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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3397-3404
Factor XI Messenger RNA in Human Platelets
By
Danko Martincic,
Vladimir Kravtsov, and
David Gailani
From the Departments of Pediatrics, Pathology, and Medicine,
Vanderbilt University, Nashville, TN.
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ABSTRACT |
The bleeding diathesis associated with congenital deficiency of
factor XI (FXI) is variable and correlates poorly with standard coagulation assays. Platelets are reported to contain FXI activity that
may substitute for the plasma protein. The presence of this platelet
activity in some patients deficient in plasma FXI could partly explain
the variable bleeding associated with the deficiency state. Polyclonal
antibodies to plasma FXI recognize a 220 kD platelet membrane protein
distinct in structure from plasma FXI. The messenger RNA (mRNA) coding
for this protein has been postulated to be an alternatively spliced FXI
message lacking the fifth exon found in the liver (wild type) message.
We analyzed RNA from platelets, leukocytes, and bone marrow for FXI
mRNA by reverse transcription polymerase chain reaction (RT-PCR)
technology. Single FXI mRNA species were identified by RT-PCR in
platelet and bone marrow RNA, but not leukocyte RNA, that are the same
size as the message from liver RNA. Sequencing of PCR products
confirmed that the FXI mRNA species in platelets is identical to the
one in liver. Wild-type FXI mRNA was also identified in three leukemia
cell lines with megakaryocyte features (MEG-01, HEL 92.1.7, and
CHRF-288-11). The data show that platelets contain wild-type FXI mRNA.
FXI protein, therefore, may be present in platelets and may be released
during platelet activation. The data do not support the premise that the 220 kD platelet protein that cross-reacts with FXI antibodies is a
product of an alternatively spliced mRNA from the FXI gene.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
FACTOR XI (FXI) IS THE zymogen of a
plasma serine protease produced primarily in the liver that contributes
to blood coagulation through activation of factor IX by limited
proteolysis.1-4 This activity is most important during
major hemostatic challenges such as trauma or surgery.5,6
Unlike bleeding associated with classic hemophilia (deficiency of
factor VIII or factor IX), hemorrhage in FXI deficiency is highly
variable and correlates poorly with standard assays of blood
coagulation such as the activated partial thromboplastin time
(aPTT).7,8 Indeed, there are individuals with extremely low
plasma levels of FXI (< 1% of normal) who do not experience bleeding
despite trauma or surgery.9,10 It has been suggested that
coinheritance of other coagulation abnormalities such as von Willebrand
disease exacerbates hemorrhage in FXI deficiency,11
although this premise has been challenged.12 Conceivably,
multiple factors contribute to the wide range of bleeding symptoms
observed in these patients.
It has been postulated that a mild phenotype in some patients with
severe FXI deficiency may be due to the presence of FXI-like activity
associated with platelets, which may bypass the requirement for plasma
FXI in certain situations.13 Platelets may contain small
amounts of FXI activity (<1% of plasma activity)14 and three groups have reported on the partial purification of a 220 kD
polypeptide from platelet extracts that is recognized by anti-FXI polyclonal antibodies.14-16 This protein appears to have a
substantially different structure than plasma FXI (a 160 kD dimeric
molecule consisting of two identical 80 kD
polypeptides).17,18 Based on polymerase chain reaction
(PCR) studies of platelet RNA, it has been proposed that the 220 kD
platelet protein is a tetramer of a 50 to 55 kD polypeptide that is the
product of an alternatively spliced FXI messenger RNA (mRNA) lacking
the fifth exon normally found in the full-length message from
liver.19,20 Consistent with this is a recent report of a
truncated FXI complementary DNA (cDNA) lacking the fifth exon, isolated
from a library for the human megakaryoblastic leukemia cell line
CHRF-288-11.20 To determine if FXI mRNA or related novel
messages are present in blood tissues, we used reverse transcription
PCR (RT-PCR) technology to analyze RNA from human platelets,
leukocytes, and bone marrow, as well as human leukemia cell lines with
megakaryocytic features, for the presence of FXI mRNA. The results show
that normal platelets and bone marrow, as well as established
megakaryoctye cell lines, contain FXI mRNA that is identical to the
mRNA found in liver, but failed to identify novel splice products of
the FXI gene.
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MATERIALS AND METHODS |
Materials and Reagents
Molecular biology.
The human liver FXI cDNA was a gift from Dr Dominic Chung (University
of Washington, Seattle, WA). TaKaRa long range DNA polymerase for PCR
was from Pan Vera Corp (Madison, WI). A Thermo-sequenase radiolabeled
terminator sequencing system for DNA sequencing was from Amersham Life
Sciences (Arlington Heights, IL). Guanidinium isothiocyanate extraction
buffer was from Promega Corporation (Madison, WI). A Ready-To-Go
T-Primed First-strand Kit for RT of RNA was from Pharmacia Biotech
(Piscataway, NJ). Omniscript RT for PCR with or without RT (Qiagen,
Valencia, CA). Oligonucleotide primers were prepared by the DNA core
facility of the Vanderbilt University Cancer Center. SeaKem LE agarose
and NuSieve agarose were from FMC Bioproducts (Rockland, ME).
Cell lines and tissue culture.
The MEG-01 human megakaryoblastic cell line (ATCC
CRL-2021)21 and the human erythroleukemia cells line HEL
92.1.7 (ATCC TIB 175)22 were from the American Tissue Type
Collection (Rockville, MD). The CHRF-288-11 human megakaryoblastic cell
line23 was a gift from Dr Michael Lieberman (University of
Cincinnati, Cincinnati, OH). The 293 human fetal kidney fibroblast cell
line transfected with a full-length FXI cDNA derived from liver was
described previously.24,25 RPMI 1640 medium was from
Mediatech (Herndon, VA), and Dulbecco's modified Eagle medium (DMEM)
and Fischer's media were from Life Technologies (Grand Island, NY).
Purification of platelets and leukocytes.
The procedure for obtaining human blood specimens was approved by the
Institutional Review Board of Vanderbilt University. Platelets were
isolated either from peripheral blood or expired platelet-pheresis
products from the Vanderbilt Hospital blood bank by differential
centrifugation. Briefly, blood (120 mL) was obtained from the
antecubital vein of healthy normal individuals into a 1/10th volume of
3.8% sodium citrate anticoagulant. Anticoagulated blood or the
platelet-pheresis product was placed in 50 mL polypropylene centrifuge
tubes and erythrocytes and leukocytes were pelleted by centrifugation
at 300g for 10 minutes in a Sorval RC5 Superspeed centrifuge fitted with an SS-34 rotor (Sorval, Inc,
Wilmington, DE). The top half of the platelet rich supernatant was
gently aspirated and placed into fresh centrifuge tubes using
polypropylene pipettes, and centrifugation was repeated as above. The
top half of the platelet-rich plasma was aspirated from the tube and
used as the source of platelets for RNA purification. The platelet count was determined using a Technicon H-3 hematology analyzer (Bayer-Miles, Tarrytown, NY). A typical platelet preparation from fresh
blood contains 5 to 10 × 109 platelets and from a
pheresis product 1 to 5 × 1011 platelets. Samples of
purified platelets were stained with Wright's stain and examined under
a microscope. Rare leukocytes or erythrocytes (<1 per 10,000 platelets) were detected in some platelet preparations, whereas others
had no detectable cells. Platelets were pelleted at 3,500g for
20 minutes, dissolved in guanidinium thiocyanate extraction buffer (5 to 10 × 109 platelets/mL of guanidinium
thiocyanate), and stored at 4°C pending RNA isolation.
Leukocytes were purified as follows: Twenty mL of peripheral blood
collected into a 1/10th volume of 3.8% sodium citrate was diluted 1:1
with RPMI 1640 medium. Eight mL of the diluted blood was layered onto 4 mL of Ficoll Hypaque (Lymphoprep Ficoll Hypaque, Nycomed Pharma AS,
Oslo) in a 15 mL tube followed by centrifugation at 800g for 30 minutes in a DPR-6000 centrifuge (Damon/International Equipment Co
Division, Needham Heights, MA). The pellet (erythrocytes and
granulocytes) and the intermediate layer halfway down the Ficoll
Hypaque (monocytes and lymphocytes) were carefully collected and
subjected to a second purification on Ficoll Hypaque as above. The
erythrocyte/granulocyte fraction (1 mL in volume) was brought up to 15 mL with 150 mmol/L NH4Cl, 10 mmol/L KHCO3 to
lyse red cells, then centrifuged at 1,000g for 5 minutes in a
Beckman TJ-6 bench-top centrifuge to pellet the granulocytes. The
monocyte/lymphocyte fraction underwent centrifugation in a similar
manner. Microscopic evaluation of the granulocyte and mononuclear cell
preparations showed contamination with some platelets (<1 per 10 leukocytes) with the contamination being somewhat greater for the
mononuclear cell preparations than the granulocytes. The majority of
platelets do not sediment through Ficoll Hypaque and remain in the
upper layer. Leukocytes were dissolved in guanidinium thiocyanate
extraction buffer (1 × 106 cells/0.2 mL extraction
buffer). Normal human bone marrow, 5 mL, was obtained from a donor
harvest for bone marrow transplantation. The cells were pelleted by
centrifugation at 3,000g for 10 minutes and the pellet was
dissolved in 2 mL of guanidinium thiocyanate extraction buffer. All
cell lysates were stored at 4°C pending RNA purification.
Cultures of MEG-01, HEL 92.1.7, and CHRF-288-11 megakaryocytic
leukemia cells and 293 cells transfected with a human liver FXI cDNA.
Cell lines were maintained in a 37°C fully humidified incubator
with a 5% CO2 atmosphere. MEG-01 was grown in RPMI 1640 containing 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L
L-glutamine, 1 mmol/L sodium pyruvate, 1.5 gm/L sodium bicarbonate, 10 mmol/L HEPES, and penicillin/streptomycin/amphotericin B. HEL 92.1.7 cells were grown in RPMI 1640 containing 10% heat-inactivated FCS, 2 mmol/L L-glutamine, and penicillin/streptomycin. CHRF-288-11 was grown
in Fischer's medium supplemented with 10% heat-inactivated FCS and
penicillin/streptomycin. Cells were grown to a concentration of
approximately 1 × 107/20 mL media, then washed four
times with phosphate-buffered saline, pelleted by centrifugation at
1,000g for 10 minutes, and dissolved in 2 mL of guanidinium
thiocyanate extraction buffer. Cells that were adherent to the culture
flasks were released by incubation with trypsin/EDTA and then treated
as above. Extracts were stored at 4°C pending RNA purification. 293 cells transfected with a wild-type FXI liver cDNA24,25 were
grown in DMEM 5% FCS, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate,
and penicillin/streptomycin/amphotericin B. These cells, which served
as a positive control for the RT/PCR process, were processed into
guanidinium thiocyanate extraction buffer as above.
Isolation of RNA and RT.
Total RNA from platelets and cell lines was isolated using standard
guanidinum thiocyantate/phenol:chloroform extraction and precipitated
with 70% ethanol.26 Leukocyte, bone marrow, and cell line
poly-A RNA were isolated from the guanidinium isothyocyante extractions
using a PolyA Tract System 1000 (Promega) according to the
manufacturer's instructions. PolyA RNA was eluted from the system
using nuclease-free water. A Ready-To-Go T-Primed first-strand RT kit
(Pharmacia) was used for RT reactions. Briefly, 5 µg of total RNA
(platelets) or 100 to 1,000 ng of poly A RNA (leukocytes, bone marrow,
or cell lines) was mixed with oligo-dT and random hexanucleotides as
recommended by the manufacturer. Samples were heated at 65°C for 5 minutes, placed on ice for 2 minutes, and then added to tubes
containing the Ready-To-Go beads. After 1 minute of incubation at room
temperature, samples were mixed, transferred to a 37°C water bath,
and incubated for 2 hours. To show that signals obtained from PCR
reactions were RT dependent and, therefore, not due to contamination of
RNA preparations with genomic or cDNA, RNA underwent RT in the presence
or absence of RT as follows: 2 µg of RNA was added to reaction
mixtures containing 1× buffer, 0.5 mmol/L of each
deoxyribonucleoside (dNTPs), 0.5 µmol/L of random hexanucleotides,
and 10 units of RNase inhibitor, and incubated at 37°C for 2 hours
either in the presence or absence of 4 units of Omniscript RT.
Reactions were then cooled on ice and 8 to 10 µL was used as template
in a 50 µL PCR reaction.
PCR amplification of RT mRNA and cDNA libraries.
Oligonucleotide primer pairs for PCR (Table
1) were designed using OSP software
27 and published sequences for human
FXI,18,28 glycoprotein IIb (GPIIb),29 and
CD-18.30 Three sets of primers were prepared for FXI to
amplify sequences represented by exons 3 through 6 of the FXI heavy
chain, exons 10-14 from the carboxy-terminus of the heavy chain and
catalytic light chain, and exons 3-15 covering the entire sequence
encoding the mature protein (Fig 1). An
additional pair of primers amplifying parts of exon 10 and 11 were
prepared (Table 1) for use in identifying a common polymorphism in exon 1131 known to be present in one of the platelet donors (see below). PCR reactions for GPIIb, CD-18, and exons 3-6, 10-14, and 10-11 of FXI were amplified in reactions containing 1× TaKaRa reaction
buffer, 1.5 mmol/L magnesium chloride, 100 µmol/L of each dNTPs, 10 pmol of each primer, 2.5 units of TaKaRa LA Taq polymerase, and 3.0 µL of RT mRNA in a total volume of 50.0 µL. PCR was performed on a
Perkin Elmer model 460 DNA Thermal Cycler (Foster City, CA). The
following cycling parameters were used: one cycle of denaturation at
94°C for 3 minutes, annealing at 60°C for 40 seconds and
extension at 70°C for 1 minute, followed by 38 cycles of
denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. Long-range PCR to
amplify the 1.6 kb long fragment covering FXI exons 3-15 was performed
in a total volume of 50 µL containing 1× TaKaRa reaction
buffer, 1.5 mmol/L magnesium chloride, 400 µmol/L of each
deoxyribonucleoside (dNTP), 10 pmol of each primer, 2.5 units of TaKaRa
LA Taq polymerase, and 3.0 µL of RT mRNA. PCR was performed on a
PTC-100 Thermal Cycler (MJ Research) using the following cycling
conditions: an initial cycle of denaturation at 94°C for 3 minutes
followed by 38 cycles of denaturation at 94°C for 40 seconds and
annealing/extension at 66°C for 2 minutes. Small PCR fragments were
size fractionated by electrophoresis on 2% NuSieve agarose gels.
Long-range PCR fragments were separated on 1% SeaKem LE agarose gels.
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Table 1.
Oligonucleotide Primers Used for Polymerase Chain
Reaction Amplification of Reverse-Transcribed RNA. Primer Sequences are
5'-3', Left-to-Right
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| Fig 1.
Strategy for PCR amplification of human FXI mRNA. The FXI
cDNA is encoded by 15 exons. Three sets of oligonucleotide primers
(Table 1) were designed to amplify a 400 bp fragment from exons 3 to 6 of the heavy chain (primers 1 and 2), a 552 bp fragment from exons 10 to 14 of the C-terminus of the heavy chain and the catalytic light
chain (primers 3 and 4), and a 1,673 bp fragment from exons 3 to 15 covering the coding region for the mature plasma protein (primers 5 and
6). A1 to A4 designate the four apple domains of the heavy chain; E,
exon.
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DNA sequencing.
The PCR fragments representing FXI exons 3-6 from normal platelets,
bone marrow, 293 control cells, MEG-01, HEL, and CHRF-288-11 RNA were
subjected to dideoxy-chain termination sequencing using a Thermo
Sequenase radiolabeled terminator cycle sequencing kit according to the manufacturers recommendations. Sequencing of the exon
10-11 product from the platelet RNA of an individual known to carry a
polymorphism in exon 11 was performed in a similar manner. Fragments
were sequenced in both directions using the oligonucleotide primers
originally used to produce the PCR fragments. Reaction products were
run on standard 7% polyacrylamide gels followed by autoradiography
overnight. Sequences were compared with the published sequence for the
human FXI cDNA.18
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RESULTS |
PCR analysis for FXI mRNA in platelets and bone marrow.
The human FXI gene contains 15 exons. Exon 1 is not translated, whereas
exon 2 encodes a signal peptide that is not present on the mature
molecule.28 Exons 3-10 encode the FXI heavy chain and exons
11-15 encode the catalytic light chain.28 Three sets of PCR
primers were used to amplify portions of the FXI message from RT RNA
(Fig 1). The expected PCR products generated from the primer pairs,
based on the published cDNA sequence for FXI18 are: 400 bp
for exons 3-6, 552 bp for exons 10-14, and 1,673 bp for exons 3-15. PCR
products of the expected sizes were generated when RT RNA from the 293 cell line transfected with a liver FXI cDNA (control cells) was used as
template (Fig 2A, lane 1). Human pancreas
has previously been showed by Northern blot analysis to express FXI
mRNA.25 PCR products similar to those from the control cell
line were obtained when a human pancreas cDNA library was used as
template for all three sets of PCR primers (data not shown). When the
PCR template was RT RNA from normal human platelets, single products
were obtained that were identical in size to those from reactions with
the 293 control cells (Fig 2A). Note, in Fig 2A the control sample in
lane one for the exon 3-15 PCR product appears very heavy. This is due
to overexposure of the film to show the PCR products from platelets,
which are faint. The exon 3-15 PCR product represents a nearly
full-length FXI message. As platelet RNA appears to be partially
degraded on ethidium-stained agarose gels (data not shown), it is
likely that there are very few full-length FXI messages in platelet
RNA, accounting for the weakness of this PCR product. Peripheral blood
platelets are anucleate cell fragments lacking the machinery for RNA
synthesis (except perhaps for mitochondrial RNA). RNA in platelets,
therefore, must be produced by megakaryocytes. We identified FXI
message by RT-PCR in RNA from normal human bone marrow (Fig
3, lane 1). Whereas the cell of origin of
the message is not determined by this experiment, the results are
consistent with a megakaryocytic origin for the FXI message identified
in platelets. The positive result with bone marrow RNA could have been
due to platelets within the bone marrow sample. However, the bone
marrow RNA was prepared from a small sample (5 mL), and in our
experience there is insufficient platelet RNA in this volume to
generate a positive signal by RT-PCR. The DNA sequences of the exon 3-6 PCR fragments from platelets, bone marrow, pancreas, and the 293 control cells were identical to the published sequence for the human
FXI cDNA.18
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| Fig 2.
PCR amplification of FXI mRNA from platelets and
megakaryocytic cell lines. PCR was performed as described in the
Materials and Methods section. (A) RT RNA extracted from human
platelets was used as template to amplify exons 3-6 (top), exons 10-14 (middle), and exons 3-15 (bottom) of the FXI message. Lanes 1, RNA from
293 cells transfected with the human wild-type FXI cDNA (positive
control); 2, RNA extracted from fresh platelets; 3 and 4, RNA extracted
from platelets obtained by platelet-pheresis; and 5, no template
(negative control). (B) RT poly-A RNA from megakaryocytic cell lines
was used as template to amplify exons 3-6 of the FXI message. Lanes 1, HEL 92.1.7 cells; 2, MEG-01 cells; 3, CHRF-288-11 cells; and 4, no
template (negative control). The positions of molecular weight markers
(MWM) in kilobases are shown at the left of the figure.
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| Fig 3.
PCR amplification of FXI exons 3-6, GPIIb, and CD-18
using RT RNA from human platelets, bone marrow, and leukocytes. PCR was
performed as described in the Materials and Methods section. Lanes 1, bone marrow; 2, 293 cells transfected with wild-type FXI cDNA; 3, fresh
platelets; 4, platelets from platelet-pheresis; 5, granulocytes; 6, mononuclear leukocytes. MWM indicates a 100 bp DNA ladder that serves
as molecular weight markers. The intense marker band is 500 bp in
size.
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A recent report describes a truncated FXI message lacking exon 5 in the
megakaryocytic leukemia cell line CHRF-288-11.20 The
absence of this exon in platelet or bone marrow mRNA should have
resulted in PCR products of 241 bp for the exon 3-6 primer pair and
1,514 bp for the exon 3-15 primers. We did not observe either of these
smaller species in any fresh platelet preparation (three separate
donors, Figs 2A and 3 and data not shown) or in platelet-pheresis
products from our blood bank (three separate donors, Figs 2A and 3).
PCR analysis for FXI mRNA in leukemia cell lines with
megakaryoblastic features.
Cell lines MEG-01,21 HEL 92.1.7,22 and
CHRF-288-1123 are derived from tissues of patients with
acute nonlymphocytic leukemia. These cell lines have been shown to
possess features and express proteins typically found in normal
megakaryocytes. An FXI cDNA lacking exon 5 and a small part of the
3'-portion of exon 4 has been isolated from a cDNA library
prepared from CHRF-288-11 RNA, and the MEG-01 line is reported to
express an FXI mRNA of 1.9 kb (the message in liver is 2.1 kb)
detectable by Northern blot analysis.20 RNA was isolated
from cells and analyzed by RT-PCR with the exon 3-6 primer pair
specific for the FXI heavy chain (Fig 2B). PCR products identical to
the one from the control cell line were obtained with RNA from all
three cell lines. No product that could represent a truncated FXI mRNA was identified in the cell lines. DNA sequences of the exon 3-6 PCR
products for all cell lines were identical to the published sequence
for the human FXI cDNA from liver.18
PCR analysis of RNA from peripheral blood leukocytes.
A major concern when isolating RNA from peripheral blood platelets is
contamination of the platelet preparation with small numbers of
leukocytes that contain relatively large amounts of RNA compared to
platelets. Although the platelet isolation technique used in this study
produced several platelet preparations with no apparent leukocyte
contamination by visual microscopic inspection, some leukocyte
contribution to all platelet RNA preparations could not be ruled out.
To address this potential problem, RNA was isolated and reverse
transcribed from granulocytes and from mononuclear leukocytes (a
combination of lymphocytes and monocytes) and along with platelet RNA
was analyzed by PCR for leukocyte- and platelet-specific markers, in
addition to FXI. The results of a representative experiment are shown
in Fig 3. All platelet preparations and bone marrow were positive for
GPIIb. As suspected, one of six platelet preparations gave a positive
signal for CD-18, a component of the leukocyte marker
LFA-1,30 indicating white cell contamination of the
platelet preparation (data not shown). Both granulocytes and
mononuclear cells were consistently positive for CD18. In addition,
some preparations of mononuclear cells were positive for GPIIb, which
is typically only found on platelets and megakaryocytes.32
This is consistent with the platelet contamination noted in the
leukocyte preparation. Most importantly, granulocytes and mononuclear
cells did not give a positive signal for FXI. It is our impression that
platelets contain very small amounts of FXI mRNA, and the low level of
platelet contamination of the mononuclear cells would not provide
sufficient platelet RNA to detect the FXI message. Although low levels
of leukocyte expression of FXI mRNA can not be ruled out by this analysis, the results show that platelets are the major source of FXI
mRNA in peripheral blood.
Control experiments for potential contamination of RNA preparations
with DNA.
An obvious concern when using PCR to detect mRNA is the generation of
a false-positive signal due to contamination with genomic or cDNA.
Analysis by a technique that does not involve amplification steps, such
as Northern blot or RNAse protection analysis can overcome this
limitation. However, we were unable to show FXI message by these
techniques in any of the RNA preparations in this study except for the
transfected 293 cells that overexpress the message (data not shown).
The amount of FXI message, therefore, is below the level of detection
for these types of assays. In our experiments, PCR primer pairs were
selected to amplify fragments with more than one exon, eliminating the
possibility of genomic DNA being responsible for the signals. Control
PCR reactions in which the template was RNA that had not been reverse
transcribed were performed for all RNA preparations (Fig
4). These reactions did not generate PCR
products indicating that RNA preparations were not contaminated with
factor XI cDNA, and that PCR products in reactions using RT RNA as
template were dependent on the RT reaction. This shows that the
template for the PCR signals is, indeed, mRNA.
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| Fig 4.
Control reactions for RT dependence. Reactions for RT of
RNA from human platelets or human megakaryocytic cell lines were
performed as described in the Materials and Methods section either in
the absence (-) or presence (+) of RT. The reaction products were
then subject to PCR amplification using the oligonucleotide primer pair
specific for exons 3-6 of human factor XI. PCR products were obtained
only for reactions in which RT was present. Lanes 1, platelet RNA; 2, HEL 92.1.7 cells; 3, MEG-01 cells; 4, CHRF-288-11 cells; 5, 293 cells
transfected with wild-type FXI cDNA. The position of the molecular
weight marker is shown at the left of the figure.
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Finally, several polymorphisms that do not result in amino acid changes
have been reported in the coding sequence for the human FXI
gene.31 One of these polymorphisms, a T to C change at base
pair 1234 within exon 11, was known to be present in one copy of the
FXI gene from one of our platelet donors. A PCR primer pair that
amplifies portions of exons 10 and 11 (Table 1) was used to generate a
216 bp fragment from the platelet RNA of this donor, and the base pair
sequence of the PCR product was determined (Fig
5). The sequence shows both a T and a C
residue at base pair 1234, consistent with the known heterozygous state
for this person. This result could not have been created by genomic DNA
contamination of the sample, as the PCR product involves parts of two
exons separated by several kilobases of intron sequence in the
gene.28 The pattern is highly unlikely to be caused by cDNA
contamination because two separate sequences are present in the PCR
product and no cDNAs containing the T to C polymorphism have been
created in our laboratory. These data also confirm that platelet RNA is the template responsible for the PCR products in our experiments.
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| Fig 5.
Nucleotide sequence of an RT-PCR amplified portion of FXI
exon 11 from platelet RNA. RT-PCR and DNA sequencing were performed as
described in the Materials and Methods section using platelet RNA from
an individual known to be heterozygous for a T to C polymorphism within
amino acid codon 379 of FXI (nucleotide position 1234 of the human cDNA
sequence18) and a person homozygous for the wild-type (T)
allele. The arrow indicates the position of the polymorphism. Note the
signal for both a T and C residue at this position for the
heterozygous individual.
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DISCUSSION |
The bleeding diathesis associated with FXI deficiency has been
perplexing since the first description of FXI-deficient patients in
1953.33 Congenital deficiency of FXI is a relatively rare disorder with a particularly high incidence in people of Ashkenazi Jewish descent.34 Excessive bleeding is typically triggered by trauma or surgical procedures, however, hemorrhage does not correlate well with plasma FXI levels determined by contact activation initiated clotting assays.7,8 In a seminal study, Asakai et
al5 presented an explanation for some of the variation when they noted that hemorrhage depends not only on the FXI level in plasma,
but also on the tissue involved. Bleeding from the oral cavity and
urinary tract are particularly common, likely due to the high levels of
fibrinolytic activity in these tissues. Nevertheless, there are
severely deficient patients who do not bleed with surgical procedures,
suggesting an alternative procoagulant activity may be present in some
individuals that substitutes for plasma FXI. It has been proposed that
the platelets of some people contain an FXI-like activity that may
modify bleeding in the absence of plasma FXI.14,15
Platelet  -granules, contain numerous proteins involved in hemostasis
and fibrinolysis that are also present in plasma.35 The
source of granule proteins appears to vary. The vWF 36 and the A chain of factor XIII37 are apparently of
megakaryocyte origin, as megakaryocytes express mRNA for these
proteins. In contrast, megakaryocytes do not express mRNA for albumin
or the and  chains of fibrinogen, indicating these proteins are
taken up into granules from the plasma.38 Our results show
FXI mRNA in human platelets and bone marrow, as well as three cell
lines with megakaryocyte features. Furthermore, platelets are the major source of FXI mRNA in peripheral blood, because leukocyte RNA does not
contain FXI message. The amount of FXI message in platelets and the
leukemia cell lines appears to be small. Nevertheless, the data suggest
FXI similar to the protein produced by the liver may be present in
platelets, and could be released at sites of platelet activation to
increase local concentrations of this clotting factor at a wound site.
It should be noted that it is difficult to postulate a role for a
platelet pool of FXI in the variable bleeding observed in FXI
deficiency, because this protein would presumably be affected by the
same mutation that produced the plasma deficiency state.
The presence of FXI mRNA in platelets does not provide definitive proof
that FXI protein is present. The issue of whether or not platelets
contain FXI protein, and the form this protein takes, is a
controversial one. Evidence for platelet FXI comes from three major
lines of investigation: (1) assays of FXI activity, (2) antibody-based
immunologic studies of FXI antigen, and (3) molecular biology
investigations of FXI mRNA. Well-washed platelets contain small amounts
of activity that shorten the abnormally long clotting time of
FXI-deficient plasma in an aPTT assay.13,39,40 The nature
of this activity is difficult to determine by this type of assay,
however, as complex platelet extracts contain factors that interact
with the clotting cascade at many levels. Indeed, Schiffman et
al41,42 and Osterud et al43 made this point when they examined platelet extracts for FXI activity and concluded no
appreciable activity was present.
Using immunoprecipitation techniques with polyclonal antihuman FXI
antibodies, three groups have reported on the partial purification of a
220 kD protein from platelets by immunoprecipitation.14-16 The protein appears to be on the platelet surface, because it copurifies with the membrane fraction of sonicated
platelets.13 The migration of this protein through
SDS-polyacrylamide gels is different from plasma FXI, a 160 kD
homodimer comprised of identical disulfide-bond-linked 80 kD
polypeptides.17 Under reducing conditions the platelet
protein migrates as a single 50 to 55 kD protein implying that the 220 kD protein is a polymer of a smaller subunit. The protein shares some
features with plasma FXI, in addition to cross-reactivity with
antibodies. Like plasma FXI, the platelet protein is cleaved by factor
XIIa, and the cleaved form appears to cleave the synthetic substrate
S-2366, indicating it is a protease.15 In contrast, unlike
plasma FXI the platelet protein is not activated by trypsin, its
proposed catalytic domain is substantially larger than that of factor
XIa, and it has not been shown that it has FXIa activity (ie, activates
factor IX). Although this protein may eventually be shown to possess
FXI activity, it is difficult to invoke it at present as an explanation
for variable bleeding in FXI deficiency because it is not known if some
people have the protein while others lack it.
Using flow cytometry, Hu et al44 detected binding of
anti-FXI antibodies to platelets from normal and FXI-deficient
patients, a finding supported by earlier studies.14 These
authors propose that the binding site for the antibody is a FXI-like
molecule encoded by a modified message from the FXI gene that may
circumvent mutations responsible for plasma FXI deficiency. The
findings are consistent with this hypothesis, however, other
possibilities must be considered. The simplest explanation is that the
antibody is cross-reacting with a protein that may have homology to
FXI, but is not a product of the FXI gene. In this circumstance it would not be expected to be affected by a mutation causing deficiency of plasma FXI. Alternatively, the antibodies could bind to normal FXI
in normal platelets, and to mutant FXI molecules in platelets from
FXI-deficient patients. A common point mutation associated with FXI
deficiency in Ashkenazi Jews is a phenylalanine to leucine substitution
at amino acid 283.5 The mutation interferes with normal
dimer formation of FXI, resulting in reduced secretion of the protein
into plasma.45 Factor XI associated with platelets may be
stored in granules and not be secreted, however, and abnormal proteins
could be retained within the platelets. Taken as a whole, the data
obtained with FXI antibodies indicate that platelets contain a protein
that is a protease and that has some homology to plasma FXI. The range
of activities of this protein and its role in hemostasis, if any,
remain to be determined.
Using PCR techniques, Hsu et al have recently provided evidence for an
alternatively spliced mRNA from the FXI gene,20 and postulate that it codes for the 50 to 55 kD subunit of the platelet protein identified by FXI antibodies. With platelet RNA as template, each exon of the FXI gene could be amplified individually by RT/PCR except for exon 5, which encodes a portion of the apple two domain of
the FXI heavy chain. This group also isolated a novel FXI cDNA lacking
exon 5 and the 3'-terminal bp of exon 4 from a leukemia cell line
cDNA library.20 This cell line, CHFR-288-11, shows megakaryocytic features including expression of platelet factor 4, vWF,
and glycoprotein IIb/IIIa, as well as induction of multinucleation and
hyperploidy upon stimulation with phorbol ester.23 We were interested to determine if the novel FXI message identified in the cDNA
library of this cell line, is present in normal human blood tissues and
in freshly prepared RNA from megakaryocyte cell lines.
We did not find evidence for truncated FXI mRNA in normal platelets or
in the three megakaryoblast cell lines tested, including CHRF-288-11.
All PCR products representing the FXI heavy chain contain normal
sequence for exon 5. The results with RT-PCR cannot exclude the
presence of low levels of alternatively spliced message, however, the
data indicate the predominant message for FXI in platelets and cell
lines is identical to the message produced in liver. Generation of FXI
mRNA lacking exon 5 that would maintain an open reading frame would
require an unusual mRNA splicing event that would ignore nearly perfect
consensus donor splice junctions at the 3'-end of exons 4 and
5.28 Our studies failed to identify this species in normal
blood cells. It is possible that some individuals may have a
polymorphism in the FXI gene that could allow this splicing event to
occur, and that we did not identify it in the platelets of the six
individuals we tested. However, this possibility would not explain the
discrepancy between our data with CHRF-288-11 mRNA and the previously
published results of the novel alternatively spliced FXI message from a
library prepared from this same cell line.20
In summary, our results show FXI mRNA in platelets, suggesting
platelets may contain FXI of megakaryocytic origin that is similar to
plasma FXI produced in the liver. Although platelets appear to contain
a membrane protein that cross-reacts with FXI antibodies, our data do
not support the premise that this protein is a product of an
alternatively spliced message from the FXI gene. This is supported by
the failure of mutations in the FXI gene causing plasma FXI deficiency
to influence the presence of the platelet membrane
protein.44
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr Jerry Ware (Scripps Inst, LaJolla, CA)
for providing cDNA libraries and information regarding HEL and
CHRF-288-11 cells, and to Dr Michael Lieberman (University of
Cincinnati, Cincinnati, OH) for providing the CHRF-288-11 cell line. We
thank Dr Mortimer Poncz (University of Pennsylvania, Philadelphia, PA)
for recommendations concerning leukocyte and platelet specific markers,
and Dr George J. Broze, Jr (Washington University, St. Louis, MO) for
his thoughtful reading of the manuscript. We also wish to thank Mr
Mao-Fu Sun for expert technical assistance.
| |
FOOTNOTES |
Submitted October 22, 1998; accepted July 12, 1999.
Supported by National Institutes of Health Grants HL02917 and HL58837.
D.G. is an Established Investigator of the American Heart Association.
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.
Address correspondence to David Gailani, MD, Division of Hematology,
Vanderbilt University, 538 MRB II, 2220 Pierce Ave, Nashville, TN
37232-6305; email: dave.gailani{at}mcmail.vanderbilt.edu.
| |
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ABSTRACT
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