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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3035-3041
RAPID COMMUNICATION
Secretable Human Platelet-Derived Factor V Originates From the Plasma
Pool
By
Rodney M. Camire,
Eleanor S. Pollak,
Kenneth Kaushansky, and
Paula
B. Tracy
From the Department of Biochemistry, University of Vermont, College
of Medicine, Burlington; the Division of Hematology, The Children's
Hospital of Philadelphia, Philadelphia, PA; and the Division of
Hematology, University of Washington School of Medicine, Seattle.
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ABSTRACT |
Factor Va (FVa), derived from plasma or released from stimulated
platelets, is the essential protein cofactor of the prothrombinase complex. Plasma-derived factor V (FV) is synthesized by the liver, whereas the source of the platelet-derived cofactor has not been unambiguously identified. Megakaryocytes, platelet precursors, are
known to synthesize platelet proteins and to endocytose proteins from
plasma (ie, fibrinogen) and then package these proteins into  -granules. To determine which mechanism accounts for FV presence in
platelets, two patients heterozygous for FVLeiden who
underwent allogeneic transplantation from homozygous FV wild-type donors (bone marrow [BM] or liver) were studied. Patient JMW, whose
skin biopsy specimen showed heterozygous FVLeiden, received
a BM transplant from a wild-type homozygous FV donor as analyzed from
posttransplant peripheral blood cells. Patient FW, whose native liver
is heterozygous for FVLeiden, received a homozygous
wild-type FV liver. Because each individual has two distinct genetic
pools of factor V in liver and megakaryocytes, it was possible to
determine whether secretable platelet-derived FV was normal or
contained the FVLeiden mutation. Platelet-derived FVa
released from thrombin-activated platelets from a normal individual, an
individual heterozygous for the FVLeiden mutation, and the
two patients was incubated with phospholipid vesicles and activated
protein C (APC). Western blotting analyses using a monoclonal antibody
that allows distinction between platelet-derived FVa and
FVaLeiden subsequent to APC-catalyzed cleavage were then
performed. Based on the accumulation of proteolytic fragments derived
from APC-induced cleavage, analyses of platelet-derived FVa from JMW
demonstrated both normal FVa and FVaLeiden consistent with
a plasma-derived origin of the secretable platelet-derived FVa. Western
blotting analyses of the APC-cleaved platelet-derived FVa from FW
showed a wild-type phenotype, despite the presence of a
FVLeiden allele in her megakaryocyte genome, also
consistent with a plasma origin of her secretable platelet-derived FVa.
Platelets do not appear to endocytose the plasma cofactor, because a
35-hour incubation of platelet-rich plasma with 125I-factor
V showed no specific association/uptake of the radiolabeled ligand with
the platelet pellet. Collectively, these results show for the first
time that the majority of secretable platelet-derived factor V is
endocytosed by megakaryocytes from plasma and is not exclusively
synthesized by these cells, as previously believed.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
BLOOD COAGULATION factor Va is a
heterodimer composed of a heavy chain (molecular weight
[Mr] = 105,000) and a light chain (Mr = 74,000) which arises from limited proteolysis of the
single-chain procofactor factor V (Mr = 330,000).1 Factor Va is the cofactor required in vivo for
the rapid generation of thrombin catalyzed by the prothrombinase
complex, a 1:1, Ca2+-dependent complex that is composed of
the serine protease factor Xa, and factor Va, bound to an appropriate
cellular membrane surface.2 The cofactor plays a central
role in this enzymatic complex and profoundly influences the amount of
thrombin generated.3 Clinically, this is underscored by the
observation that deficiencies of the procofactor factor V can lead to
severe hemorrhage and possibly death,4 whereas inefficient
inactivation of the cofactor (ie, factor VaLeiden;
Arg506  Gln) by activated protein C (APC) can
lead to venous thrombosis.5,6 Interestingly, recent studies
using gene knockout technology indicate that factor V deficiency in
mice is embryonic-lethal, suggesting that a complete deficiency in
humans may lead to a similar fate.7
Factor V, the precursor of factor Va, is distributed between two blood
pools. Approximately 80% of the total factor V circulates in plasma (7 µg/mL; 20 nmol/L) as the single-chain procofactor, and the remaining
20% is found within the -granules of platelets ( 4,600 to 14,000 molecules/platelet) in a partially proteolyzed state exhibiting
significant cofactor activity after its release by a variety of
agonists.8,9 Plasma-derived factor V is synthesized in the
liver10,11; however, the origin of the platelet-derived
cofactor has not been unambiguously defined. Because platelets retain
only limited biosynthetic capacity,12 platelet-derived
factor V might originate from plasma through endocytosis, like
fibrinogen, albumin, and IgG,13-15 or be synthesized by the
precursor of platelets, the megakaryocyte, like platelet factor 4,  -thromboglobulin, and von Willebrand factor (vWF).16,17
A few studies have suggested that megakaryocytes may synthesize factor
V. Chiu et al,18 using isolated guinea pig megakaryocytes, demonstrated biosynthesis of factor V by in vitro incubation with radiolabeled amino acids and isolation of biosynthesized factor V. Gewirtz et al,19 using freshly isolated human
megakaryocytes and megakaryocytes cloned from their colony-forming
units, showed that these cell populations both bind and synthesize
factor V. Expression of these traits appears to be related to cell
maturation, because cellular binding of factor V appears earlier than
their ability to synthesize the protein.19 In addition,
this same group of investigators was able to demonstrate factor V mRNA
in both megakaryocytes and platelets using reverse
transcriptase-polymerase chain reaction (RT-PCR).20
However, it is unclear from these studies if the amount of factor V
synthesized by megakaryocytes is sufficient to account quantitatively
for the amount of factor V contained within platelets and, therefore,
does not rule out endocytosis of factor V from plasma by megakaryocytes
as a potential mechanism.
To elucidate more conclusively whether platelet-derived factor V is
taken up by megakaryocytes or is synthesized by these cells, two
patients, each heterozygous for the factor VLeiden
mutation, who underwent allogeneic transplantation from a wild-type factor V donor (bone marrow [BM] or liver) were studied. Our results provide the first clear evidence that secretable platelet-derived factor V is predominately taken up from plasma by megakaryocytes before
platelet formation.
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MATERIALS AND METHODS |
Materials and Reagents
Tris[hydroxymethyl]aminomethane (Trizma-Base),
L--phosphatidyl-L-serine [bovine brain] (PS),
L--phosphatidylcholine [egg yolk] (PC), Tween-20,
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
prostaglandin E1 (PGE1), heparin (bovine lung), and glycine were purchased from Sigma (St Louis, MO). Nitrocellulose membrane sheets (0.45 µm) were purchased from Bio-Rad (Hercules, CA).
The chemiluminescent substrate, Luminol, Reflection autoradiography film, and 125I were purchased from DuPont, NEN Research
Products (Boston, MA). Crystallized bovine serum albumin was purchased
from ICN ImmunoBiologicals (Aurora, OH). The -thrombin inhibitor,
hirudin, was obtained from Calbiochem (La Jolla, CA). The fluorescent
-thrombin inhibitor, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide
(DAPA),21 and human APC were purchased from Haematologic
Technologies Inc (Essex Junction, VT). Phospholipid vesicles composed
of 75% (% wt/wt) PC and 25% (%wt/wt) PS (PCPS) were prepared as
previously described,22 and their concentration determined
by phosphorous assay.23
Patient Information
Patient no. 1.
JMW is a 52-year-old woman diagnosed with acute myelogenous leukemia in
1995. After several cycles of intensive chemotherapy she entered a
hematologic remission but relapsed within 6 months. She then underwent
BM transplantation (BMT), and her donor was an HLA-identical brother.
Her four hospital courses were complicated by four venous thromboses,
always in the circulatory bed in which a central venous catheter had
been inserted. Neither her parents nor any of her seven siblings gave a
history of thrombosis. Nevertheless, because of this history of
catheter-induced thrombosis, she underwent evaluation for a
hypercoagulable state, including tests of the lupus anticoagulant,
protein C, protein S, anti-thrombin III, and APC resistance. Both with
standard coagulation tests and with factor V-deficient plasma, the
patient was found to display APC resistance. Posttransplantation
peripheral blood leukocytes were then tested by PCR (35 cycles) for
factor VLeiden and found to be homozygous wild type,
whereas DNA obtained from a skin biopsy performed to confirm the
diagnosis of skin graft-versus-host disease showed her to be
heterozygous for factor VLeiden. In addition, incubation of
her plasma (posttransplant; 1:10 dilution with HBS [20 mmol/L HEPES,
0.15 mol/L NaCl, pH 7.4]) with 10 µmol/L PCPS vesicles
and 2 nmol/L APC,24 followed by Western blotting for factor
Va with HFVaHC#17,25 demonstrated an
APC-induced cleavage pattern consistent with a heterozygous factor
VLeiden phenotype (data not shown). Thus, JMW's
plasma-derived factor V is an equal mixture of wild-type factor V and
factor VLeiden. Her leukocytes, megakaryocytes, and
platelets, derived from the marrow transplant donor, express a
homozygous wild-type factor V genotype with no evidence of factor
VLeiden allelic expression remaining due to retention of
the recipient's marrow. Our inability to detect a factor
VLeiden allele after PCR (35 cycles) of DNA extracted from
her peripheral blood cells argues strongly against any significant
contribution of recipient cells to her marrow megakaryocytes subsequent
to BMT.
Patient no. 2.
FW is a 35-year-old woman postorthotopic liver transplantation for
Budd-Chiari syndrome, who presented with left arm swelling 1 month post
liver transplant. An ultrasound of the arm showed partially obstructing
thrombi in the distal jugular/subclavian junction of the
bronchopulmonary vein. The patient was placed on heparin for proper
anticoagulation. A workup for risk factors contributing to
hypercoagulability showed normal plasma levels of plasminogen,
fibrinogen, functional antithrombin, functional protein C, free protein
S antigen, and a normal activated protein C resistance test. A
genotypic analysis from the patient's peripheral blood cells showed
that the patient was heterozygous for factor VLeiden.
Because of the discrepant results between the patient's APC resistance
assay (2.2 with normal being > 2.0) and the presence of heterozygous
factor VLeiden from the patient's peripheral blood,
further analyses were performed. DNA was extracted from histologic
specimens from the patient's native liver and from the donor's gall
bladder. Results revealed homozygous wild-type FV in the genome of the
donor gall bladder, and FVLeiden in FW's original liver.
In addition, incubation of her plasma (posttransplant; 1:10 dilution
with HBS) with 10 µmol/L PCPS vesicles and 2 nmol/L
APC,24 followed by Western blotting for factor Va with
HFVaHC#17,25 showed an APC-induced cleavage
pattern consistent with a homozygous factor V wild-type phenotype (data not shown). Thus, FW's plasma-derived factor V is normal, wild-type whereas her leukocytes and megakaryocytes are heterozygous for factor
VLeiden.
DNA Extraction From Tissue Blocks
Formalin-fixed paraffin-embedded tissue (PET) blocks were used as the
DNA source.26 After thorough cleaning of the microtome and
installation of a new disposable knife, 15- to 20-µm sections providing a 1- to 2-cm tissue area were cut from a paraffin block containing no tissue, followed by sectioning of the PET block. Sections
were deparaffinized subsequently with xylene:ethanol (1:1, vol/vol).
Deparaffinized tissue sections were incubated in 100 µL Proeinase K
solution (6 µg Proteinase K, 10 mmol/L Tris HCl pH 8.3, 50 mmol/L
KCl, 1.5 mmol/L MgCl2, 0.01% gelatin) at 37°C for 1 to
3 hours, and then at 100°C for 10 minutes. After microcentrifugation at 14,000 rpm for 10 minutes, the supernatant containing the DNA was transferred to a clean microfuge tube for PCR
analysis.
Isolation of Platelets
Platelets were isolated from consenting individuals essentially as
described previously.24 Briefly, 26 mL of blood were collected into a 30-mL syringe (4 syringes, 100 mL of blood) containing 4 mL of ACD (0.022 mol/L citrate, 0.014 mol/L dextrose, final concentrations) and 5 µmol/L PGE1 (final
concentration). The blood in each syringe was transferred to a 50-mL
conical polypropylene centrifuge tube, everted twice, divided into two
tubes, and centrifuged (190g, 15 minutes) at ambient
temperature to obtain platelet-rich plasma (PRP). The PRP (7.5 mL)
from two centrifuge tubes was combined and centrifuged at
1,100g for 15 minutes at ambient temperature. The platelet-poor
plasma (PPP) was removed and the remaining platelet pellet was gently
resuspended in a small volume (10 mL) of PPP. All platelets from the
same donor were pooled (30 to 40 mL) to generate a platelet
concentrate. This platelet concentrate was then shipped from either
Seattle, WA (JMW) or Philadelphia, PA (FW) to Burlington, VT, via
express mail (18 to 36 hours). Upon receipt, the platelet suspensions
were placed immediately at 37°C and platelets were isolated as
previously described.27,28 Platelets were counted on a
Coulter counter (Coulter Electronics, LTD, Hialeah, FL) and brought to
a final platelet concentration of 1 × 109/mL in 5 mmol/L HEPES-Tyrode's (0.14 mol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L
NaHCO3, 0.42 mmol/L NaH2PO4 · H2O, 1 mmol/L MgCl2, 2 mmol/L
CaCl2, 5 mmol/L dextrose) buffer pH 7.4 for all
experiments. Control studies performed on site and detailed in previous
studies24 indicated that the time of shipment and the
presence of PGE1 had no influence on the rate or mechanism
of platelet-derived factor Va inactivation by APC.
APC-catalyzed inactivation and proteolysis of platelet-derived
factor Va and factor VaLeiden.
The inactivation of platelet-derived factor Va by APC was performed in
the presence of PCPS vesicles. Platelet-derived factor Va release and
activation was accomplished by platelet incubation (1 × 109/mL) with 50 nmol/L -thrombin (5 NIH U/mL) for 5 minutes at ambient temperature, followed by the addition of 60 nmol/L
hirudin. Activated platelets were removed from platelet-derived factor
Va by gentle centrifugation (1,100g, 5 minutes) and PCPS
vesicles (20 µmol/L) were then added. In all experiments, the initial
concentration of platelet-derived factor Va was donor dependent, and
ranged from 1.0 nmol/L to 3.0 nmol/L.
Functional analyses and Western blotting.
After platelet activation with -thrombin (50 nmol/L, 5 minutes), the
inactivation of platelet-derived factor Va/VaLeiden was
initiated immediately by APC addition (0.25 nmol/L). To monitor inactivation of the cofactor, samples of the reaction mixture were
withdrawn at various time intervals and placed in a prothrombinase assay using purified protein components with saturating amounts of
factor Xa (5 nmol/L) and PCPS vesicles (20 µmol/L) as the membrane surface as previously described.3,28 At the same time
intervals, samples of the reaction mixture were prepared for sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by
addition of 62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5%
-mercaptoethanol, 0.001% bromophenol blue (final concentrations).
After heating at 95°C for 3 minutes, SDS-PAGE analysis was
performed on 5% to 15% gradient slab gels according to the method of
Laemmli.29 Following electrophoresis, the proteolytic
fragments resulting from APC-catalyzed cleavage of factor Va were
transferred to nitrocellulose using electroblotting techniques as
described by Towbin et al.30 Transfer was performed at 500 mA for 2 hours at 4°C.31 Nitrocellulose was blocked
with 5% nonfat dry milk in 20 mmol/L Tris, 0.15 mol/L NaCl, 0.05%
Tween-20 (TBS-T) at pH 7.4. The platelet-derived factor Va antigen
(50 ng/lane) was probed with a mouse anti-human factor Va
heavy-chain IgG monoclonal antibody
HFVaHC#17,25 which recognizes an epitope
between amino acids 307-506 in the factor Va heavy chain. The secondary
antibody used was a horse anti-mouse IgG coupled to horseradish
peroxidase (HRP; Southern Biotechnologies, Birmingham, AL). Detection
of factor Va was performed by enhanced chemiluminescence (Western Blot
Chemiluminescence Detection Kit; Dupont NEN, Boston, MA) by exposure of
blots (5 to 30 seconds) to Reflection autoradiography film and
developed in a Kodak M35A X-OMAT processor (Eastman Kodak, Rochester,
NY).
125I-labeling of factor V and incubation with PRP.
Plasma-derived factor V was isolated by immunoaffinity chromatography
as described.32 Factor V was radioiodinated using the
IODO-GEN (Pierce, Rockford, IL) transfer technique and characterized as
previously detailed.31 125I-factor V was
greater than 97% precipitable with 10% trichloroacetic acid, remained
as a single-chain protein as determined by SDS-PAGE and
autoradiography, could be activated by thrombin to yield the characteristic heavy and light chains, and expressed a specific radioactivity of 3,000 to 5,000 cpm/ng (0.2 to 0.4 mol of iodine/mol of
factor V). The labeled protein was stored in 50% glycerol/2 mmol/L
CaCl2 at  20°C.
To assess factor V uptake by platelets, 125I-factor V (used
as a trace, 200,000 cpm/mL; 0.1 nmol/L) was incubated with PRP (factor V = 20 nmol/L; platelet concentration = 2 to 3 × 108/mL) in the presence or absence of 5 µmol/L
PGE1 for 12, 21, and 35 hours. Specific
binding/internalization of 125I-factor V was determined as
previously described33 after layering of 0.5 mL of the
PRP-125I-factor V mixture over 0.5 mL n-butyl phthalate and
centrifugation at 12,000g, 2 minutes. Nonspecific
binding/entrapment was determined using a 25-fold molar excess of cold
factor V (1.0 µmol/L, final) added to the PRP mixture.
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RESULTS |
Studies were initiated to determine whether factor V in platelet
-granules originates through endogenous synthesis in the megakaryocyte, through uptake from plasma, or through both mechanisms. To differentiate between these processes, we studied the inactivation of platelet-derived factor Va by APC on synthetic phospholipid vesicles
from two patients heterozygous for factor VLeiden who
underwent allogeneic transplantation from homozygous factor V wild-type
donors (BM or liver). Patient JMW, whose skin biopsy sample revealed
heterozygous factor VLeiden, underwent BMT and subsequent
DNA analyses indicated that her peripheral blood cells now exclusively
express the wild-type factor V genotype, because factor
VLeiden allelic expression was ablated completely before
transplant. Patient FW, whose marrow cell population showed
heterozygous factor VLeiden, underwent liver
transplantation, and DNA analyses on a sample of the new liver
indicated that her factor V was homozygous wild type. Therefore, if
platelet-derived factor V is exclusively synthesized by megakaryocytes,
then platelet-derived factor Va from JMW should be homozygous wild type
whereas that derived from FW should be heterozygous for the factor
VLeiden mutation. Alternatively, if platelet-derived factor
V is endocytosed by megakaryocytes (and/or platelets), then
platelet-derived factor Va from JMW should be a mixture of wild type
and factor VaLeiden and FW should be homozygous wild type.
Wild-type factor Va versus factor VaLeiden is easily
distinguished based on the heavy-chain products observed subsequent to
APC-induced cleavage using monoclonal antibody
HFVaHC#17, which is specific for an epitope located
between amino acids 307-506 within the factor Va heavy chain (see
Fig 1).25 Thus, platelets
isolated from a normal individual, a heterozygous factor
VLeiden individual, and the two transplant patients were
incubated with thrombin as described in Materials and Methods to both
activate the platelet and to release and activate the platelet-derived cofactor. After centrifugation to remove platelets, phospholipid vesicles were added to the supernatant to provide an appropriate membrane surface required for the APC-catalyzed inactivation of the
cofactor. Following the addition of APC to initiate the reaction, samples were removed at selected time intervals and prepared for SDS-PAGE/Western blotting to detect the proteolytic fragments accompanying inactivation. As shown in Fig
2A, APC initially cleaved normal platelet-derived factor Va within the
heavy chain at Arg506 generating an Mr = 75,000 intermediate, which was further cleaved at Arg306
generating a fragment migrating at Mr = 30,000. The
inactivation of platelet-derived factor Va by APC derived from an
individual heterozygous for factor VLeiden is depicted in
Fig 2B. As expected, APC cleaved the normal pool of platelet-derived
factor Va initially at Arg506, followed by cleavage at
Arg306; however, the mutant pool of platelet-derived factor
Va, which lacks the cleavage site at Arg506, was initially
cleaved by APC at Arg306 generating Mr = 60/58,000 fragments, followed by cleavage at Arg679,
generating a fragment that migrates at Mr = 54,000 and is
resistant to any further APC-induced proteolysis.
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| Fig 1.
Schematic representation of membrane-bound factor
Va/VaLeiden heavy-chain inactivation by APC. Normal plasma
factor Va is inactivated following three ordered and sequential
cleavages in the heavy chain at Arg506, Arg306,
and Arg679.45 Cleavage at Arg506,
which gives rise to an Mr = 75,000 fragment and an
Mr = 28/26,000 doublet, is necessary to optimally expose
the site at Arg306. Further cleavage at Arg306
yields an Mr = 45,000 fragment and an Mr = 30,000 fragment.45 Individuals with the Arg506
Gln mutation (factor VLeiden) no longer have a
cleavage site at position 506, which slows the rate of cleavage at
Arg306.46 Cleavage at Arg306 yields
an Mr = 45,000 fragment and an Mr = 60/58,000 doublet. Further cleavage of the Mr = 60/58,000
doublet at Arg679 yields an Mr = 54,000 fragment.
Fragments that are recognized by the monoclonal antibody
(HFVaHC#17) used in this study are indicated by the
shaded boxes.25
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| Fig 2.
APC-catalyzed inactivation of platelet-derived factor Va
and factor VaLeiden bound to phospholipid vesicles.
Platelets (1 × 109/mL) were treated with 5 NIH U/mL (50 nmol/L) of -thrombin for 5 minutes to both activate the platelets
and release and activate the platelet-derived factor V. Hirudin (60 nmol/L) was then added to inhibit thrombin. The activated platelets
were immediately removed from suspension by gentle centrifugation
(1,100g, 5 minutes), and PCPS vesicles (20 µmol/L) were added
to the supernatant to provide an appropriate alternate anticoagulant
surface. APC (0.25 nmol/L) was then added. At selected time points,
samples of the reaction mixture were withdrawn and subjected to
SDS-PAGE using a 5% to 15% gradient gel. After transfer to
nitrocellulose, fragments were visualized using a monoclonal antibody,
HFVaHC#17, as described.25 Each of the
panels represents the inactivation of secretable platelet-derived
factor Va on phospholipid vesicles by APC: (A) normal platelet-derived
factor Va, (B) platelet-derived factor Va derived from a heterozygous
factor VLeiden individual, (C) platelet-derived factor Va
derived from JMW, and (D) platelet-derived factor Va derived from FW.
The time course of inactivation by APC is given at the top of each
immunoblot. In (A) and (C), 145* indicates the platelet factor Va/APC
mixture after a 145-minute incubation, with an additional 20 nmol/L APC
incubated for 5 minutes. In (B) and (D), following a 45-minute
incubation with 0.25 nmol/L APC, platelet-derived factor Va was
incubated with increasing concentrations of APC (2, 10, 20, 40 nmol/L)
for an additional 15 minutes, which is indicated in the last four lanes
of these immunoblots. The position of the molecular weight markers are
indicated at the left of the immunoblots. Arrows to the right of the
immunoblots represent residue numbers corresponding to factor Va
fragments derived from APC-induced cleavage.
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As seen in Fig 2C, the APC-induced cleavage pattern of platelet factor
Va derived from JMW is consistent with a heterozygous factor
VaLeiden phenotype based on the accumulation of factor Va
fragments migrating at Mr =30,000 and 54,000 to 60,000. These results clearly indicate that a significant portion of
platelet-derived factor V must be endocytosed by megakaryocytes
(and/or platelets) from plasma, because posttransplantation her
plasma is the only source of the factor VLeiden
protein, because no factor VLeiden allele could be
detected in DNA isolated from her marrow-derived cells after 35 cycles
of PCR.
To more firmly establish the contribution that megakaryocyte
endocytosis of plasma factor V makes to the platelet factor V pool, FW
was studied. Western blotting analyses of the APC-cleaved platelet-derived factor Va from FW indicated a wild-type phenotype (Fig
2D), despite the presence of a factor VLeiden allele in her
marrow cell populations. As previously shown by our laboratory, normal
platelet-derived factor Va can be initially cleaved by APC at either
Arg506 or Arg306.24,28 It is
evident from Fig 2A and D, both of which represent normal
platelet-derived factor Va, that there was some initial cleavage of the
cofactor at Arg306 which generated fragments migrating at
Mr = 60/58,000. Because both of these factor Va molecules
lack the factor VLeiden mutation, these fragments were
subsequently cleaved by APC at Arg506 to generate a
30,000-dalton fragment. No accumulation of a 54,000-dalton fragment was
observed (Fig 2D) since the cleavage site at position 506 remained
intact, in contrast to that observed with platelet-derived factor
VaLeiden (Fig 2B and C). In fact, prolonged exposure of the
blot shown in Fig 2D did not allow detection of any 54,000-dalton
factor VaLeiden subsequent to APC addition. Thus, because
factor VLeiden protein was undetectable in FW's platelet
pool, the combined results are consistent with endocytosis of
platelet-derived factor V by megakaryocytes (and/or platelets)
from plasma and argues against synthesis as the primary and only
mechanism responsible for the presence of factor V in platelets.
Studies investigating the nature of platelet fibrinogen have indicated
that this protein is taken up by megakaryocytes through a process
involving receptor-mediated endocytosis most likely involving GP
IIb/IIIa.13-15,34,35 In addition, it was also established that platelets, as well as megakaryocytes, are involved in fibrinogen uptake.14,35 To establish whether platelets are involved in the endocytosis of factor V from plasma, single-chain, human
plasma-derived factor V was radiolabeled with 125I and
subsequently incubated with PRP (200,000 cpm/mL of PRP) from a normal
donor. Nonspecific binding of 125I-factor V was assessed by
incubating PRP with trace label (200,000 cpm/mL) plus a 25-fold excess
of cold single-chain factor V (final concentration in PRP = 1.0 µmol/L). Even after a 35-hour incubation period, we were unable to
detect any specific association of 125I-factor V with the
platelet pellet (data not shown), suggesting that uptake of factor V
most likely takes place exclusively in the megakaryocyte. However,
these in vitro conditions may not adequately mimic the environment of
the normal platelet circulating in vivo, a condition that may be more
conducive to the uptake of factor V by platelets.
| |
DISCUSSION |
The purpose of this present study was to determine whether
platelet-derived factor V originates from endogenous synthesis by
megakaryocytes, endocytosis by megakaryocytes (and/or
platelets), or through both mechanisms. Results from our present study
provide strong evidence that platelet-derived factor V originates from plasma by endocytosis from the megakaryocyte and indicates that endogenous synthesis of the procofactor by megakaryocytes contributes little to the total pool of factor V contained within a platelet.
The mechanism of acquisition of factor V within megakaryocytes or
platelets had been established previously as due to endogenous synthesis in the megakaryocyte. The biosynthesis of factor V has been
shown in isolated guinea pig megakaryocytes18 and human megakaryocytes19 by in vitro incorporation of radiolabeled
amino acids into the procofactor. In addition, specific mRNA for factor V has been identified in both human megakaryocytes and platelets by
RT-PCR.20 Although each of these studies show that
megakaryocytes may synthesize factor V, there has been no report to
date demonstrating whether such synthesis accounts quantitatively for
megakaryocyte- and platelet-derived factor V. In addition, neither of
the studies provide any data showing that biosynthesis of factor V is
the only mechanism of factor V acquisition in megakaryocytes or
platelets. Thus, although these previous studies demonstrate some level
of factor V synthesis by megakaryocytes, our present results clearly show that the principal mechanism by which platelets acquire factor V
is via its endocytosis from plasma by megakaryocytes.
Interestingly, Gewirtz et al19 noted that while mature
megakaryocytes can both bind and synthesize factor V, immature
megakaryocytes do not appear to synthesize the procofactor. However,
these same immature megakaryocytes contain large amounts of
immunochemically detectable factor V, suggesting that the ability of
these megakaryocytes to bind factor V develops early and in addition to
binding factor V, megakaryocytes may also endocytose this protein early
in development. Clearly, the data presented in the current study
strongly support the latter mechanism. Additional studies are planned
to determine quantitatively the extent to which megakaryocyte
endocytosis of plasma factor V versus its endogenous synthesis
contributes to the cofactor pool within the platelet.
Several studies have now clearly established that the platelet
-granule is a unique type of secretory granule whose contents can
originate by endogenous synthesis in the megakaryocyte, or by
endocytosis and pinocytosis at both the megakaryocyte and circulating platelet level.16,36 Platelet-specific proteins, like
platelet factor 4 and -thromboglobulin, appear to be synthesized
solely by the megakaryocyte.16,36 However, the origin of
platelet proteins which have plasma counterparts is less obvious. For
example, albumin, IgG, and fibrinogen are endocytosed by
megakaryocytes, and to a certain extent by platelets, and incorporated
into -granules.13-15 Alternatively, platelet and
megakaryocyte vWF originates from endogenous synthesis and is
independent of plasma vWF.17
The mechanism(s) by which factor V is endocytosed by megakaryocytes is
currently unknown. George16 has hypothesized that the
different origins of -granular proteins are suggested by investigating their relative platelet-plasma concentration. For example, -thromboglobulin and platelet factor 4, proteins that are
known to be exclusively synthesized in the megakaryocyte, have greater
concentrations in platelets than in plasma (platelet/plasma ratio
>250,000).16 Alternatively, albumin and IgG, proteins, which are thought to enter megakaryocytes and/or platelets
though fluid-phase endocytosis (pinocytosis), have much higher
concentrations in plasma than in platelets (platelet/plasma ratios
<0.07). A protein with an intermediate platelet to plasma ratio is
fibrinogen (2.5). Recent data from several laboratories have
established that fibrinogen enters megakaryocytes (and possibly
platelets) through receptor-mediated endocytosis via glycoprotein (GP)
IIb-IIIa.34,35,37 Interestingly, factor V has a platelet to
plasma ratio of 60, much lower than that of -thromboglobulin and
platelet factor 4, and much higher than albumin and IgG. Thus, simply
by looking at the concentration of factor V in platelets to that in
plasma, it is tempting to hypothesize that factor V as well as
fibrinogen enters megakaryocytes through receptor-mediated endocytosis.
Several studies investigating platelet IgG, which is taken up from
plasma by fluid phase endocytosis, have shown that the concentration of
platelet IgG mirrors the concentration and composition of plasma IgG in
both normal subjects as well as in a wide range of abnormal plasma IgG
concentrations.16 As with platelet IgG, the concentration
of platelet-derived factor V also appears to mirror the concentration
of plasma-derived factor V. Our laboratory has shown that
platelet-derived factor V consistently comprises 20% of the total
factor V contained in whole blood from normal donors as determined by
radioimmunoassay.8 Although there have been several reports
documenting factor V deficiency with respect to plasma factor
V,4 unfortunately none of these studies simultaneously measured the factor V content in platelets. However, our laboratory has
recently studied an individual with mild factor V deficiency. Our
results indicate that the patient has 30% factor V activity in
plasma, as determined by a clotting assay, and 37 % platelet factor
Va activity, as determined by a purified prothrombinase assay
(unpublished observations, September 1996). In addition, Weiss and Lages38 have recently described a patient with
congenital factor V deficiency, whose plasma- and platelet-derived
factor V concentrations were 14% and 12% of normal,
respectively, both based on activity assays. Thus, like endocytosed
platelet IgG, the concentration of platelet-derived factor V is also
very sensitive to the concentration of its plasma counterpart and
suggests an endocytotic mechanism of acquisition of the procofactor by
megakaryocytes.
Additional insight into the nature of proteins endocytosed by
megakaryocytes can be made by investigating the gray platelet syndrome
(GPS). GPS is a rare congenital bleeding disorder in which
megakaryocytes and platelets are deficient in -granule secretory
proteins.39 Studies into the GPS indicate that some -granule proteins appear reduced to a lesser extent than other -granule proteins, for example, albumin compared with platelet factor 4.39 Why this occurs is currently not known;
however, it has been hypothesized that the functional abnormality in
GPS may result from the defective targeting of endogenously synthesized secretory proteins to developing -granules in
megakaryocytes.39,40 Consistent with the notion that
endocytosed proteins are affected to a lesser extent than endogenously
synthesized proteins, Chenu and Delmas41 have demonstrated
that levels of bone sialoprotein, which is endocytosed by
megakaryocytes, were normal in platelets of a patient with GPS.
Interestingly, our laboratory has shown that lysed platelets from
a patient with GPS contain near-normal amounts of platelet-factor V
antigen, again consistent with an endocytotic mechanism.42
Our combined data support endocytosis by megakaryocytes as the major
mechanism by which platelet-derived factor V is acquired, because we
could not show direct platelet uptake of factor V from plasma. Since
platelet-derived factor V is stored within the platelet as a partially
proteolyzed molecule9 and is a different substrate for
proteases24,28,43 and platelet kinases44 when
compared with its plasma counterpart, we hypothesize that the
platelet-derived molecule may be physically altered during endocytosis
and packaging in the -granule.
| |
FOOTNOTES |
Submitted June 25, 1998;
accepted August 10, 1998.
Supported by Grant No. HL P01-46703, Project 4 (to P.B.T.).
Address reprint requests to Paula B. Tracy, PhD, Given C409, Department
of Biochemistry, University of Vermont College of Medicine, Burlington,
VT 05405-0001; e-mail: ptracy{at}salus.med.uvm.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We acknowledge Rama Kudaravalli, PhD, for her expert technical
assistance regarding DNA extraction and testing for the factor VLeiden mutation, and Ilka Warshawsky, MD, for her clinical
service work. The Blood Drawing Services of the General Clinical
Research Center at Fletcher Allen Health Care in Burlington, VT, are
gratefully acknowledged as well.
| |
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Abstract
Introduction
Abstract