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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3800-3807
Tissue-Specific Expression of Functional Platelet Factor XI Is
Independent of Plasma Factor XI Expression
By
Chang-jun Hu ,
Frank A. Baglia,
David C.B. Mills,
Barbara A. Konkle, and
Peter N. Walsh
From the Departments of Biochemistry and Medicine, The Sol Sherry
Thrombosis Research Center, Temple University School of Medicine,
Philadelphia, PA; and the Cardeza Foundation, Thomas Jefferson
University, Philadelphia, PA.
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ABSTRACT |
Platelet factor XI is an alternatively spliced product of the factor
XI gene expressed specifically within megakaryocytes and platelets as
an approximately 1.9-kb mRNA transcript (compared with ~2.1 kb in
liver cells) lacking exon V. Flow cytometry with an affinity-purified
factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on
activated platelets), and with S12 antibody (to P-selectin, an
 -granule membrane protein expressed on the platelet surface during
secretion) on platelets activated with ADP, thrombin, thrombin receptor
peptide (SFLLRN amide), or collagen at various concentrations exposed
platelet factor XI and PAC1 antibody binding in parallel. Unactivated
platelets expressed approximately 40% of total platelet factor XI but
no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI
is independent of -granule secretion, because ADP and collagen
exposed platelet factor XI but no S12 binding sites. Platelets from
four patients with plasma factor XI deficiency (<0.04 U/mL) had
normal constitutive and activation-dependent expression of platelet
factor XI. Well-washed platelets from normal and from factor
XI-deficient donors incubated with low concentrations of thrombin (0.05 to 0.1 U/mL) corrected the clotting defect observed with factor
XI-deficient plasma. Thus, functionally active platelet factor XI is
differentially expressed on platelet membranes in a tissue-specific
manner both constitutively and in a concentration-dependent fashion by
various agonists in the absence of detectable plasma factor XI.
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INTRODUCTION |
COAGULATION FACTOR XI is a glycoprotein
(GP) present in plasma as a zymogen and is required for normal
hemostasis.1,2 Factor XI circulates in plasma noncovalently
bound to high molecular weight kininogen.1 When a
surface-associated complex of factor XI, high molecular weight
kininogen, and factor XIIa is formed, it results in the formation of
factor XIa.2 Human platelets participate in the interaction
of intrinsic coagulation proteins, including factor XII, high molecular
weight kininogen, and factor XI.3-5 However, in addition to
providing a surface that promotes coagulation, platelets also contain a
number of coagulation proteins, including fibrinogen, factor V, high
molecular weight kininogen, and factor XI.6
Previous studies have demonstrated that factor XI is present in
platelets3,4,7-10 in a form that is structurally different from plasma factor XI and that is referred to as platelet factor XI.
Platelet factor XI migrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight (Mr)
of 220 kD (55 kD after reduction),7-9 compared with plasma factor XI, which has a Mr of 160 kD and a subunit with an Mr of 80 kD.
Factor XI coagulant activity and antigen in well-washed platelet
suspensions constitutes about 0.5% of the factor XI activity in normal
plasma.4,7-9,11,12 Assuming a specific activity similar to
that of plasma factor XI, this represents approximately 300 molecules
of platelet factor XI per platelet. The physiologic role and importance
of platelet factor XI is unknown, but the fact that factor XI activity
is associated with the platelet plasma membrane7 suggests
the possibility that the protein can participate in blood coagulation.
Moreover, the observation that a few patients with severe plasma factor
XI deficiency but no evidence of hemostatic abnormality have normal
amounts of platelet factor XI suggests that platelet factor XI may
substitute for plasma factor XI in hemostasis.7-10 This
suggestion is supported by reports of several hemostatically abnormal
patients with no detectable factor XI either in plasma or
platelets.4,13 Confirmation of the existence and
biochemical nature of platelet factor XI has come from recent studies
in our laboratory (Hsu et al, manuscript submitted) that demonstrate that platelet factor XI is an alternatively spliced form of
the factor XI gene, lacking exon V and expressed specifically within
megakaryocytes.14
To determine the subcellular localization of expressed platelet factor
XI and to analyze the mechanisms of exposure of platelet factor XI in
response to a variety of agonists, we have developed a flow cytometric
assay using an affinity-purified antihuman plasma factor XI antibody.
For these studies, we have used two additional antibodies, PAC1 and
S12. PAC1 is a monoclonal IgM antibody that binds only to the activated
form of the GPIIb/IIIa complex and recognizes an epitope on the
GPIIb/IIIa complex critical for fibrinogen binding.15 The
S12 antibody recognizes a 140-kD -granule membrane protein16 P-selectin17 (previously known as
GMP-140 or PADGEM) that becomes associated with the platelet surface
during secretion. Our studies indicate that platelet factor XI is
expressed constitutively on unactivated platelets from both normal
donors and donors with severe plasma factor XI deficiency and that the
exposure of platelet factor XI is increased by various platelet
agonists in a concentration-dependent manner.
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MATERIALS AND METHODS |
Materials.
Fluorescein-5-isothiocyanate (FITC), Sephadex G-25 (PD10) gel
filtration resin, CNBr-activated Sepharose 4B, p-nitrophenylphosphate, alkaline-phosphatase-conjugated rabbit antigoat IgG, apyrase, prostaglandin E1, and bovine thrombin were purchased from
Sigma Chemical Co (St Louis, MO). S12 antibody was purchased from
Centocor Inc (Malvern, PA). FITC-labeled PAC1 antibody was purchased
from the Cell Center of the University of Pennsylvania (Philadelphia, PA). Plasma factor XI was purchased from Enzyme Research Laboratories (South Bend, IN). Nitro-blue tetrazolium chloride and
5-bromo-4-chloro-3 -indolylphosphate p-toluidine salt
(NBT/BCIT) phosphatase substrate were purchased from Kirkegaard & Perry
Laboratories, Inc (Gaithersburg, MD). D-Phe-Pro-Arg chloromethyl ketone
(PPACK) was purchased from Calbiochem-Novabiochem Corp (La Jolla, CA).
Collagen (type 1) was purchased from Chrono-Log Corp (Havertown, PA).
FITC-conjugated F(ab)2 fragment rabbit antigoat IgG
antibody was purchased from Accurate Chemical & Scientific Corp
(Westbury, NY). Thrombin receptor peptide (TRP; SFLLRN amide) was
synthesized using 9-fluorenylmethyloxycarbonyl (FMOC) chemistry on a
Applied Biosystems 430A Synthesizer (Applied Biosystems, Foster City,
CA) and reverse-phase high-performance liquid
chromatography (HPLC) purified to greater than 99.9%
homogeneity.
Fluorescence labeling of S12 antibody.
The labeling procedure was modified according to the method of
Dachary-Prigent et al.16 In brief, the purified S12
antibody was dialyzed overnight at 4°C against 20 mmol/L borate
buffer, pH 9.5, containing 150 mmol/L NaCl. S12 was incubated at room temperature for 4.5 hours with FITC at a 15 to 1 molar ratio of FITC to
antibody. FITC-labeled S12 was separated from unbound FITC by gel
filtration on a Sephadex G-25 gel filtration column.
Affinity purification of goat antihuman plasma factor XI antibody.
Antihuman plasma factor XI antibody was prepared by Hugh Hoogendoorn
and Alan R. Giles (Queen's University, Kingston, Ontario, Canada) in a goat injected with highly purified human plasma factor XI
prepared in our laboratory as previously described.18 The antibody was partially purified by caprylic acid precipitation and
ammonium sulfate precipitation. The antibody was then affinity-purified using a column of human factor XI coupled to CNBr-activated Sepharose 4B, according to the procedure described by the manufacturer (Pharmacia LKB Biotechnology, Uppsala, Sweden).
Titration of factor XI antibody using enzyme-linked immunosorbent
assay (ELISA).
A 96-well microtiter plate (Falcon, Lincoln Park, NJ) was coated with
purified plasma factor XI in 15 mmol/L Na2CO3,
35 mmol/L NaHCO3 buffer, pH 9.6. The wells were then
incubated overnight at 4°C with 3% bovine serum
albumin-phosphate-buffered saline (BSA-PBS) to block nonspecific
binding. After incubation with various concentrations of either
preaffinity-purified antibody, affinity-purified antibody, or preimmune
goat IgG, the plate was washed with PBS containing 0.02% Tween 20. Alkaline phosphatase-conjugated rabbit antigoat IgG was placed in each
well. After incubation and three washes, substrate solution was added.
After incubation at room temperature for a suitable time period, the
reaction was stopped by the addition of 3 mol/L NaOH, and absorption at
405 nm was read on a microplate reader (Thermo Max; Molecular Devices, Menlo Park, CA).
SDS-PAGE and Western blot.
PAGE of plasma was performed according to the procedure of
Laemmli.19 The electrophoretic transfer to polyvinylidene
difluoride (PVDF; Millipore Corp, Bedford, MA) membrane was performed
using a Transphor apparatus (Model TE 50; Hoefer Scientific
Instruments, San Francisco, CA). The membrane strips were incubated
with anti-factor XI antibody, washed, incubated with alkaline
phosphatase-conjugated rabbit antigoat IgG, and developed with NBT/BCIT
substrate.
Blood collection and preparation of platelets.
Fresh blood was obtained from healthy volunteers or factor XI-deficient
patients and anticoagulated with acid-citrate-dextrose (ACD) solution
(2.5% trisodium citrate, 1.5% citric acid, 2.0% dextrose; 1 part
anticoagulant:6 parts blood). Platelet-rich plasma (PRP) was prepared
by centrifugation at 180g for 20 minutes at room temperature.
Apyrase (1 U/mL), PPACK (20 nmol/L), and prostaglandin E1 (PGE1; 1 µmol/L) were added to PRP that
was then incubated at room temperature for 15 minutes. PRP was
gel-filtered on Sepharose CL-2B (Sigma Chemical Corp) equilibrated in
HEPES-Tyrode's buffer (126 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L
MgCl2, 0.38 mmol/L NaH2PO4, 5.6 mmol/L dextrose, 15 mmol/L HEPES, pH 7.4) containing 2 mg of BSA per
milliliter. Platelets eluting in the void volume were adjusted to 2 × 108/mL in the same buffer.
Platelet activation and fixation.
Gel-filtered platelet suspensions containing 2 mmol/L CaCl2
were incubated without stirring for 3 minutes at 37°C in the
presence of one of the following agonists: thrombin (0.2 to 10 nmol/L), SFLLRN amide (0.2 to 10 µmol/L), ADP (1 to 15 µmol/L), and collagen (2.5 to 50 µg/mL). Activated platelets were then added to an equal volume of 2% formalin buffered with PBS and incubated for 30 minutes at 37°C. Fixed platelets were washed three times with PBS-1% BSA and stored in the same buffer at 4°C until assay. The concentration of fixed platelets was adjusted to 1 × 108/mL.
The measurement of platelet factor XI on platelets and the binding
of FITC-S12, FITC-PAC1 to platelets using flow cytometric assay.
Twenty-five microliters of fixed platelets was mixed with 25 µL of
affinity-purified goat antihuman factor XI antibody or preimmune goat
IgG, respectively, at a final concentration of 200 µg/mL and
incubated for 60 minutes at 4°C. After being washed twice,
platelets were incubated with FITC-conjugated F(ab)2
fragment rabbit antigoat IgG at a final concentration of 5 µg/mL for
60 minutes at 4°C. Platelets were then washed twice and suspended in 0.4 mL of 1% BSA-PBS for flow cytometry.
Ten microliters of a fixed platelet suspension containing 1 × 106 platelets was incubated at room temperature with 40 µL of FITC-S12 (20 µg/mL) or 40 µL of FITC-PAC1 (100 µg/mL) for
30 minutes and then diluted with 0.35 mL of 1% BSA-PBS for flow
cytometry.
The platelet samples were analyzed in a Coulter Epics Elite Flow
Cytometer (Coulter Corp, Miami, FL) equipped with a Coherent Innova 300 Argon laser that was calibrated daily with 2-µm calibrate beads
(Coulter Corp).
Coagulation assays.
One hundred microliters of factor XI-deficient plasma or normal plasma
was incubated with either 50 µL of 30 µmol/L phosphatidylserine and
L--dioleoyl-phosphatidylcholine (1:3 molar ratio) from bovine brain
in 20 mmol/L PBS or washed platelets (50 µL, 2 × 108 platelets/mL) and 50 µL of CaCl2 (25 mmol/L). In some assays, goat antihuman factor XI was added (14.6 µmol/L) without preincubation. Thereafter, thrombin at various
concentrations was added to initiate clot formation. In the absence of
added thrombin, the clotting times of all samples were greater than 5 minutes.
Patients and normal donors.
Four hemostatically normal donors and four patients with moderately
severe plasma factor XI deficiency (<0.04 U/mL) were studied, none of
whom had received drugs known to affect platelet function for 2 weeks
before blood donation. Clinical data were obtained and evaluations of
factor XI-deficient patients were performed by an individual (B.A.K.)
without knowledge of the results of the studies. The studies were
performed without knowledge of the clinical data or evaluations. The
factor XI-deficient patients were females of Ashkenazi Jewish
extraction; were 41, 77, 37, and 59 years of age; had plasma factor XI
levels of 0.02, <0.01, 0.02, and 0.03 U/mL; and had plasma partial
thromboplastin times (normal, 25 to 35 seconds) of 68.2, 91.1, 70.5, and 55 seconds, respectively. All were documented to have no evidence
of a factor XI inhibitor, and prothrombin times and other coagulation
assays and platelet counts were normal. Two of the four patients gave a
history of easy bruising and two gave a history of melena (1 on one
occasion and 1 recurrently) in the past, but none had experienced epistaxis, hematuria, menorrhagia, hematemesis, muscle hematomata, central nervous system bleeding, or hemarthrosis. Each of the four
factor XI-deficient patients had experienced surgical operations and/or traumatic injury: one had abdominal surgery with
prophylactic factor XI replacement (plasma) without excessive bleeding;
one had multiple dental extractions without prophylaxis or excessive bleeding and both a hysterectomy and surgical repair of facial trauma,
both with prophylaxis and both without excessive bleeding; one had
experienced mild excessive bleeding, in no instance requiring either
prophylactic or therapeutic factor XI or blood replacement therapy,
after a hysterectomy, after a breast biopsy and after trauma associated
with an automobile accident; and one had experienced no excessive
bleeding and no prophylactic factor XI replacement therapy after a
tonsillectomy and after a breast biopsy.
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RESULTS |
Characterization of affinity-purified goat anti-factor XI antibody.
Our initial studies were aimed at preparing, purifying, and
characterizing a monospecific polyclonal anti-factor XI antibody. An
ELISA was performed in which the titer of affinity-purified antibody
was increased greater than sixfold compared with preaffinity-purified antibody. It was also confirmed by the Western blot that the titer of
goat anti-factor XI antibody was enhanced greater than sixfold after
affinity purification (data not shown).
The specificity of affinity-purified goat antiplasma factor XI was
further determined by Western blot. Normal pooled plasma or factor
XI-deficient plasma was subjected to SDS-PAGE under either reducing or
nonreducing conditions. After electrophoretic transfer, the PVDF
membrane, probed with anti-factor XI antibody, showed a single band at
a molecular weight of approximately 160 kD (nonreduced), which migrated
at approximately 80 kD under reduced conditions (data not shown). No
visible bands were detected in lanes to which factor XI-deficient
plasma was applied. These results indicate that the affinity-purified
anti-factor XI antibody is monospecific.
Comparison of the exposure of platelet factor XI, GPIIb/IIIa, and
P-selectin on platelets.
To detect platelet factor XI on the surface of normal platelets,
unactivated platelets and platelets activated by various concentrations
of thrombin, thrombin receptor peptide, ADP, or collagen were incubated
with affinity-purified goat anti-factor XI antibody or with preimmune
goat IgG as a control. After incubation with
FITC-F(ab)2 fragment rabbit antigoat IgG antibody,
flow cytometry was performed. Figure 1
shows representative fluorescence histograms of affinity-purified
anti-factor XI antibody binding to unactivated platelets (Fig 1A) and
to platelets activated by 10 µmol/L thrombin receptor peptide (Fig
1B) and of preimmune goat IgG nonspecifically bound to unactivated (Fig
1C) and activated platelets (Fig 1D). Shown in
Table 1 are the numerical data obtained for
analysis of this representative example. The log mean fluorescence intensity of the sample incubated with affinity-purified anti-factor XI
antibody minus that of the sample incubated with preimmune goat IgG is
the net log mean fluorescence intensity associated with anti-factor XI
antibody bound to platelet factor XI on platelets. The total platelet
factor XI exposure (100%) is defined as the net log mean fluorescence
intensity of platelets activated by 10 µmol/L thrombin receptor
peptide. As shown in Table 1, these values for one representative
normal sample are 1.64 fluorescence intensity units for activated
platelets (100%) and 0.42 fluorescence units (25.6%) for unactivated
platelets. It is clear from Fig 1A that unactivated platelets represent
a bimodal distribution of platelets with 43% of the platelets present
in the C gate (ie, positive for binding factor XI antibody) showing a
log mean fluorescence intensity of approximately 2.8 fluorescence
intensity units, whereas the remainder represent a population of
platelets that are negative for factor-XI antibody binding, displaying
a log mean fluorescence intensity of approximately 0.5 fluorescence
intensity units. A small proportion of activated platelets (<10%)
showed high levels of factor XI expression (log mean fluorescence
intensity, ~25 units) in this experiment, but this was not a
consistent finding.
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| Fig 1.
Flow cytometric assay for the detection of platelet
factor XI on the surface of normal platelets. The experiment was
performed as described in the Materials and Methods. The Y axis
displays the number of platelets at any specific fluorescence intensity noted on the X axis as a log scale. The C gate was set at the edge of
the background of platelet fluorescence intensity without adding any
primary antibody or preimmune goat IgG. A representative result is
shown, in which affinity-purified anti-factor XI antibody was bound to
unactivated platelets (A) and to platelets activated by 10 µmol/L
thrombin receptor peptide (B). Results in (C) depict preimmune goat IgG
nonspecifically bound to unactivated and (D) to activated platelets.
The mean fluorescence intensity in (A) minus that in (C) or (B) minus
(D), respectively, represent the net mean fluorescence intensity due to
anti-factor XI antibody binding to platelet factor XI.
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Unactivated platelets expressed a mean of 39.07% (1.91% SEM; n = 4;
Fig 2) of total platelet factor XI. The
exposure of platelet factor XI on platelets was increased
with increasing concentrations of thrombin, thrombin receptor peptide,
ADP, and collagen (Fig 2). Concentrations of 0.8 nmol/L (~0.1 U/mL)
thrombin, 0.8 µmol/L thrombin receptor peptide, 15 µmol/L ADP, or
50 µg/mL of collagen stimulated exposure of platelet factor XI almost
as great as that of 10 µmol/L thrombin receptor peptide. These
results indicate that platelet factor XI is expressed constitutively on
unactivated platelets and that the expression of platelet factor XI on
activated platelets is increased by various platelet agonists in a
concentration-dependent manner.
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| Fig 2.
Comparison of the exposure of platelet factor XI,
GPIIb/IIIa, and P-selectin on platelets. One hundred percent binding of anti-factor XI antibody, FITC-PAC1, or FITC-S12 to platelets was defined as the mean fluorescence intensity of platelets activated by 10 µmol/L thrombin receptor peptide (SFLLRN amide) affinity-purified factor XI antibody and incubated with PAC1 antibody as described in the
Materials and Methods. The results shown are the means (±SEM) of data
obtained with platelets from four normal donors probed for platelet
factor XI ( ), GPIIb/IIIa ( ), or P-selectin ( ) .
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To determine the subcellular localization of platelet factor XI in
platelets, two activation-dependent monoclonal antibodies, PAC1 and
S12, were used in parallel with anti-factor XI antibody to examine both
unactivated platelets and platelets activated by various agonists.
Figure 3 shows the binding of PAC1 (Fig 3A and B) and S12 (Fig 3C and D) to unactivated platelets (Fig 3A and C)
and to platelets activated by 10 µmol/L thrombin receptor peptide
(Fig 3B and D). Whereas 39% of total platelet factor XI was expressed
on unactivated platelets, no detectable PAC1 or S12 binding to
unactivated platelets was observed (Fig 2), confirming that neither the
GPIIb/IIIa epitope for PAC1 nor the P-selectin epitope for S12 is
exposed on the surface of unactivated platelets. With increasing
concentrations of thrombin, thrombin receptor peptide, ADP, and
collagen, close correlations were found between the exposure of
platelet factor XI and the expression of GPIIb/IIIa (fibrinogen binding
site) indicated by binding of PAC1 (Fig 2). In contrast very low levels
of exposure of P-selectin were observed in unstirred platelet
suspensions incubated with ADP and collagen (Fig 2C and D), in
agreement with similar observations of Shattil et al15 and
with the conclusion that "unstirred platelets stimulated with weak
agonists undergo minimal secretion yet are capable of expressing most
of their fibrinogen receptors."15,20 Under these
conditions, concentrations of ADP and collagen shown to be optimal for
the exposure of platelet factor XI caused no significant exposure of
S12 binding sites (Fig 2), thereby indicating that the enhanced
exposure of platelet factor XI is independent of -granule secretion.
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| Fig 3.
Flow cytometric assay for the detection of GPIIb/IIIa or
P-selectin in activated platelets using PAC1 and S12 antibodies. Two
FITC-labeled activation-dependent monoclonal antibodies, PAC1 (A and B)
or S12 (C and D), were incubated with unactivated platelets (A and C)
or to platelets activated by 10 µmol/L thrombin receptor peptide (B
and C), and flow cytometry was performed as described in the Materials
and Methods. The Y axis displays the number of platelets at any
specific fluorescence intensity noted on the X axis as a log scale. The
C gate was set at the edge of the background of platelet fluorescence
intensity without adding any antibody. The maximal binding of PAC1 or
S12 to platelets was defined as the mean fluorescence intensity of
platelets activated by 10 µmol/L thrombin receptor peptide and
incubated with FITC-PAC1 (80 µg/mL) or FITC-S12 (16 µg/mL). The
mean fluorescence intensity obtained in the absence of antibody was
similar to that shown in (A) and (C) and was subtracted from that
obtained in (B) and (D) to obtain the net mean fluorescence intensity
due to PAC1 binding to GPIIb/IIIa or S12 binding to P-selectin on
activated platelets.
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Platelet factor XI in platelets from factor XI-deficient donors.
Because previous studies from our laboratory have shown that the
platelets from selected patients with severe plasma factor XI
deficiency possess normal quantities of platelet factor
XI,7,8 we selected four normal donors and four patients
with severe plasma factor XI deficiency to study constitutive and
agonist-dependent exposure of platelet factor XI by flow cytometry. The
results (Fig 4) show normal constitutive
expression of platelet factor XI (~30% to 60% of total) in the
plasma factor XI-deficient subjects, compared with the normal subjects
(~30% to 50% of total) and normal responses of the patients'
platelets to high concentrations of thrombin (100 nmol/L), thrombin
receptor peptide (SFLLRN amide, 10 µmol/L), ADP (100 µmol/L), and
collagen (50 µg/mL) in the agonist-dependent exposure of platelet
factor XI. Platelets obtained from both normal and factor XI-deficient
platelets also had normal responses to all agonists in exposure of
GPIIb/IIIa and P-selectin in flow cytometry performed with PAC-1 and
S12 antibodies (data not shown).
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| Fig 4.
Effects of platelet agonists on the exposure of platelet
factor XI in normal and factor XI-deficient donors. Four normal donors and four patients with plasma factor XI deficiency were tested for the
exposure of platelet factor XI on unactivated platelets and on
platelets activated by ADP, thrombin, collagen, or TRP as described in
the Materials and Methods. The percentage of exposure of platelet
factor XI was calculated as described in the text and in the legends to
Figs 1 and 3. The total exposure (100%) of platelet factor XI was
defined as the net mean fluorescence intensity of normal platelets
activated with 10 µmol/L TRP. (A) depicts the mean percentage of
platelet factor XI exposure in four normal platelets, whereas (B)
depicts results obtained with platelets from four patients with plasma
factor XI deficiency.
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Functional assay of platelet factor XI.
To establish a functional assay for platelet factor XI, normal or
factor XI-deficient plasma was titrated with various concentrations of
thrombin in the presence of either phospholipids or platelets (Fig 5). In the presence of phospholipids
and in the absence of factor XI a prolongation of clotting time at low
concentrations of thrombin (0.05 to 0.5 U/mL) is apparent (Fig 5A)
compared with results in the presence of factor XI (Fig 5B). This
difference is reduced or eliminated at high concentrations of thrombin
due to the direct conversion of fibrinogen to fibrin by thrombin. The
conclusion that the procoagulant effect of low concentrations of
thrombin is factor XI-dependent is confirmed by the observation that
the affinity-purified factor XI antibody prolongs the clotting times of
normal plasma to levels observed with factor XI-deficient plasma (Fig
5A and B). When normal platelets (activated with the thrombin receptor
peptide) are substituted for phospholipids in factor XI-deficient
plasma (Fig 5C), the clotting times at low concentrations of thrombin
were indistinguishable from those obtained in normal plasma (Fig 5D),
indicating that the amount of platelet factor XI added with activated
platelets at a concentration similar to that in plasma (200,000 platelets/µL) is sufficient to correct the clotting defect of factor
XI-deficient plasma observed with low concentrations of thrombin. The
conclusion that this correction by platelets of the defect in factor
XI-deficient plasma (Fig 5C) is due to platelet factor XI (and not some
other coagulant activity of platelets) is confirmed by the observation
that the defect is reproduced in the presence of activated platelets by the affinity-purified anti-factor XI antibody (Fig 5C and D). Finally,
platelets obtained from the four factor XI-deficient donors were
compared with normal platelets and found to give identical results (Fig
5C and D), from which it can be concluded that the platelets of these
four patients with less than 0.04 U/mL of plasma factor XI have normal
amounts of platelet factor XI.
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| Fig 5.
Effects of platelets and factor XI in thrombin-activated
coagulation assays. Coagulation assays were performed as described in
the Materials and Methods. Either factor XI-deficient plasma (A and C)
or normal plasma (B and D) was incubated with phospholipid vesicles
(PS:PC 1:3 ratio, 10 µmol/L; A and B) or gel-filtered platelets
(200,000 platelets/µL; C and D) activated with the thrombin receptor
peptide, SFLLRN-amide (5 µmol/L), and CaCl2 (5 mmol/L). In some assays, affinity-purified goat antihuman factor XI (14.6 µmol/L) was added, followed by thrombin at various concentrations (0.05, 0.1, 0.25, 0.5, and 1.0 U/mL) to initiate clot formation. Data
shown are the means (±SEM) for four experiments, each performed in
triplicate. In the absence of added thrombin, the clotting times of all
samples were greater than 5 minutes. (A) Factor XI-deficient plasma and
phospholipid vesicles in the absence () or presence () of
anti-factor XI antibody. (B) Normal plasma and phospholipid vesicles in
the absence () or presence () of anti-factor XI antibody. (C)
Factor XI-deficient plasma and normal activated platelets () or
platelets from factor XI-deficient patients ( ) or normal platelets
in the presence of anti-factor antibody ( ). (D) Normal plasma and
normal activated platelets () or platelets from factor XI-deficient
patients () or normal platelets in the presence of anti-factor XI
antibody ().
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DISCUSSION |
Early evidence for the presence of a contact factor associated with
washed platelets21-23 was subsequently reinterpreted as evidence of platelet-associated factor XI
activity7,11-13,24-26 and antigen.8,25 Platelet
subcellular fractionation studies demonstrated that platelet factor XI
is associated with the platelet plasma membrane,7 and three
separate groups8-10 have immunoprecipitated and partially
purified a protein from platelets with factor XI activity, having an Mr
of approximately 220,000 to 240,000 (nonreduced) and Mr of
approximately 50,000 to 55,000 (reduced). Despite the consistency of
these observations, questions about the existence, biochemical nature,
and functional significance of platelet factor XI have
remained.6 The possibility that platelet factor XI may have
a role in the maintenance of normal hemostasis and may substitute for
plasma factor XI has been suggested4,6-11,25,27 on the
basis of previous reports of small numbers of selected patients with no
detectable plasma factor XI and normal hemostasis with normal amounts
of platelet factor XI7,25 and reports of hemostatically
abnormal factor XI-deficient patients whose platelets contained no
detectable factor XI activity.4,13
Questions about the existence and biochemical nature of platelet factor
XI have recently been addressed in our laboratory by the use of the
polymerase chain reaction with reverse transcriptase to amplify 12 of
13 exons coding for mature plasma factor XI (ie, exons III-IV and
VI-XV, lacking exon V) from platelet mRNA with sequences identical to
those encoding plasma factor XI.14 Northern hybridization
demonstrated the presence of a factor XI mRNA transcript of
approximately 1.9 kb in Meg-01 cells (compared with ~2.1 kb in liver
cells), and in situ amplification and hybridization confirmed the
presence of factor XI mRNA lacking exon V in platelets but not other
blood cells.28,29 Finally, we have cloned a full-length sequence of platelet factor XI from a cDNA library of a human megakaryoblastic cell line (CHRF-288-11), confirming the conclusion that platelets but not other blood cells contain an alternatively spliced product of the factor XI gene expressed in megakaryocytes that
encodes a platelet protein lacking the aminoterminal portion of the
Apple 2 domain encoded by exon V (Hsu et al, manuscript submitted).
The purpose of the present studies was to determine the mechanisms and
site of exposure of platelet factor XI and to determine whether the
expression of functional platelet factor XI can occur in the absence of
plasma factor XI. Our results demonstrate that platelet factor XI is
constitutively expressed on the surface of unactivated platelets and
that, in response to a variety of physiologically relevant platelet
agonists (thrombin, thrombin receptor peptide, ADP, and collagen),
platelet factor XI is further exposed in an agonist
concentration-dependent manner in parallel with the expression of the
platelet membrane protein, GPIIb/IIIa, but not in parallel with the
-granule protein, P-selectin. These observations are consistent with
our previous demonstration that platelet factor XI is a platelet-plasma
membrane-associated protein that is expressed in functional form on the
surface of both unactivated and activated platelets.7
To determine whether the platelet factor XI expressed on the platelet
surface is functionally active, we have developed a coagulation assay
in which normal or factor XI-deficient plasma is titrated with low
concentrations of thrombin in the presence or absence of platelets (Fig
5). The results demonstrate that activated normal platelets correct the
coagulation defect of factor XI-deficient plasma when trace
concentrations of thrombin are added to activated platelet factor XI.
The concentration of platelets added in this assay is similar to that
present in normal blood (~2 × 108/mL), from which
it can be concluded that the amount of platelet factor XI present in
normal blood is sufficient to correct the coagulation defect of factor
XI-deficient plasma observed with low concentrations of thrombin. This
is especially interesting, because it is estimated that, because
activated platelets contain approximately 2.5 U of factor XI activity
per 1011 platelets,7 the final concentration of
factor XI added with 200,000 platelets per milliliter is only 0.5% of
the factor XI present in normal plasma. Thus, only a trace of platelet
factor XI appears to be required to correct the factor XI-dependent
functional defect observed with low concentrations of thrombin (0.05 to
0.5 U/mL).
To address the question of whether the tissue (ie, platelet)-specific
expression of platelet factor XI is independent of the expression of
factor XI in plasma, we have studied the platelets of patients with
severe plasma factor XI deficiency using both flow cytometry and our
functional assay that examines the capacity of low concentration of
thrombin to activate factor XI in plasma. The results of both assays
demonstrate that both platelet factor XI antigen and activity are
present in the platelets of four unrelated patients with moderately
severe (<0.04 U/mL) plasma factor XI deficiency. We conclude from
this observation that platelet factor XI is differentially expressed on
platelet membranes in a tissue-specific manner both constitutively and
in concentration-dependent fashion by various agonists in the absence
of detectable plasma factor XI. This demonstration does not formally
address the hypothesis that the presence of platelet factor XI in
patients with plasma factor XI deficiency might explain the absence of
severe bleeding complications in some factor XI-deficient
individuals.7-10,30 A formal test of this hypothesis would
require a study in which independent assessments of clinical bleeding
histories, platelet factor XI and plasma factor XI determinations, and
molecular genetics of factor XI deficiency would be performed in
cohorts of patients with normal and abnormal hemostasis.
Our observation that the tissue (ie, platelet)-specific expression of
functional platelet factor XI is independent of plasma factor XI
expression raises interesting questions about the molecular genetics of
factor XI deficiency. A number of DNA mutations responsible for factor
XI deficiency have been identified and are especially common among
Ashkenazi Jewish families, including one of the most frequent, a
mutation (F283L) in which a TTC in exon 9, coding for Phe 283, is
mutated to CTC (Leu).31 This missence mutation is located
within the Apple 4 domain, which mediates dimer formation of the factor
XI molecule.32 The F283L mutant has been constructed by
site-directed mutagenesis and demonstrated to cause diminished secretion of factor XI that was attributed to defective intracellular dimerization of the molecule.33 It is therefore
postulated33 that the deficiency of plasma factor XI that
occurs in patients with the homozygous F283L mutation is due to failure
of intracellular dimer formation and the consequent defect of secretion
of the protein from liver cells into plasma. The mechanism of synthesis of platelet factor XI is unknown as is the role of dimerization and
secretion in the synthesis of the protein. If dimer formation and
secretion are not required for synthesis of platelet factor XI, then it
is possible that patients with severe plasma factor XI deficiency due
to the homozygous F283L mutation could have normal constitutive and
agonist-dependent exposure of platelet factor XI. Although all four of
the plasma factor XI-deficient patients we have studied are of reported
Jewish extraction, the molecular genetics have not yet been determined
in any of them.
The other major common mutation producing factor XI deficiency in Jews
is a nonsense mutation in exon 5 (E117X) that converts GAA (Glu 117) to
TAA (stop), leading to premature polypeptide termination.31
However, in platelet factor XI, exon V is spliced out.14,28,29 Therefore, patients with the E117X mutation
would be expected to lack plasma factor XI, but to produce platelet factor XI normally, because the stop codon in the spliced out exon V
would not be expressed. Therefore, it will be important to define the
molecular genetics of plasma factor XI deficiency with normal platelet
factor XI and also to determine whether patients exist with
deficiencies of both plasma factor XI and platelet factor XI, which
have been postulated to cause a more severe hemostatic deficiency state
than plasma factor XI deficiency with normal platelet factor
XI.4,6-8,11,24,26
| |
FOOTNOTES |
This paper is lovingly dedicated to the memory of Chang-jun Hu, MD,
whose untimely death on March 2, 1998 has deeply saddened his family,
friends, and colleagues.
Submitted October 6, 1997; accepted January 12, 1998.
Supported by research grants from the National Institutes of Health
(Grants No. HL55407, HL46213, and HL56153) to P.N.W.
Address reprint requests to Peter N. Walsh, MD, PhD, Sol Sherry
Thrombosis Research Center, Temple University School of Medicine, 3400 N Broad St, Philadelphia, PA 19140; e-mail:
pnw{at}astro.ocis.temple.edu.
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 |
The authors are grateful to Patricia Pileggi for her assistance in
manuscript preparation.
| |
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Abstract
Introduction
Abstract