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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1542-1547
Protein C Inhibitor Acts as a Procoagulant by
Inhibiting the Thrombomodulin-Induced Activation of Protein C in
Human Plasma
By
Marc G.L.M. Elisen,
Peter A.Kr. von dem Borne,
Bonno N. Bouma, and
Joost C.M. Meijers
From the Department of Haematology, University Hospital and Institute
of Biomembranes, Utrecht University, Utrecht, The Netherlands.
 |
ABSTRACT |
Protein C inhibitor (PCI), which was originally identified as an
inhibitor of activated protein C, also efficiently inhibits coagulation
factors such as factor Xa and thrombin. Recently it was found, using
purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited by PCI. The paradoxical inhibitory effect of
PCI on both coagulant and anticoagulant proteases raised questions
about the role of PCI in plasma. We studied the role of thrombomodulin
(TM)-dependent inhibition of thrombin by PCI in a plasma system.
Clotting was induced by addition of tissue factor to recalcified plasma
in the absence or presence of TM, and clot formation was monitored
using turbidimetry. In the absence of TM, PCI-deficient plasma showed a
slightly shorter coagulation time compared with normal plasma.
Reconstitution with a physiologic amount of PCI gave normal clotting
times. Addition of PCI to normal plasma and protein C-deficient plasma
resulted in a minor prolongation of the clotting time. This suggested
that PCI can act as a weak coagulation inhibitor in the absence of TM.
TM caused a strong anticoagulant effect in normal plasma due to
thrombin scavenging and activation of the protein C anticoagulant
pathway. This effect was less pronounced when protein C-deficient
plasma was used, but could be restored by reconstitution with protein
C. When PCI was added to protein C-deficient plasma in the presence of
TM, a strong anticoagulant effect of PCI was observed. This
anticoagulant effect was most likely caused by the TM-dependent
thrombin inhibition by PCI. However, when PCI was added to normal
plasma containing TM, a strong procoagulant effect of PCI was observed,
due to the inhibition of protein C activation. PCI-deficient plasma was
less coagulant in the presence of TM. A concentration-dependent
increase in clotting time was observed when PCI-deficient plasma was
reconstituted with PCI. The combination of these results suggest that
the major function of PCI in plasma during coagulation is the
inhibition of thrombin. A decreased generation of activated protein C
is a procoagulant consequence of the TM-dependent thrombin
inhibition by PCI. We conclude that TM alters PCI from an anticoagulant
into a procoagulant during tissue factor-induced coagulation.
| |
INTRODUCTION |
PROTEIN C INHIBITOR (PCI) is a plasma
glycoprotein that belongs to the SERPIN superfamily of serine protease
inhibitors, of which  1-protease inhibitor is the
prototype.1,2 PCI was initially identified in blood plasma
by Marlar and Griffin3 and isolated by Suzuki et
al4 as a major regulator of the anticoagulant protease
activated protein C (APC). Recently it has been shown that this member
of the SERPIN superfamily has a broad target specifity, capable of
inhibiting various proteases in coagulation, fibrinolysis, and
reproduction.3-12 The lack of documented patients with an
abundancy, deficiency, or specific mutation of PCI makes it difficult
to determine the true physiologic function for PCI in plasma.
Like the other protease inhibitors, antithrombin (AT) and heparin
cofactor II (HCII), PCI is classified as a heparin-binding serpin.
Glycosaminoglycans, including heparin, can accelerate the inhibition
rate of serine proteases by these serpins (for review see Pratt and
Church13).
Two regions of PCI were previously identified as a heparin binding site
by homology to a consensus glycosaminoglycan recognition site, and
their involvement was demonstrated in protease inhibition assays.14-17 Glycosaminoglycans present either on the cell
surface of the endothelial cell or on its basement membrane are
believed to be a site of physiologic activity for heparin
binding-serpins.
Thrombomodulin (TM) is an endothelial cell receptor (for review see
Esmon18), which plays an important modulating role in the
anticoagulant response after vascular injury. Binding of thrombin to TM
enables thrombin to rapidly cleave the protein C zymogen into the
anticoagulant APC. Furthermore, by binding to TM, thrombin is no longer
able to clot fibrinogen or to activate platelets. TM possesses a
covalently linked glycosaminoglycan chain, which stabilizes the
interaction with thrombin.19
Hemostasis involves a series of complex interactions between
procoagulant, anticoagulant, and fibrinolytic mechanisms and in each
system a role for PCI has been suggested.3,5-7
Rezaie et al20 recently described that PCI is a potent
inhibitor of the thrombin-TM complex, thereby providing new clues to
the elucidation of a possible physiologic function of PCI in plasma.
Although PCI has mostly been implicated to be an inhibitor of APC, it
is a much better inhibitor of thrombin. The role of PCI in plasma is
therefore intriguing because it can act as a procoagulant by inhibiting
APC and as an anticoagulant by inhibiting thrombin. This study was
undertaken to investigate this dualistic character of PCI. We studied
the effect of the TM-mediated inhibition of thrombin by PCI on tissue
factor-induced coagulation in a plasma system. Our results indicate
that a main function of PCI in plasma is the inhibition of thrombin,
with TM as a cofactor during coagulation.
| |
MATERIALS AND METHODS |
All reagents used were analytical grade. Rabbit lung TM and Spectrozyme
TH were obtained from American Diagnostica Inc, Greenwich, CT. A
recombinant fragment of TM containing the EGF domains 4-6 (TM4-6) was a
kind gift of Dr Evan Sadler (Washington University School of Medicine,
St Louis, MO). PCI was isolated from fresh frozen plasma. The
concentration of plasma PCI was determined by active-site titration and
immunoassay as described.16 Protein C was isolated and
activated as described by Hackeng et al.21 Recombinant
human tissue factor (Innovin) was obtained from Baxter (Unterschleissheim, Germany). Bovine serum albumin (fraction V) was
purchased from Sigma (St Louis, MO). The chromogenic substrate S2366
was purchased from Chromogenix (Mölndal, Sweden). Purified human
thrombin was a generous gift of Dr Walter Kisiel (University of New
Mexico, Albuquerque, NM).
Normal, control, PCI-deficient, and protein C-deficient plasma.
Normal plasma samples were obtained from a donor pool of 40 healthy
volunteers. Blood was taken from the antecubital vein, collected into
citrate, centrifuged twice for 15 minutes at 2,500g, and stored
at  70°C until use. For the preparation of protein C and
protein C inhibitor-deficient plasmas, a plasma pool of four healthy
donors was prepared as above and subsequently passed over a Sepharose
column to which monoclonal antibodies against either protein C (4D1) or
PCI (API-39) were immobilized. Control plasma was obtained by passing
of plasma over a Sepharose column to which bovine serum albumin had
been coupled. The undiluted flow through of these columns was collected
separately and used as deficient plasmas. PCI-deficient plasma was
tested using an enzyme-linked immunosorbent assay (ELISA) and was found
to contain less than 0.5% of PCI. Protein C-deficient plasma was
tested with a protein C ELISA and contained less than 1% of protein C. In all assays used, the control plasma was fully comparable to normal plasma, demonstrating that passage of plasma over a Sepharose column
had not affected the functional quality of the plasma.
Protease inhibition by PCI was measured using a discontinuous assay
method as described by Rezaie et al20 with minor
modifications. APC (0.5 nmol/L final concentration [f.c.]) or
thrombin (0.5 nmol/L f.c.) were incubated with at least a 10-fold
excess of PCI at 37°C in the presence or absence of TM (50 nmol/L).
Using the same reaction conditions, the inhibition of thrombin by PCI
was also monitored in the presence of 100 nmol/L TM4-6. After
incubation for a period of time varying between 15 seconds to 60 minutes, Spectrozyme TH (0.25 mmol/L f.c.) or S2366 (1.0 mmol/L f.c)
was added for monitoring the thrombin or APC activity, respectively. Color development was followed at 405 nm using a Spectramax 340 kinetic
microplate reader (Molecular Devices, Menlo Park, CA). All experiments
were performed in HEPES buffer (25 mmol/L HEPES, pH 7.4, 137 mmol/L
NaCl, 3.5 mmol/L KCl, 3 mmol/L CaCl2, 0.1% bovine serum
albumin). Rate constants were calculated using the equation: k2=(-ln
a)/t[I], where a represents residual protease activity,
t is time, and [I] is the PCI concentration.
In all experiments the substrate utilization was less than 10% and
control assays showed that thrombin, TM, and PCI were stable during the experiments.
Turbidimetry was used to monitor the TM-mediated thrombin inhibition by
PCI. The change in turbidity during fibrin formation was monitored at
405 nm in a microplate reader.22-24 An increase in
turbidity indicated gel assembly. All experiments were performed in
citrated plasma recalcified with CaCl2 (final concentration of 17 mmol/L) resulting in a free Ca2+ concentration of 2.3 mmol/L. A mixture of recombinant tissue factor (Innovin, final dilution
3 × 104) and calcium necessary for recalcification
was added to 67.5 µL of plasma to initiate clotting. The volume was
adjusted to 125 µL with HEPES buffer, resulting in a final plasma
concentration of 54%. After mixing, 100 µL of the reaction mixture
was transferred to a microplate and turbidity at 405 nm was monitored
at 37°C using a Spectramax 340 kinetic microplate reader.
| |
RESULTS |
TM-mediated inhibition of APC and thrombin by PCI.
Time courses of inhibition of APC and thrombin by PCI in the absence
and presence of an excess of TM in a system with purified components
are shown in Fig 1. In
agreement with the observations of Rezaie et al,20 we found
an inhibition of TM-bound thrombin by PCI. For both proteases identical
ratios of protease:inhibitor:thrombomodulin were used. In the absence
of TM, thrombin was more rapidly inactivated by PCI compared with APC.
Even a 100-fold molar excess of PCI resulted in a minor inhibition of
APC (Fig 1A), whereas residual thrombin activity was decreased to 55%
under the same conditions (Fig 1B). Addition of rabbit lung TM resulted
in modest inhibition of APC by PCI, whereas a strong enhancement of the
inactivation rate of thrombin by PCI was observed. The addition of the
TM fragment TM4-6 also accelerated thrombin inhibition by PCI (Fig 1C),
but to a lesser extent as was observed for rabbit-lung TM.
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| Fig 1.
Inhibition of APC and thrombin by PCI in the absence and
presence of TM. APC 0.5 nmol/L (A) or thrombin 0.5 nmol/L (B and C)
were incubated with 10 ( , ), 25 ( , ), or 50 ( , ) nmol/L PCI, respectively, in the absence (open symbols) or presence (closed symbols) of TM (50 nmol/L rabbit lung [A and B] and 100 nmol/L TM4-6
[C]). At indicated time points, the chromogenic substrates S2366 (1 mmol/L f.c.) or Spectrozyme TH (0.25 mmol/L f.c.) were added to monitor
the remaining activity of APC and thrombin, respectively. Please note
the difference in x-axis scales in A, B, and C.
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A 50% decrease of thrombin activity was observed within 30 seconds
when a 50-fold molar excess of PCI was added in the presence of rabbit
lung TM or TM4-6, whereas a 15% decrease of the APC activity was
observed after 30 minutes under the same conditions. The second order
rate constants for the APC and thrombin inhibition by PCI depicted in
Table 1 show that rabbit lung TM stimulated the APC
inhibition by PCI twofold, whereas a 33-fold enhancement was achieved
for the inhibition of thrombin by PCI. When TM4-6 was used, a 24-fold
stimulation of the thrombin inhibition by PCI was observed.
In the absence of TM, the inhibition of thrombin by PCI was 18 times
more effective compared with the APC-PCI reaction. The addition of TM
enhanced this difference in rate inactivation constants of APC-PCI and
thrombin-PCI to a factor of 267, favoring the TM-mediated inhibition of
thrombin by PCI.
The effect of TM on tissue factor-induced coagulation.
We first studied the effect of TM on tissue factor-induced coagulation
in normal plasma to measure its scavenging effect on thrombin and
protein C activation. Normal plasma was incubated for 15 minutes with
8.3, 16.7, or 25 nmol/L TM. After recalcification, tissue factor was
added and the rate of fibrin formation was followed by measuring the
increase in turbidity at 405 nm. Addition of TM to normal plasma
resulted in a concentration-dependent delay in coagulation
(Fig 2A), which may be due to thrombin
scavenging and/or activation of the protein C anticoagulant
pathway.
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| Fig 2.
Effect of TM on tissue factor-induced coagulation. Normal
plasma (A) and protein C-deficient plasma (B) were incubated
with 8.3 (), 16.7 (), or 25 nmol/L ( ) TM or with buffer ().
Clotting was initiated by adding recombinant tissue factor
(Innovin, diluted by a factor 3 × 104) and calcium
required for recalcification, and the formation of fibrin was measured
in time as the change in turbidity at 405 nm.
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To monitor the thrombin scavenging effect of TM alone, we repeated the
experiment with protein C-deficient plasma. A similar, but less
dramatic pattern, was observed suggesting that the anticoagulant effect
of TM is indeed partly caused by thrombin scavenging and partly by
protein C activation (Fig 2B). Reconstitution of protein C-deficient
plasma had only a minor effect on coagulation in the absence of TM,
whereas in the presence of TM, a concentration-dependent anticoagulant
effect of protein C was observed (Fig 3).
These results indicated that TM was needed for activation of the
protein C anticoagulant pathway during tissue factor-induced
coagulation and excluded the possibility that the presence of traces of
APC in our preparations were responsible for the observed effects.
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| Fig 3.
Reconstitution of protein C-deficient plasma in the
absence or presence of TM. Plasma deficient in protein C was incubated with buffer (,) or 40 (,), 80 (,) or 160 ( ,)
nmol/L protein C in the absence (A, open symbols) or presence (B,
closed symbols) of TM (25 nmol/L). Fibrin formation was monitored as
the change in turbidity at 405 nm after addition of recombinant tissue
factor (Innovin, diluted by a factor 3 × 104) and calcium
required for recalcification. Please note the difference in x-axis
scales in (A and B).
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Although the maximal turbidity signal was affected by the addition of
TM in a concentration-dependent manner, this effect was the same in
normal and deficient plasmas and did, therefore, not exclude comparison
of turbidity measurements between the different plasmas when the same
concentration of TM was used.
The role of PCI in tissue factor-induced fibrin formation in plasma.
The role of PCI in tissue factor-induced coagulation was studied using
turbidimetry. In this assay, PCI-deficient plasma was reconstituted
with 80 or 160 nmol/L PCI and compared with normal plasma. The results
shown in Fig 4 demonstrate that PCI has a concentration-dependent anticoagulant effect on tissue factor-induced coagulation. PCI-deficient plasma displayed a slightly shorter coagulation time that could be restored to normal by the addition of a
plasma concentration of PCI. To rule out any effects due to complex
formation between APC and PCI, the experiments were repeated with
protein C-deficient plasma. Addition of 40, 80, and 160 nmol/L PCI to
protein C-deficient plasma resulted in a minor delay in coagulation
identical to the delay observed when normal plasma was used (data not
shown), indicating that PCI acts as an inhibitor of coagulation under
these conditions.
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| Fig 4.
Effect of protein C inhibitor on tissue factor-induced
coagulation. Normal plasma (closed symbols) and PCI-deficient plasma (open symbols) were incubated with buffer (,), 80 (), or 160 () nmol/L of protein C inhibitor. After incubation, coagulation was
started by the addition of recombinant tissue factor (Innovin, diluted
by a factor 3 × 104) and calcium. The formation of fibrin
was measured in time as the change in turbidity at 405 nm.
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The role of PCI in tissue factor-induced fibrin formation in plasma
in the presence of TM.
Fibrin formation was monitored after addition of tissue factor to
recalcified plasma, which was preincubated for 15 minutes with 10 nmol/L TM. In contrast to the observed anticoagulant effect of PCI in
the absence of TM, the addition of 80 or 160 nmol/L PCI to normal
plasma resulted in a procoagulant effect in the presence of TM
(Fig 5A). To study the possibility that the
procoagulant PCI effect was caused by a TM-stimulated inhibition of APC
by PCI, the experiment was repeated using protein
C-deficient plasma. The effect of PCI was monitored by enriching
protein C-deficient plasma with 80 and 160 nmol/L PCI, followed by
monitoring the tissue factor-induced fibrin formation (Fig 5B).
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| Fig 5.
Effect of protein C inhibitor on tissue factor-induced
coagulation in the presence of TM. Normal plasma (A) and protein
C-deficient plasma (B) were incubated with buffer (), 80 (), or
160 () nmol/L protein C inhibitor in the presence of TM (10 nmol/L). After incubation, recombinant tissue factor and calcium were added, and
the change in turbidity was monitored at 405 nm.
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The addition of PCI to protein C-deficient plasma in the presence of
TM resulted in a PCI-dependent delay in fibrin formation, making PCI an
inhibitor of coagulation under these conditions, in contrast to the
procoagulant PCI effect in the presence of TM observed in normal plasma
(Fig 5A). This indicated that PCI plays a major role in the inhibition
of the protein C pathway by either preventing the activation of protein
C or by inhibiting APC.
Comparison of the TM-mediated PCI reactivity between normal plasma
and PCI-deficient plasma.
The effect of PCI was also monitored using PCI-deficient plasma, which
was directly compared with normal plasma, both in the absence or
presence of rabbit lung TM (Fig 6A) or TM4-6 (Fig 6B). In the absence of TM, fibrin formation occurred earlier in
PCI-deficient plasma compared with normal plasma, as was already
observed (Fig 4). However, in the presence of 10 or 25 nmol/L TM,
PCI-deficient plasma displayed a delay in coagulation compared with
normal plasma. The same phenomenon was observed when normal plasma was
compared with PCI-deficient plasma in the presence of TM4-6. Figure 6B shows that addition of the TM fragment resulted in a
concentration-dependent delay in coagulation. Compared with rabbit lung
TM, higher concentrations of the recombinant TM4-6 fragment (50 and 100 nmol/L) were necessary to enable comparison between TM and TM4-6.
Figure 6A and B show that the overall effect of PCI in tissue
factor-induced coagulation is procoagulant in the presence of TM or
TM4-6.
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| Fig 6.
The effect of TM on tissue factor-induced coagulation of
normal plasma and PCI-deficient plasma. Normal plasma (open symbols) and PCI-deficient plasma (closed symbols) were incubated with buffer
(,) or 10 (,) or 25 (,) nmol/L rabbit lung TM (A) or
50 ( , ) or 100 (,) nmol/L TM4-6 (B). Clotting was initiated by adding recombinant tissue factor and calcium, and the formation of
fibrin was measured in time as the change in turbidity at 405 nm.
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| |
DISCUSSION |
In this report, we studied the inhibition of thrombin and APC by PCI in
the absence and presence of TM in a purified system and in a plasma
system. The interaction between PCI and TM was studied to search for an
explanation for the dual role of PCI in plasma, in which it displays
both procoagulant and anticoagulant properties. Previous studies
indicated that binding of thrombin to TM induces a change in the
conformation, which prevents thrombin to clot fibrinogen and to
activate platelets,18,25,26 but which results in a
stimulation of protein C activation. Rezaie et al20
suggested that this TM-induced change in thrombin conformation stimulated the inhibition of thrombin by PCI.
Our data show that thrombin, both in the absence and presence of TM, is
a much better target for PCI than APC. In the absence of TM, the rate
inactivation constant for thrombin by PCI was 18 times more favorable
compared with the APC-PCI reaction using identical stoichiometry
between protease and PCI. In the presence of rabbit lung TM, we
observed a modest (twofold) stimulation of the APC-PCI reaction
compared with a 33-fold enhancement of the thrombin inhibition by PCI.
When the human TM fragment TM4-6 was used, a 24-fold stimulation was
observed for the inhibition of thrombin by PCI. Comparison of the
second order rate constants of the thrombin inhibition by PCI in the
presence of rabbit lung TM or TM4-6 suggests that the inhibition
reaction is merely dependent on conformational changes in thrombin when
bound to TM.
Earlier studies indicated stimulating effects varying between 2- and
140-fold20,27 for the inhibition of TM-bound thrombin by
PCI. Our data indicated that the net result of the rate inactivation constant for the TM-mediated thrombin-PCI reaction was 267 times more
favorable compared with the TM-dependent APC-PCI reaction. Because
thrombin, especially in the presence of TM, was a much better target
for PCI than APC, we studied the role of PCI on tissue factor-induced
coagulation in plasma in the presence and absence of TM. In the absence
of TM, PCI acted as a modest inhibitor of tissue factor-induced
coagulation. Comparison of the plasma concentration of PCI to the
concentration of antithrombin (80 nmol/L and 2.3 µmol/L,
respectively), another thrombin inhibitor with similar affinity for
thrombin in the absence of a glycosaminoglycan, confirmed the minor
role of PCI in coagulation in the absence of TM. In the presence of TM,
however, addition of PCI exhibited a strong procoagulant effect on
tissue factor-induced coagulation, provided protein C was present. This
suggests that PCI either inhibits the activation of protein C by
TM-bound thrombin or that PCI inhibits APC, although the latter
mechanism is unlikely because of the unfavorable kinetics. In both
scenarios, PCI inhibits the anticoagulant activity of the protein C
pathway. When generation of APC cannot take place such as in normal
plasma in the absence of TM or vice versa in the presence of TM, but in
protein C-deficient plasma, PCI exhibits an anticoagulant effect
presumably due to the inhibition of thrombin. The overall impact of PCI
is procoagulant because the impact of the inhibition of protein C
activation is stronger than the modest direct inhibitory effect of
thrombin.
The similarity between the reactive site regions of PCI and ATIII may
be viewed as an additional indication that PCI reactivity is directed
towards the inhibition of TM-bound thrombin. The P1 residue of the
serpin primarily dictates serpin-serine protease interactions with
additional contributions of neighboring residues.28-30 Whereas the reactive site bonds for PCI and
1-antitrypsin, two major inhibitors of APC differ, both
PCI and ATIII possess an Arg-Ser reactive site
bond.28,29,31 In addition, mutagenesis studies have shown
that glycine at the P2 position as in ATIII, is preferred, but that
phenylalanine at the P2 of PCI is tolerated for thrombin
inhibition.28,29 On the other hand, a P2 glycine is not
well tolerated for APC inhibition in contrast to the hydrophobic phenylalanine at P2 of PCI.28,29 This may explain why PCI
can inhibit both APC and thrombin, two enzymes with apparent opposite functions. Whereas ATIII is mainly responsible for the inactivation of
free thrombin, the role of PCI as thrombin inhibitor is directed to the
inactivation of TM-bound thrombin. The ability of TM4-6 to enhance the
inhibition of thrombin by PCI shows that the inhibition reaction is not
primarily dictated by the chondroitin sulfate. This is an important
distinction between the reactivities of PCI and ATIII towards thrombin,
as it has been demonstrated that the inhibition of TM-bound thrombin by
ATIII is dictated by this glycosaminoglycan.32 The
inhibitory role for PCI on APC and TM-bound thrombin reflects the
importance of PCI as regulator of the anticoagulant protein C pathway.
In conclusion, a major function of PCI in plasma during coagulation is
the inhibition of TM-bound thrombin. The modest anticoagulant behavior
of PCI as a thrombin inhibitor is counteracted by the downregulation of
protein C activation resulting in a procoagulant effect.
| |
FOOTNOTES |
Submitted July 21, 1997;
accepted October 13, 1997.
Supported in part by Grant No. 92.306 from The Netherlands Heart
Foundation, and a fellowship from the Royal Netherlands Academy for
Arts and Sciences. J.C.M.M. is an Established Investigator of The Netherlands Heart Foundation.
Address reprint requests to Marc G.L.M. Elisen, PhD,
Department of Haematology (G03.647), University Hospital
Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr A.R. Rezaie for helpful suggestions, Dr J.E. Sadler for the
gift of TM4-6, Dr W. Kisiel for the gift of human thrombin, and the
personnel of the Red Cross Blood Bank for providing us the donor
plasma.
| |
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Abstract
Introduction
Abstract