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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Active tissue factor pathway inhibitor is expressed on the surface
of coated platelets
Susan A. Maroney,1 Sandra L. Haberichter,2 Paul Friese,3 Maureen L. Collins,1 Josephine P. Ferrel,1
George L. Dale,3 and Alan E. Mast1
1Blood
Research Institute, Blood Center of Wisconsin, Milwaukee; 2Department of Pediatrics, The Medical College of Wisconsin, Milwaukee;
of Medicine, University of Oklahoma School of Medicine, Oklahoma City
3Department
The incorporation of blood-borne forms
of tissue factor (TF) into a growing blood
clot is necessary for normal fibrin generation and stabilization of the blood clot.
Tissue factor pathway inhibitor (TFPI) is
the primary physiologic inhibitor of tissue factor and is present within platelets.
Expression of TFPI on the platelet surface
may be the optimal location for it to
abrogate blood-borne TF activity that in-
corporates within the blood clot, balancing the need for adequate hemostasis
while preventing development of occlusive thrombosis. TFPI is produced by
megakaryocytes but is not expressed on
the platelet surface. Activation of platelets with thrombin receptor activation peptide does not cause release or surface
expression of TFPI, demonstrating that
TFPI is not stored within platelet ␣ gran-
ules. TFPI is expressed on the platelet
surface following dual-agonist activation
with convulxin plus thrombin to produce
coated platelets. In association with its
expression on the surface of coated platelets TFPI is also released in microvesicles
or as a soluble protein. (Blood. 2007;109:
1931-1937)
© 2007 by The American Society of Hematology
Introduction
Vascular injury results in the exposure of collagen and tissue factor,
present within the vessel wall, to flowing blood. Platelets rapidly
adhere to the collagen within the subendothelial tissue of the
injured vessel wall where they are activated; subsequently, platelet
aggregation provides an initial barrier against blood loss. Tissue
factor within the subendothelial tissue binds to factor VIIa, leading
to the production of thrombin, which further activates platelets and
generates fibrin. Platelets exposed to dual-agonist stimulation with
collagen plus thrombin form a distinct subpopulation of platelets,
called coated platelets, that express high levels of procoagulant
proteins, including factor V, fibrinogen, fibronectin, and VWF,
providing a procoagulant surface that supports further fibrin
generation.1-3 In this manner, collagen and tissue factor work
synergistically at the site of vascular injury to produce a plateletfibrin plug that closes the wound and prevents severe hemorrhage.
In addition to its presence within the subendothelial tissues of
the vessel wall, tissue factor is also present on circulating microvesicles released from activated leukocytes or endothelial cells.
Incorporation of these tissue factor–bearing microvesicles into the
blood clot is thought to be necessary for effective stabilization
within the vasculature.4-6 The recruitment of tissue factor–bearing
microvesicles into growing thrombi is mediated by interactions
between P-selectin glycoprotein ligand-1 present on the microvesicles and P-selectin expressed on the surface of activated
platelets within the blood clot.7 It has been demonstrated that the
tissue factor–bearing microvesicles can fuse with activated
platelets.8 In addition, splicing of tissue factor pre-mRNA
within activated platelets is another potential mechanism for
expression of tissue factor on the platelet surface.9 However,
overexpression of tissue factor or inadequate regulation of its
procoagulant activity can lead to propagation of a hemostatic
plug to an occlusive thrombus.
The primary regulator of tissue factor activity is tissue factor
pathway inhibitor (TFPI). TFPI is a trivalent Kunitz-type serine
protease inhibitor that is produced primarily by endothelial cells,10
but it also is present within platelets11,12 and monocytes13 and
circulating in plasma.14 It rapidly inhibits both factor Xa and the
factor VIIa/tissue factor catalytic complex with its second and first
Kunitz domains, respectively.15 TFPI present in platelets may play
a critical role in down-regulating tissue factor activity within a
growing blood clot and preventing formation of an occlusive
thrombus. Here, we have characterized the production, surface
expression, and activity of platelet TFPI, demonstrating that TFPI
is produced by megakaryocytes but retained intracellularly in
quiescent platelets. TFPI is then expressed on the platelet surface
following dual activation with convulxin plus thrombin to produce
coated platelets.
Submitted July 25, 2006; accepted October 22, 2006. Prepublished online as
Blood First Edition Paper, November 2, 2006; DOI 10.1182/blood-200607-037283.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2007 by The American Society of Hematology
BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
Materials and methods
Reagents
Ethyl methanesulfonate, 3,4-dichloroisocoumarin (DCI), PGE1, and 1-(Ltrans-epoxysuccinylleucylamino)-4-guanidinobutane (E-64) were obtained
from Sigma Chemical (St Louis, MO). The chromogenic factor Xa
substrate (methoxycarbonyl-d-cyclohexylglycyl-glycyl-arginine p-nitroanilide acetate) and anti–mouse TFPI polyclonal antibody were from American
Diagnostica (Greenwich, CT). The monoclonal antibody against human
CD59 was from the Blood Center of Wisconsin core laboratory, and
anti–GP-IIb/IIIa was from Dr Thomas Kunicki. The anti-TFPI 2H8
1931
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BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
MARONEY et al
monoclonal antibody and the TFPI polyclonal antibodies directed against
the entire protein and the C-terminus were from Dr George Broze, Jr. The
sheep polyclonal anti–rat VWF was from Cederlane Laboratories (Ontario,
Canada). Rat anti–mouse Lamp-1, ID4B, was from BD Pharmingen (San
Diego, CA). The mouse IgG1␬ (MOPC-21), anti–rabbit IgG (whole
molecule) peroxidase conjugate, and anti–mouse phycoerythrin conjugate
were from Sigma. The recombinant human tissue factor was from Ortho
Diagnostic Systems (Toronto, Ontario, Canada). Human factor VIIa was
from Haematologic Technologies (Essex Junction, VT); human factor X
was from Enzyme Research Laboratories (South Bend, IN). Deglycosylation enzymes were from Prozyme (San Leandro, CA). Human
recombinant TPO and IL-11 and mouse recombinant IL-3 were from
PeproTech (Rocky Hill, NJ); streptavidin magnetic beads were from
Miltenyi Biotech (Auburn, CA).
Buffers
ACD was 38.1 mM citric acid, 74.8 mM sodium citrate, 136 mM glucose;
BSGC was 129 mM NaCl, 13.6 mM sodium citrate, 11.1 mM glucose, 1.6
mM KH2PO4, 8.6 mMNaH2PO4, pH 6.5. PBS was 150 mM NaCl, 10 mM
NaH2PO4, pH7.5. CHAPS lysis buffer was 30 mM CHAPS, 10 mM EDTA,
PBS. HBSA was 50 mM HEPES ⫹ 100 mM NaCl ⫹ 0.1% BSA, pH 7.4, ⫹
10 mM CaCl2.
Platelets
The use of human donors to provide whole blood for platelet isolation was
approved by the Blood Center of Wisconsin Institutional Review Board,
and all donors gave informed consent in accordance with the Declaration of
Helsinki. Human platelets were obtained from whole blood drawn from the
anticubital vein. Mouse platelets were obtained from whole blood drawn
from the inferior vena cava. Human and mouse blood were collected in
ACD and diluted 1:2 into BSGC. Platelet-rich plasma (PRP) was prepared
immediately by centrifugation at 170g for 8 minutes at 23°C. Platelets were
washed in BSCG or gel filtered by layering 2 mL PRP onto a 25 ⫻ 60 m
(30 mL) column of Sepharose CL-2B.
Activation of platelets
In flow cytometry and Western blot experiments, platelets were activated
with 8 ␮M thrombin receptor activation peptide, SFLLRN (TRAP), for 10
minutes in PBS. In activity assays, platelets were activated with 8 or 20 ␮M
TRAP containing 10 mM EDTA, 25 ␮M DCI, and 50 ␮M E-64 to prevent
adventitious proteolysis of TFPI from the platelet surface. Control experiments demonstrated that the presence of 10 mM EDTA has no effect on
surface expression of P-selectin following activation of platelets with either
TRAP or thrombin, demonstrating that ␣ granule secretion by TRAP does
not require calcium. For all experiments, coated platelets were prepared in
10 mM HEPES, pH 7.5, 1 mg/mL BSA, 2 mM CaCl2, 1 mM MgCl2,
140 mM NaCl. Platelets were stimulated with 0.5 U/mL thrombin (final)
and either 500 ng/mL convulxin or 2 ␮M A23187. After 2 minutes, 10
U/mL hirudin was added, and the incubation continued for 8 minutes at
37°C.
Flow cytometry
Flow cytometry was performed using CELLQUEST software on a Becton
Dickinson FACSCalibur. For TRAP-activated platelets, gel-filtered platelets were suspended in primary antibodies (anti–TFPI-FITC and anti–
P-selectin-PE) in PBS with 1% BSA for 30 minutes at 4°C. The platelets
were washed and analyzed. For coated platelets, gel-filtered platelets were
activated with various agonists in the presence of biotin-fibrinogen as
previously described.16 After fixation with 1% formalin for 25 minutes,
platelets were diluted in PBS with 0.1% BSA and centrifuged at 1500g for
15 minutes. Primary antibody was applied to the cells, followed by washing
and the addition of FITC anti–mouse IgG and PE-streptavidin secondary
antibody for 30 minutes at room temperature. The platelets were washed
and analyzed.
TFPI precipitation, SDS–polyacrylamide gel electrophoresis,
and Western blot analysis
Platelets were lysed in CHAPS lysis buffer. The platelet lysate was centrifuged at
16 000g for 10 minutes to remove cellular debris. TFPI was precipitated from the
ultracentrifuged supernatant and CHAPS pellet lysate using bovine factor
Xa-agarose for 1 hour at room temperature. The agarose beads were washed 3
times with PBS/0.5% Tween, washed once with PBS, and boiled for 3 minutes in
SDS sample buffer. The sample was then subjected to 10% SDS–polyacrylamide
gel electrophoresis (PAGE) and transferred to nitrocellulose for Western blot
analysis. Nitrocellulose was immunostained for TFPI with the indicated primary
antibodies and with antirabbit conjugated to horseradish peroxidase. Proteins
were visualized using chemiluminescent substrate Western blotting reagents. In
some experiments TFPI was precipitated from human plasma or the factor
Xa–precipitated platelet TFPI was subjected to deglycosylation as described
previously.17 To quantify the relative amount of platelet and plasma 45 kDa TFPI,
defined amounts of platelet lysate and plasma from the same person were
precipitated with multiple rounds of bovine factor Xa agarose to ensure
precipitation of all TFPI, subjected to SDS-PAGE, Western blot for TFPI, and
analyzed by quantitative densitometry.
Differential centrifugation of platelets
Quiescent or dual-agonist–activated platelets were subjected to 1500g for
15 minutes; the supernatant was removed and subjected to 10 000g for 15
minutes; the supernatant was removed and subjected to 200 000g for 1 hour.
The pellets from each step, as well as the supernatant from the 200 000g
supernatant, were subjected to Western blot analysis for TFPI.
Real-time PCR
These experiments were performed using leukocyte-reduced platelet concentrates obtained from the Blood Center of Wisconsin. Following gel filtration
to remove plasma, the platelets (3 ⫻ 106/mL) were incubated with biotinylated anti-IIb/IIIa for 30 minutes at room temperature. Platelets were further
purified by positive selection using streptavidin magnetic beads. Total RNA
was isolated from these highly purified, leukocyte-reduced platelets using
RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNA was produced with
Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). Real-time
polymerase chain reaction (PCR) primers were selected using PrimerQuest
(Integrated DNA Technologies, Coralville, IA). Primers specific for fulllength TFPI were 5⬘-ATTTCACGGTCCCTCATGGTGTCT-3⬘ and 5⬘GGCGGCATTTCCCAATGACTGAAT-3⬘. Primers specific for TFPI␤ were
5⬘-GAAGGAACAAATGATGGTTGGAAGAATGCG-3⬘ and 5⬘-ATGGATGCATGAATGCAGAAGGCG-3⬘. Assays were performed in triplicate and
normalized to ribosomal protein L-19 on a 7500 Real-Time PCR System
using SYBR Green (Applied Biosystems, Foster City, CA).
Factor VIIa/tissue factor activity assays
Functional platelet TFPI activity was determined via kinetic assay. The
reaction mix contained 0.2 nM human factor VIIa, 20 nM human factor X,
and a 1:10 000 dilution of recombinant tissue factor, all diluted in HBSA,
plus the platelet sample. After a 30-minute incubation period in which the
samples generate Factor Xa, the reaction was quenched with 20 mM EDTA.
Factor Xa generated was determined by monitoring the cleavage of 500 ␮M
factor Xa substrate. The amount of TFPI in the samples was determined by
comparison to a standard curve generated using known amounts of TFPI.
PIPLC treatment
Coated platelets were incubated with 1 U/mL phosphatidyl inositol–specific
phospholipase C (PIPLC; Molecular Probes, Eugene, OR) for 1 hour at
37°C. Following treatment the cells or platelets were analyzed for
surface-associated TFPI by flow cytometry.
Isolation and growth of mouse megakaryocytes
Mouse bone marrow cells were harvested from the femur and humerus. Cells
were cultured at 5 ⫻ 106/mL in DMEM media with 10% human sera supplemented with rIL-3, rIL-11, and rTPO.18 Cells were fed every other day for 7 days.
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BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
Cytospin preparations of the cultured megakaryocytes were fixed with 3.7%
buffered formalin in preparation for staining and confocal microscopy.
Confocal microscopy
Cytospin preparations of mouse megakaryocytes/platelets and human platelets
were washed with PBS and fixed with 3.7% buffered formalin. Cells were
washed with PBS, blocked with 2% goat serum in PBS, and incubated for 1 hour
in primary antibodies. Nonimmunized mouse, rabbit, sheep, or rat IgGs were
used as negative controls. Some cells were permeablized in 1% Triton–X-100 in
20 mM HEPES, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, pH 7.0.
Secondary antibodies used for immunofluorescence detection included goat
anti–rabbit, anti–mouse, anti–rat, or anti–sheep IgG conjugated with Alexa Fluor
488 or Alexa Fluor 568 (Invitrogen, Eugene, OR). Images were acquired using a
Leica TCS SP2 confocal Laser Imaging System (Leica, Heidelberg, Germany)
equipped with a Leica 100⫻/1.4 numerical aperature oil objective as previously
described.19 The imaging solution was ProLong Gold (Molecular Probes,
Eugene, OR). Images were acquired electronically and were processed using
Leica confocal software (LCS), version 2.61, Build 1537. The antibodies and
stains are as described in “Reagents” and in the figure legends.
Results
Full-length TFPI is located within human platelets
It has previously been reported that TFPI is present within
platelets.11 This observation was confirmed by Western blot
analysis of platelets following lysis in 30 mM CHAPS. Platelet
TFPI migrates as a single 45-kDa band in SDS-PAGE, a finding
that contrasts with the multiple plasma forms of TFPI (Figure 1). In
addition, platelet TFPI reacts with an antibody directed against the
C-terminal 12 amino acids of TFPI, indicating that it is not
truncated within the C-terminal region (Figure 1). Quantitative
densitometry of the 45-kDa band in standardized amounts of
plasma and platelets from a single person indicates that about 10%
of the TFPI in whole blood is within platelets and 90% in plasma
(data not shown), consistent with the findings of Novotny et al11
who reported that 3 ⫻ 108 platelets have about 7% the amount of
TFPI found in 1 mL serum.
TFPI␤ is not detected within platelets
TFPI␤ is an alternatively spliced form of TFPI that is truncated
prior to the third Kunitz domain and contains a direct GPI-anchor
attachment site within its C-terminal region.20 TFPI␤ is highly
glycosylated and migrates at the same molecular weight as
full-length TFPI on SDS-PAGE.21 Western blot analysis of platelet
PLATELET TFPI
1933
TFPI performed following O- and N-linked deglycosylation demonstrates 2 bands. One band has undergone only partial deglycosylation and migrates at the approximate molecular weight of
nondeglycosylated TFPI, whereas the second band has undergone
extensive deglycosylation and migrates at approximately 35 kDa
(Figure 1). Both of these bands react with an antibody directed
against the C-terminal region of TFPI, indicating that they represent full-length TFPI. A band migrating at 21 kDa, the expected
molecular weight of deglycosylated TFPI␤, is not observed,
indicating that full-length TFPI is the predominant form of TFPI
present within platelets.
TFPI is synthesized by mouse megakaryocytes and TFPI
mRNA is present within human platelets
The presence of full-length TFPI in platelets, but not the Cterminally truncated forms of TFPI found in plasma, suggests that
TFPI is either produced by megakaryocytes or that only the
full-length form of TFPI is efficiently adsorbed from plasma by the
platelet. Mouse TFPI was detected by immunofluoresence within
permeablized, cultured mouse megakaryocytes grown in fetal
bovine serum, demonstrating that mouse TFPI is synthesized by the
megakaryocyte (Figure 2). Nonpermeablized megakaryocytes do
not stain for TFPI, indicating that it is not expressed on the cell
surface (Figure 2). TFPI production by human megakaryocytes was
examined through isolation of mRNA from highly purified human
platelets followed by real-time PCR analysis to quantify TFPI and
TFPI␤ expression. Signal for TFPI message was readily detected
and present at approximately 46% the level of housekeeper gene,
RPL-19. In contrast, message for TFPI␤ was present at 0.082% that
of RPL-19, essentially the same as the no-template negative
control. This is quite different than expression in cultured endothelial cell lines in which TFPI␤ mRNA is present at levels 5- to
10-fold lower than TFPI mRNA.17,21 Thus, it appears that human,
as well as mouse, megakaryocytes produce TFPI, whereas TFPI␤
mRNA is not produced, consistent with our inability to detect
TFPI␤ protein within platelets by Western blot analysis (Figure 1).
TFPI is not present on the surface of quiescent platelets and is
not expressed on the platelet surface or secreted
following TRAP stimulation
Similar to mouse megakaryocytes (Figure 2), TFPI is not expressed
on the surface of quiescent human platelets when analyzed using
flow cytometry (Figure 3A). When platelets are activated with
TRAP to induce release of ␣ granule proteins, P-selectin, a
membrane-associated ␣ granule protein is consistently present on
the surface of the vast majority of platelets. In contrast, less than
1% of the activated platelets express surface TFPI (Figure 3A).
Western blot analysis of TRAP-stimulated platelets subjected to
differential centrifugation demonstrates that this agonist does not
induce release of TFPI (Figure 3B).
TFPI is not localized within ␣ granules or lysosomes
Figure 1. Western blot analysis demonstrates the presence of full-length TFPI
in platelets. TFPI from plasma or from platelets lysed in 30 mM CHAPS buffer were
precipitated with bovine factor Xa agarose and subjected to SDS-PAGE and Western
blot analysis with either an antitotal TFPI antibody or an anti-TFPI antibody
recognizing only the C-terminal 12 amino acids. Lanes 1 to 3 are stained for total
TFPI; lanes 4 to 5 are stained for the C-terminal 12 amino acids. Lane 1 indicates
platelet TFPI; lane 2, deglycosylated platelet TFPI; lane 3, plasma TFPI; lane 4,
platelet TFPI; lane 5, deglycosylated platelet TFPI.
The lack of surface expression or secretion of TFPI from TRAPactivated platelets strongly suggests that platelet TFPI is not
localized within the ␣ granules. Confocal microscopy of human
and mouse platelets and mouse megakaryocytes was performed to
identify the intracellular location of TFPI. TFPI was present in all
platelets inspected. In human platelets stained for TFPI and
fibrinogen, used as a ␣ granule marker, the 2 proteins do not
colocalize, demonstrating that TFPI is not localized within platelet
␣ granules (Figure 4A). Similar data were obtained, demonstrating
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1934
MARONEY et al
Figure 2. Fluorescence microscopy demonstrates TFPI within mouse
megakaryocytes but not on their surface. Nonpermeablized and Triton–X-100–
permeablized mouse megakaryocytes were immunostained for TFPI. Top panels are
stained cells; bottom panels are bright field images of the cells.
that TFPI does not colocalize with VWF in ␣ granules within
mouse platelets (Figure 4B) or megakaryocytes (Figure 4C). TFPI
also does not colocalize with LAMP-1 within lysosomes of mouse
megakaryocytes (Figure 4D).
BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
Figure 4. Confocal microscopy of mouse and human platelets and mouse
megakaryocytes demonstrate that TFPI does not colocalize with either ␣
granule or lysosomal protein. (A) Human platelets stained for TFPI (green) and
fibrinogen as an ␣ granule marker (red). (B) Mouse platelets stained for TFPI (green)
and VWF as an ␣ granule marker (red). (C) Mouse megakaryocytes stained for TFPI
(green) and VWF as a ␣ granule marker (red). (D) Mouse megakaryocytes stained for
TFPI (green) and LAMP1 as a lysosome marker.
TFPI is expressed on the surface of coated platelets
Dual-agonist stimulation of platelets with thrombin and convulxin,
an activator of the platelet collagen receptor GPVI, produces a
highly activated population of platelets known as coated platelets,
in reference to the high levels of procoagulant proteins that coat the
surface of these platelets.22 Coated platelets were produced as
described in “Materials and methods” and analyzed for surface
TFPI expression using flow cytometry. In contrast to TRAPstimulated platelets, coated platelets consistently express TFPI on
their surface (Figure 5A). Following activation with thrombin and
convulxin only 35% to 50% of the platelets become coated.1
Surface expression of TFPI occurs exclusively on the coatedplatelet population identified by the presence of fibrinogen on their
surface (Figure 5A). When platelets are stimulated with thrombin
and calcium ionophore greater than 95% of the platelets become
coated1 and express surface TFPI (Figure 5A). Stimulation of
platelets with a variety of other single agonists, including thrombin,
calcium ionophore, and convulxin, did not produce significantly
elevated cell-surface expression of TFPI (Figure 5B).
TFPI is released from coated platelets on microvesicles
and as a soluble protein
Figure 3. Stimulation of platelets with TRAP does not cause surface expression
or secretion of TFPI. (A) Flow cytometry analysis of platelets stained for TFPI and
P-selectin. Quiescent platelets are in the upper left panel. Platelets stimulated with
8 ␮M TRAP are in the other 3 panels. Dot blot analysis of double-stained cells
following activation indicates that 89% of the platelets have surface P-selectin,
whereas less than 1% has surface TFPI. (B) Western blot analysis of quiescent and
TRAP (8 ␮M) activated platelets subjected to differential centrifugation. Lane 1,
1500g pellet; lane 2, 10 000g pellet; lane 3, 200 000g pellet; lane 4, 200 000g
supernatant.
Quiescent and dual-agonist–activated platelets were compared via
differential centrifugation at 1500g, 10 000g, and 200 000g23
(Figure 5C). In quiescent platelets essentially all of the TFPI is
found in the 1500g pellet, consistent with its presence within intact
platelets. Experiments with dual-agonist–activated platelets produced results that varied depending on the persons donating the
platelets and the day of the experiment. TFPI is always present
within the 1500g pellet, consistent with its presence on the surface
of coated platelets. In some experiments TFPI was found in the
10 000g pellet, whereas in others most of the TFPI was within the
200 000g supernatant. TFPI in the 10 000g pellet represents that
being released in microvesicles, whereas TFPI in the 200 000g
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BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
PLATELET TFPI
1935
Figure 5. Active TFPI is expressed on the surface of coated platelets and secreted. (A) Platelets were simultaneously activated with convulxin plus thrombin (top 4 panels)
or calcium ionophore and thrombin (bottom 2 panels) to make coated platelets and analyzed for surface TFPI expression using flow cytometry. Fibrinogen staining (y-axis)
identifies the coated platelets. Staining on the x-axis is unstained cells (top left panel), MOPC (top right panel; negative control), factor V (middle and lower left panels; positive
control), and TFPI (middle and lower right panels). (B) The relative fluorescence intensity of platelets determined by flow cytometry of platelets stained for TFPI following
stimulation with different single agonists as indicated. Error bars represent the standard deviation of 3 or more experiments. (C) Western blot analysis of TFPI in quiescent and
coated platelets produced by stimulation of platelets with thrombin and calcium ionophore following differential centrifugation. Lane 1, 1500g pellet; lane 2, 10 000g pellet; lane
3, 200 000g pellet; lane 4, 200 000g supernatant. Western blots from 3 coated-platelet preparations are shown to indicate the variable amounts of TFPI found in the 10 000g
pellet and 200 000g supernatant. (D) Comparison of TFPI activity (surface and secreted) in quiescent platelets, platelets stimulated with 8 ␮M TRAP, 20 ␮M TRAP or coated
platelets produced by stimulation with thrombin and calcium ionophore. TFPI activity was measured by inhibition of factor Xa generation by factor VIIa/TF. The number of
persons tested in each category is indicated. Error bars represent the standard deviation of 3 or more experiments. (E) TFPI activity is reversed by a monoclonal antibody
directed against the first Kunitz domain of TFPI. (F) No platelets; (f) calcium ionophore plus thrombin activated platelets; (Œ) calcium ionophore plus thrombin activated
platelets with 100 ␮M anti-TFPI antibody.
supernatant represents that released either in exosomes or as a
soluble protein.23
TFPI expressed by dual-agonist–activated platelets
inhibits tissue factor
Fresh platelets were stimulated with thrombin and calcium ionophore to produce coated platelets and were compared using TFPI
activity assays to either quiescent platelets or platelets stimulated
with 8 ␮M or 20 ␮M TRAP. Total platelet TFPI activity (surface
and secreted) was measured in platelets from multiple persons in
assays measuring the rate of factor X activation by factor VIIa/
tissue factor. Comparison of quiescent and dual-agonist–activated
platelets demonstrates a significant increase in the amount of TFPI
activity in the dual-agonist–activated platelets (1.2 ⫾ 0.7 versus
3.9 ⫾ 1.3 pmole/10 million platelets; P ⫽ .006), but no significant
increase in platelets activated with 8 or 20 ␮M TRAP (Figure 5D).
In addition, stimulation of platelets with 2 ␮M A23187 did not
produce significantly increased TFPI activity when compared with
quiescent platelets (data not shown). The factor VIIa/tissue factor
inhibitory activity is reversed by addition of an anti-TFPI monoclonal antibody, demonstrating that TFPI is responsible for the
inhibitory activity observed (Figure 5E).17,24
TFPI on the surface of coated platelets is relatively
protected from removal by PIPLC
TFPI on the surface of endothelial cells21,25,26 or fresh placenta24 is
readily removed (80%-95%) by treatment with PIPLC, indicating
that TFPI associates with the endothelial surface through a
GPI-anchor. In contrast, analysis of coated platelets by flow
cytometry before and after treatment with PIPLC revealed that only
22% of the surface TFPI is removed by PIPLC under conditions
that remove 56% of the CD59 from the platelet surface (data not
shown). Thus, compared with TFPI on the endothelial cell surface,
platelet TFPI is relatively protected from removal by PIPLC.
Previously the impact of transglutaminase inhibitors on coatedplatelet formation was documented.2 For this study, we observed
that 2 mg/mL acetyl-casein2 totally inhibited TFPI retention on the
surface of coated platelets (data not shown), suggesting that the
transglutaminase activity required for coated-platelet formation is
also essential for TFPI expression. Whether this indicates direct
derivatization of TFPI or derivatization of a cofactor required for
TFPI retention is unclear at this time. However, this observation
supports the finding that PIPLC was ineffective in removing TFPI
from coated platelets because a PI-linkage would not be expected
to be influenced by acetyl-casein.
Discussion
The platelet surface may be the optimal location for TFPI-mediated
inhibition of the procoagulant activity of blood-borne forms of TF,
thereby maintaining the appropriate procoagulant and anticoagulant balance that allows for adequate hemostasis without development of occlusive thrombosis. Novotny et al11 first demonstrated
the presence of TFPI in platelets. Those investigators reported that
platelets release soluble TFPI following stimulation with either
thrombin or calcium ionophore but did not identify TFPI on the
platelet surface. Since this initial description of platelet TFPI there
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1936
MARONEY et al
have been many advances in our understanding of platelets and the
biology of TFPI. These include the identification of proteaseactivated receptors on platelets and the production of artificial
ligands for these receptors, such as TRAP, that allow platelet
activation without active proteolysis by thrombin27,28; the description of a distinct population of highly procoagulant coated platelets
that is produced following dual activation of platelets with collagen
and thrombin22; the finding that TFPI is a cell-surface–associated
protein bound indirectly to the endothelial surface through tight
association with a GPI-anchored coreceptor25,26; and the identification of TFPI␤, an alternatively spliced, truncated form of TFPI that
is directly GPI-anchored.20,21
Full-length TFPI is present within platelets, whereas the truncated forms of TFPI present in plasma are not. Thus, it appears that
either platelet TFPI is produced by megakaryocytes or full-length
TFPI is selectively adsorbed from plasma. TFPI␤ protein is not
detectable within platelets. Real-time PCR performed using mRNA
from highly purified platelets readily detected full-length TFPI
mRNA, but not TFPI␤ mRNA, indicating that full-length TFPI is
produced by megakaryocytes and is the predominant form in
platelets. The presence of TFPI in megakaryocytes was confirmed
by immunofluoresence microscopy. Platelet TFPI is not expressed
on the surface of megakaryocytes or quiescent platelets.
Stimulation of platelets with thrombin, TRAP, convulxin, or
calcium ionophore does not cause expression of TFPI on the
platelet surface. This finding is consistent with those of Steiner et
al29 who also did not observe TFPI on the surface of platelets in
whole blood stimulated with TRAP. Although platelets have
previously been reported to secrete soluble TFPI following activation with either thrombin or calcium ionophore,11 we did not
observe secretion of TFPI following TRAP stimulation. The
absence of TFPI surface expression or release following stimulation of platelets with TRAP strongly suggests that TFPI is not
stored within platelet ␣ granules. This was confirmed by confocal
microscopy studies demonstrating that TFPI does not colocalize
with the ␣ granule proteins VWF or fibrinogen. Thus, the intracellular localization of TFPI remains unclear. The lack of colocalization of TFPI with LAMP-1 suggests that TFPI is not routed to the
platelet lysosome. However, lysosomes in platelets are heterogenous in nature and may contain the membrane proteins LAMP-1,
LAMP-2, or LAMP-3, as well as acid hydrolases and cathepsins.30
TFPI could potentially be localized to a lysosome that does not
contain the membrane protein LAMP-1 or in a distinct, uncharacterized secretory vesicle. The studies presented here also do
not exclude the possibility that TFPI is localized in multivesicular bodies that may be the precursor to ␣ granules and dense
granules.31 Activated platelets have also been shown to release
exosomes, and it is possible that TFPI could be localized in an
exosome.23
Platelets activated simultaneously with collagen and thrombin
and examined by flow cytometry reveal 2 distinct populations of
cells; one population expresses high levels of several procoagulant
proteins on the cell surface, including factor V, fibrinogen, fibronectin, and VWF, whereas the other does not.1,3 Those cells retaining
high levels of procoagulant proteins and phosphatidylserine are
referred to as coated platelets because they are “coated” with these
procoagulant proteins. Formation of coated platelets also is associated with secretion of both dense and ␣ granules, transglutaminase
activation, and glycoprotein IIb/IIIa activation.22 In contrast to
stimulation of platelets with single agonists, platelets stimulated
with thrombin plus convulxin or thrombin plus calcium ionophore
to produce coated platelets consistently expressed TFPI on their
BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
surface. In platelets activated with thrombin plus convulxin, in
which 35% to 50% of the platelets become coated, surface
expression of TFPI occurred exclusively on the population of
coated platelets (Figure 5A). Once on the platelet surface, TFPI is
relatively resistant to removal by PIPLC, with only about 20%
removed following 1 hour of incubation with the phospholipase.
This is a somewhat unexpected result because TFPI expressed on
the surface of both cultured endothelial cells and fresh placenta is
highly susceptible to PIPLC with 80% to 95% removed during a
1-hour incubation.24-26 This relative protection of platelet TFPI
from PIPLC cleavage suggests either that it is not GPI-anchored, as
the inhibition of surface expression by acetyl-casein suggests, or
that it is GPI-anchored but in a different lipid microenvironment
than endothelial TFPI. It is also possible that there are structural
differences between the GPI-anchor of platelet and endothelial
TFPI. For example, in some proteins the inositol ring of the
GPI-anchor is modified by palmitoylation or acylation, rendering it
resistant to PIPLC.32,33
Platelet TFPI is transferred to the platelet surface and released
following dual-agonist activation with thrombin plus collagen
where it effectively inhibits tissue factor activity in factor Xa
generation assays. This finding is consistent with that of other
investigators who have observed TFPI activity in platelets that is
reversed by anti-TFPI antibodies.34,35 Because TFPI is released
from dual-agonist–activated platelets and dual-agonist–activated
platelets release high amounts of microvesicles,36 differential
centrifugation experiments23 were performed to characterize TFPI
released from dual-agonist–activated platelets. These experiments
produced variable results that were dependent on the platelet donor
and on the day the experiment was performed. However, they
suggest that TFPI is released in microvesicles and exosomes or as a
soluble protein. The expression of TFPI on the surface of dualagonist–activated platelets suggests that platelet TFPI may have a
physiologic role in the inhibition of tissue factor activity at the
edges of a vascular lesion where platelets are exposed to both
collagen and thrombin. Further, in vivo experimentation will be
necessary to confirm this hypothesis and to explore the physiologic
roles for platelet TFPI in inhibition of the circulating forms of
tissue factor that incorporate into a growing thrombus.
Platelet TFPI is not located within ␣ granules, and the mechanism for its transfer to the platelet surface on activation is distinctly
different from that of platelet ␣ granule proteins such as P-selectin.
Identification of the biochemical properties involved in its transfer
from an intracellular location to the platelet surface may represent a
new mechanism for how proteins can be expressed on the platelet
surface. It has been demonstrated that simultaneous activation of
platelets with thrombin plus collagen produces a sustained increase
in platelet calcium influx in subpopulations of platelets that also
have exposed surface phosphatidylserine,37 suggesting that the
expression of TFPI on the platelet surface may result from changes
in platelet membranes and/or cytoskeleton that occur only following sustained elevation of intracellular calcium that result from
activation of both collagen and thrombin-signaling pathways.
Acknowledgments
This work was supported by the National Institutes of Health/
National Heart, Lung, and Blood Institute (Bethesda, MD) (grant
HL68835, A.E.M.) and the American Heart Association Scientist
Development Grant (0435466N, S.L.H.). A.E.M. is an Established
Investigator of the American Heart Association.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
PLATELET TFPI
Authorship
Contribution: S.A.M. designed, performed, supervised, and interpreted the real-time PCR, Western blot, confocal microscopy, and
flow cytometry studies and wrote the paper; S.L.H. performed the
confocal microscopy studies; P.F. performed the confocal microscopy and flow cytometry studies; M.L.C. performed the flow
cytometry and Western blot studies; J.P.F. performed the deglycosy-
1937
lation and Western blot studies; G.L.D. designed the experiment,
interpreted the results, and performed the flow cytometry experiments; and A.E.M. supervised the overall project, designed the
experiment, interpreted the results, performed the TFPI activity
assays, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Alan E. Mast, Blood Research Institute, PO Box
2178, Milwaukee, WI 53201-2178; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2007 109: 1931-1937
doi:10.1182/blood-2006-07-037283 originally published
online November 2, 2006
Active tissue factor pathway inhibitor is expressed on the surface of
coated platelets
Susan A. Maroney, Sandra L. Haberichter, Paul Friese, Maureen L. Collins, Josephine P. Ferrel,
George L. Dale and Alan E. Mast
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