collagen
I
t is generally appreciated that thrombin-mediated feedback
activation of factor XI contributes to the consolidation
phase of clot formation.1–3 In this process, thrombin that is
initially generated via the tissue factor (TF) pathway, activates factor XI on the surface of (thrombin-activated) activated platelets.4 The accelerating role of activated platelets in
thrombin-catalyzed factor XI activation is thought to result
from the colocalization of thrombin and factor XI on the
platelet membrane glycoprotein (GP) Ib␣ within the GP
Ib-IX-V complex.5,6 Thrombin-activated factor XI, therefore,
could be critical to the propagation of thrombin generation
once the TF pathway is inhibited by TF pathway inhibitor
(TFPI).
Hypothetically, thrombin-catalyzed factor XI activation
could play a major role in the development of growing
thrombi. Besides circulating cell-bound TF, the continuous
supply of factor XI by blood flow to a thrombin-containing
thrombus surface might result in a factor XIa-driven thrombin
production that in turn will accelerate the rate of thrombus
growth.7 An additional argument for an important contribution of a factor XI-dependent thrombus growth is found in the
observation that factor XI activation seems to be confined to
the surface of an activated platelet.8 The latter optimizes the
thrombin generating potency because of maintaining high
local concentrations of reactants and the inability to be
inactivated by plasma protease inhibitors.
The present work was undertaken to explore the role of
thrombin in initiating its own production by feedback activation of factor XI under physiological conditions as in platelet-rich plasma (PRP) and to know how effective collagen
and factor VIIa are in promoting thrombin generation under
these conditions. In an attempt to answer these questions, we
used a continuous thrombin generation assay9,10 in which
thrombin generation is not driven by factor XIIa and TF.
Methods
Preparation of PRP and Washed
Platelet Suspensions
Blood (9 volumes) from healthy donors was collected on 0.13 mol/L
trisodium citrate (1 volume). PRP was obtained by centrifugation of
the blood at 260g for 15 minutes at 20°C. The platelets were counted
(Beckman Coulter Counter) and adjusted to 9⫻107 platelets/mL
using autologous platelet-poor plasma (PPP) that was prepared from
citrated blood by centrifuging twice at 2900g for 10 minutes at 20°C.
Suspension of washed platelets (6.6⫻108 platelets/mL) were prepared as previously described.11
Received December 23, 2003; revision accepted March 1, 2004.
From the Cardiovascular Research Institute Maastricht (S.J.H.W., S.B., H.C.H., T.L.) and the Department of Biochemistry (T.L.), Maastricht
University, Maastricht, The Netherlands.
Correspondence to Dr T. Lindhout, Department of Biochemistry, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. E-mail
[email protected]
© 2004 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1138
DOI: 10.1161/01.ATV.0000128125.80559.9c
Wielders et al
Factor XI Feedback Activation
1139
Preparation of Plasma Factor XI–Deficient and
Plasma Factor VIII–Deficient PRP
Factor XI-deficient PPP (60 ␮L) and factor VIII-deficient PPP (60
␮L) were mixed with a suspension of washed platelets (10 ␮L;
6.6⫻108 platelets/mL) that were isolated from normal PRP. Severe
factor XI-deficient patient PPP (homozygote for type II mutation
with factor XI level of ⬍1%) was a kind gift from Dr Uri
Selighsohn,3 and congenital factor VIII-deficient plasma (factor VIII
level of ⬍1%) was purchased from George King Bio-Medical Inc.
Continuous Thrombin Generation Assay
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Wells of Immulon 2 HB, round-bottom 96-well plates (Thermo
Labsystems), were supplied with 20 ␮L 2.4 mmol/L fluorogenic
substrate Z-Gly-Gly-Arg-AMC.HCl (Bachem) in HEPES buffer (pH
7.45; 10 mmol/L HEPES, 136 mmol/L NaCl, 2 mmol/L MgCl2,
2.7 mmol/L KCl, 1 mg/mL bovine serum albumin), 70 ␮L PRP
(9⫻107 platelets/mL), and 10 ␮L HEPES buffer containing 0.6
mg/mL corn trypsin inhibitor (CTI; Hematologic Technologies), and
other agents when indicated. Final platelet concentration in all
experiments was 5⫻107/mL. Blood coagulation was started by
adding thrombin12 or recombinant factor VIIa (Novo Nordisk
Pharma) in 20 ␮L HEPES buffer containing CaCl2 (0.1 mol/L).
Fluorescence tracings were recorded in a 96-well plate spectrofluorometer (Molecular Devices, Spectra Max Gemini XS) with
␭ex⫽368 nm and ␭em⫽460 nm. A cutoff filter of 455 nm was used.
The fluorescence intensity after correction for the inner filter effects and
substrate depletion were converted into molar concentrations thrombin
as described.10 Thrombin generation curves are the mean of three
independent experiments. All procedures were performed at 37°C.
Continuous Thrombin Generation Assay in Plasma
Factor VIII–Deficient PRP
A suspension of washed platelets (10 ␮L, 6⫻108/mL) was added to
a mixture containing 60 ␮L factor VIII-deficient PPP, 6 ␮L CTI (1
mg/mL), 4 ␮L collagen (0.15 mg/mL; Horm, Nycomed Pharma), and
20 ␮L fluorogenic substrate (2.4 mmol/L). Reaction was started by
adding 20 ␮L CaCl2 (0.1 mol/L) containing 120 nmol/L factor VIIa
or 30 nmol/L thrombin.
Detection of Platelet-Associated TF
A suspension of washed platelets (100 ␮L; 6⫻108/mL) was incubated for 10 minutes at 37°C with thrombin (5 nmol/L) and CaCl2
(3 mmol/L) in the presence of 20 ␮g/mL of a neutralizing polyclonal
rabbit antibody against human TF (American Diagnostics) or 24
pmol/L TF (Innovin, Dade Behring) either in the presence or in the
absence of anti-TF (20 ␮g/mL). A similar incubation was performed
with 20 ␮g/mL rabbit immunoglobulin (Dako) as a negative control
for rabbit antibodies. An aliquot (10 ␮L) of the incubation mixture
was transferred to a solution containing PPP (60 ␮L), CTI (10 ␮L,
0.6 mg/mL), and 20 ␮L fluorogenic substrate (2.4 mmol/L). The
reaction was started by the addition of 20 ␮L CaCl2 (0.1 mol/L).
Thrombin generation was monitored as described.
Results
Thrombin Initiates Thrombin Generation in PRP
We hypothesized that thrombin-catalyzed factor XI activation
at the platelet surface is the sole contributor to thrombin
generation in PRP when TF is not available. To test this
hypothesis, we added increasing amounts of thrombin to
recalcified PRP and continuously monitored thrombin generation using a fluorogenic thrombin substrate.10 Initial experiments showed that recalcification of PRP resulted after a lag
phase of ⬇20 minutes in a sudden onset of thrombin
generation, which could be fully inhibited by CTI (50
␮g/mL), a selective factor XIIa inhibitor.13 Therefore, all next
Figure 1. Plasma factor XI and reciprocal thrombin generation in
PRP. A, Citrated PRP containing the fluorogenic thrombin substrate Z-Gly-Gly-Arg-AMC (0.4 mmol/L) was incubated with
varying thrombin concentrations (from top to bottom: 5, 2, 1,
and 0.5 nmol/L thrombin and no thrombin) and 17 mmol/L
CaCl2. The time course of thrombin was calculated from fluorescence tracings (see Methods). B, PRP was reconstituted from
normal PPP and washed normal platelets and activated with 1
nmol/L thrombin and 17 mmol/L CaCl2 (F). The same experiment was performed with PRP reconstituted from factor
XI-deficient PPP and washed normal platelets (Œ).
experiments were performed in the presence of CTI to avoid
contact activation-driven thrombin generation.
The addition of increasing amounts of thrombin (0 to 5
nmol/L) to PRP yielded increasing amounts of in situ-formed
thrombin (Figure 1A). That is, thrombin dose-dependently
decreased the time to thrombin peak and increased the
thrombin peak values. Platelets appeared to be essential
because in PPP, thrombin was not formed. We note that the
thrombin that was added to initiate thrombin generation in
PPP was also not detectable. It is assumed that this exogenous
thrombin is neutralized by its plasma inhibitors within the
mixing time and first read out by the fluorometer (typically
⬍30 s). These findings thus indicate that thrombin initiates
and dose-dependently propagates thrombin generation only in
PRP. Indeed, when plasma is activated with thrombin (5
nmol/L), thrombin peak values increased with the platelet
concentration and reached a maximum with 3⫻108 platelets/mL (data not shown).
To investigate the role of factor XI, thrombin generation
was monitored in PRP that was reconstituted from factor
XI-deficient plasma and washed normal platelets and compared with thrombin generation in PRP reconstituted from
normal plasma and washed normal platelets. In reconstituted
normal PRP, thrombin generation showed a short lag phase of
⬇5 minutes and reached after 40 to 50 minutes incubation
with thrombin (1 nmol/L) an apparent peak value of 20
nmol/L (Figure 1B). In PRP, lacking plasma factor XI, the
1140
Arterioscler Thromb Vasc Biol.
June 2004
Figure 2. Dependency on platelet-associated tissue factor.
Washed platelets (6⫻108/mL) were incubated for 10 minutes
with 5 nmol/L thrombin and 3 mmol/L CaCl2 without further
addition (Œ) and in the presence of 20 ␮g/mL rabbit immunoglobulin (▫), 20 ␮g/mL rabbit anti-human TF (‚), 2 pmol/L TF
and 20 ␮g/mL rabbit anti-human TF (E) or 2 pmol/L TF (F).
Thrombin generation was started with the addition of PPP as
described in Methods.
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time of onset of thrombin appearance was ⬇30 minutes and
thus greatly prolonged. At the end of the experiment, a
thrombin level ⬇2 nmol/L was measured. Thrombin generation could be restored by mixing factor XI-deficient patient
plasma with 10% normal PPP (data not shown).
In view of a report demonstrating that platelet ␣-granules
contain TF,14 the question had to be addressed whether a
thrombin-induced release of functional TF could be (partly)
responsible for thrombin generation in PRP. To explore a
possible role of platelet-associated TF in initiating thrombin
generation, a suspension of washed normal platelets was
incubated with thrombin in the absence and presence of a TF
activity neutralizing antibody. It is apparent that the anti-TF
antibody shortened to some extent the lag phase (defined as
the time to generate 2 nmol/L thrombin), and increased the
thrombin and the thrombin peak value (Figure 2). However,
incubation with a negative control for rabbit antibodies
resulted in a similar stimulation of thrombin generation. We
assume that the slightly stimulated thrombin generation is
caused by an antibody-induced platelet activation reaction.15
To verify that the polyclonal anti-TF antibody does neutralize
TF activity under the conditions of the experiment, thrombinstimulated platelets were incubated with 24 pmol/L recombinant TF (Innovin, Dade Behring) in the presence and absence
of anti-TF. Thrombin generation was then started by adding
PPP. Figure 2 shows that TF greatly enhanced thrombin
generation in PRP and that this TF-driven thrombin generation could be fully abolished by anti-TF. Altogether, these
experiments strongly support the notion that PRP does not
contain sufficient amounts of functional TF to contribute to
thrombin generation in PRP.
Collagen Enhances Thrombin-Initiated and Factor
VIIa–Initiated Thrombin Generation in PRP
We next investigated the ability of collagen to synergistically
enforce the platelet-dependent function of thrombin in its
own initiation and propagation. The time of onset of thrombin
generation, initiated with 0.5 nmol/L thrombin, was dramatically shortened, and the thrombin peak value was greatly
increased when PRP was preincubated with 5 ␮g/mL colla-
Figure 3. Dependency of reciprocal thrombin generation on
thrombin-driven and factor VIIa-driven pathways. A, Recalcified
PRP was incubated with 0.5 nmol/L (triangle) and 5 nmol/L (circle) thrombin in the absence (white symbol) and presence (black
symbol) of 5 ␮g/mL collagen. B, Recalcified PRP was activated
with 2 nmol/L (triangle) and 20 nmol/L (circle) factor VIIa in the
absence (white symbol) and presence of 5 ␮g/mL collagen
(black symbol). C, Factor VIIa (20 nmol/L), collagen (5 ␮g/mL)
and CaCl2 (17 mmol/L) were added to PRP reconstituted from
normal washed platelets (6⫻108/mL) and normal PPP without
further addition (F), with 20 ␮g/mL rabbit anti-human TF antibody (‚) or with 20 ␮g/mL control rabbit immunoglobulin (E),
and to PRP reconstituted from normal washed platelets (6⫻108/
mL) and factor XI-deficient PPP (Œ) or factor VIII-deficient
PPP (▫).
gen 15 minutes before the initiation of the coagulation
reaction with thrombin (Figure 3A). The same, but to a lesser
extent, was found when PRP was activated with 5 nmol/L
thrombin. In the absence of exogenous thrombin, collagen did
not initiate thrombin generation. It is assumed that collagen
induces via its platelet receptor glycoprotein (GP) VI a
procoagulant response, of which the exposure of anionic
phospholipids in the outer leaflet of the platelet plasma
membrane is an essential part. We confirmed that factor
XI-dependent reciprocal thrombin generation could be fully
inhibited with 3 ␮g/mL annexin A5 (NeXins), a protein16 that
binds with very high affinity to phosphatidylserine and
phosphatidylethanolamine (data not shown).
Wielders et al
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We next addressed the question whether platelet-dependent
propagation of thrombin generation by factor VIIa, as reported by Monroe et al,17 is also amplified by feedback
activation of factor XI. Figure 3B shows that factor VIIa
dose-dependently initiates thrombin generation in PRP containing CTI. However, a supraphysiological amount of factor
VIIa, namely twice the plasma factor VII level, was required
to generate rather small amounts of thrombin (ie, ⬇5 nmol/L
thrombin after 60 minutes). Interestingly, collagen dramatically stimulated the factor VIIa-initiated thrombin generation.
Under these conditions, a relatively low concentration of
factor VIIa (2 nmol/L) rapidly initiates and sustains an
extensive amount of thrombin, as indicated by a greatly
reduced lag phase and increased maximal thrombin activity.
A previous experiment with a neutralizing TF antibody
(see Figure 2) ruled out that platelet-associated TF is not
involved in thrombin-initiated thrombin generation in PRP.
Because factor VIIa-driven thrombin generation could be
more sensitive to the presence of TF, the anti-TF antibody
was used to investigate a contribution of TF in collagenenhanced factor VIIa-driven thrombin generation. Figure 3C
demonstrates that anti-TF did not inhibit factor VIIa-driven
thrombin generation. As a matter of fact, both the anti-TF and
a control antibody stimulated thrombin generation as previously observed in thrombin-initiated thrombin generation
experiments (Figure 2). Evidently, TF, if present in PRP, does
not contribute to factor VIIa-driven thrombin generation in
collagen-stimulated PRP.
To dissect the contribution of the intrinsic (ie, thrombinactivated factor XI) and the extrinsic pathway (ie, factor VIIa
activated factor X) in factor VIIa-initiated thrombin generation, thrombin generation was monitored in PRP reconstituted
from normal platelets and congenital factor VIII-deficient
plasma and in PRP reconstituted from factor XI-deficient
plasma and normal platelets. Although thrombin generation
was absent when plasma factor XI-deficient PRP was activated with thrombin (see Figure 1B), in the presence of 20
nmol/L factor VIIa and 5 ␮g/mL collagen a considerable
amount of thrombin was formed (Figure 3C). In factor
VIII-deficient PRP, thrombin did not initiate its own generation, whereas factor VIIa induced thrombin generation to
approximately the same extent as observed in plasma factor
XI-deficient PRP. Because factor VIII and factor XI deficiency resulted in similar but reduced thrombin generation, it
is apparent that a large part of thrombin is directly generated
via a factor VIIa-catalyzed activation of factor X, and an
additional amount is supplied by the factor XI-dependent
pathway.
Discussion
Extensive work from Walsh et al has revealed much of the
molecular basis of thrombin-catalyzed for XI activation at the
surfaces of activated platelets.18 Yet the precise implications
of this process for ex vivo and in vivo thrombin generation
remain to be clarified. Our work demonstrates that low
amounts of thrombin (⬍ 1 nmol/L), despite thrombin’s short
half life (⬍30 s), initiate and amplify thrombin generation in
PRP. This reciprocal thrombin generation is solely mediated
by factor XI and operates in the absence of platelet-derived
Factor XI Feedback Activation
1141
TF. Yet platelets play a critical role, because initiation and
propagation was not observed in PPP. In contrast, TFindependent factor VIIa-driven thrombin generation is only
partly dependent on factor XI, suggesting that factor VIIacatalyzed factor X activation initiates and contributes largely
to thrombin generation in the propagation phase.
That (platelet-derived) TF is not involved in plateletenhanced thrombin-initiated thrombin generation is supported by the finding that when contact activation is inhibited
no thrombin is formed. This notion was further corroborated
by experiments in which platelets were incubated with
thrombin in the presence of a polyclonal antibody against TF.
Although this antibody inhibits TF-driven thrombin generation in PRP, it did not inhibit reciprocal thrombin generation.
Furthermore, experiments with PRP that was reconstituted
from factor XI-deficient plasma and platelets isolated from
normal PRP clearly indicated that plasma factor XI is a
prerequisite for a TF-independent thrombin generation. A
factor XI-dependent pathway in thrombin generation was
finally confirmed in similar experiments; however, in factor
VIII-deficient plasma, thrombin-initiated thrombin formation
could not be detected.
Intriguingly, in the presence of platelets, a factor XI plasma
level of ⬇10% is already sufficient to normalize factor
XI-dependent reciprocal thrombin generation. Yet no significant thrombin generation was measured in PRP that was
reconstituted from normal platelets and congenital factor
XI-deficient plasma, indicating that platelet factor XI, under
the conditions of the experiment, does not compensate for the
lack in plasma factor XI. This finding is in good agreement
with earlier observations from our laboratory3 but apparently
contradicts the notion that platelet-derived factor XI may be
important in maintaining normal hemostasis in patients with
a severe plasma factor XI deficiency.19
Pretreatment of resting platelets in PRP with collagen
shortened the lag phase of thrombin generation as well as the
thrombin peak value that was induced by thrombin or factor
VIIa. The lag phase reflects a threshold mechanism of
thrombin-dependent feedback reactions, eg, platelet activation and the activation of the essential cofactors factor V and
VIII.20 The thrombin peak value is determined by the rate of
thrombin production, which in turn reflects the concentration
of the prothrombinase complex at the surface of activated
platelets and the rate of thrombin inactivation.9 The interaction of collagen with its platelet receptor, GPVI, induces a
signal pathway leading to exposure of anionic phospholipids
that support blood coagulation.21 In addition, (in situ-formed)
thrombin synergistically enhances the generation of platelet
procoagulant phospholipid.22 Thus, a picture emerges in
which the surface of collagen-treated platelets exert a dual
role: (1) thrombin-activated platelets offer binding sites
(GPIb␣) for both factor XI and thrombin; and (2) collagen/
thrombin-activated platelets provide procoagulant anionic
phospholipids that support the assembly of prothrombin and
factor X converting enzyme complexes on platelet surfaces.
That these phospholipids alone or in combination with
protein receptors on the platelet surface mediate thrombin
generation is consistent with the fact that a protein with high
1142
Arterioscler Thromb Vasc Biol.
June 2004
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affinity for anionic phospholipids, ie, annexin A5, prohibited
in full factor XI-dependent reciprocal thrombin generation.
There is much debate about the role and function of
platelets in factor VIIa-mediated clot formation.23,24 The
question has been addressed to what extent high-dose factor
VIIa supports thrombin generation in a TF-independent
manner. Our results confirm that supraphysiological factor
VIIa concentrations increase dose-dependently thrombin generation only in the presence of platelets. Collagen dramatically accelerated the initiation and propagation of factor
VIIa-driven thrombin generation. Using plasma factor XIdeficient or plasma factor VIII-deficient reconstituted PRP,
we could demonstrate that factor VIIa-initiated thrombin
generation was partly driven by the factor XIa-activated
intrinsic pathway. The factor XI-dependent consolidation
phase of the blood coagulation process is likely triggered by
thrombin that is initially generated by factor VIIa-activated
factor X. Furthermore, in these in vitro experiments, no
evidence was found that TF is required in factor VIIainitiated and factor XI-dependent propagation of thrombin
formation at the surface of activated platelets. It is evident
that care should be exercised in predicting the in vivo
implications of these findings. The primary initiator of
coagulation in vivo is TF, either located in the vessel wall
and/or present in circulating blood.25–27 However, the question to what extent functional TF, if incorporated into a
thrombus, determines thrombus growth has yet to be answered.28,29 Alternatively, FXI-dependent thrombus propagation could, after the initiation phase, limit the availability of
TF at the blood/thrombus interface.7 It is an interesting
thought that the continuous supply of factor XI to a thrombus
that contains fibrin-bound thrombin is a potential alternative
pathway to mediate thrombus growth once TF is no longer
available because of its rapid neutralization by TFPI or the
increasing distance between blood–thrombus interface and
vessel wall-located TF.
Collectively, our investigations suggest an important role
of thrombin-activated factor XI in maintaining normal hemostasis and driving thrombus growth. Our results may also
explain how pharmaceutical doses of factor VIIa function in
maintaining or restoring normal hemostasis.
Acknowledgments
Part of this study was supported by a grant from the Dutch
Thrombosis Foundation (TSN).
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Factor XI−Dependent Reciprocal Thrombin Generation Consolidates Blood Coagulation
when Tissue Factor Is Not Available
Simone J.H. Wielders, Suzette Béguin, H. Coenraad Hemker and Theo Lindhout
Arterioscler Thromb Vasc Biol. 2004;24:1138-1142; originally published online April 8, 2004;
doi: 10.1161/01.ATV.0000128125.80559.9c
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