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Granules of Platelets1
Department of Growth and Development, Davies Medical Center, San Francisco, California 94114
Address all correspondence and requests for reprints to: Kam Chan, Ph.D., Davies Medical Center, Laboratory of Growth and Development, Room B-200, Castro and Duboce Streets, San Francisco, California 94114. E-mail: igf{at}itsa.ucsf.edu
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-granules, which
contain growth factors, including insulin-like growth factor I (IGF-I)
and IGF-binding protein-3 (IGFBP-3). We investigated the mechanism by
which IGF-I and IGFBP-3 appeared in the -granules with a goal of
modulating their levels in platelets to affect platelet functions.
Reverse transcription-PCR was initially used to test whether
megakaryocytes contained IGFBP-3 and IGF-I messenger RNA transcripts.
We found that megakaryocytes did not express the IGFBP-3 gene, but did
have IGF-I messenger RNA. We subsequently investigated whether they
incorporated IGFBP-3 and IGF-I by the process of endocytosis and
packaged them into the -granules. This hypothesis was tested in two
ways. 1) We examined whether during pregnancy in the rat the
-granule content for IGFBP-3 paralleled the changes in plasma
IGFBP-3 levels caused by the pregnancy-induced IGFBP-3 protease. The
-granule contents of both IGFBP-3 and IGF-I declined in parallel to
the plasma changes in pregnant rats and returned to normal postpartum.
As the binding protein protease acts extracellularly, endocytosis of
the IGF-I:IGFBP-3 complex from the extracellular fluid by
megakaryocytes was suggested. 2) We tested whether an IGF-I:IGFBP-3
complex comprised of human IGF-I and IGFBP-3 (recombinant 28.7 kDa)
injected iv appeared in rat platelet -granules. Hypophysectomized
rats were injected iv with 5.24 mg of a 1:1 complex of IGF-I:IGFBP-3.
After 24 h, platelet lysates were prepared and analyzed for
IGFBP-3 by Western ligand blotting, and IGF-I was determined by RIA.
Platelet lysates of the treated animals showed a prominent new band at
approximately 28 kDa, whereas control rats were negative. In addition,
the -granule IGF-I concentration increased from 0.38 to 1.9
ng/1 x 109 platelets.
These results indicate that the IGF-I:IGFBP-3 complex is taken up by
megakaryocytes and packaged into the -granules of platelets and
demonstrate how the contents of IGF-I and IGFBP-3 in platelets can be
modulated by their plasma concentrations. As reverse transcription-PCR
has shown that the IGF-I, but not the IGFBP-3, gene is expressed by
megakaryocytes, there may be two mechanisms for directing IGF-I into
the -granules of platelets.
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Introduction |
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-granules contents into the injured
tissue. Both IGF-I and IGFBP-3 are released from the granules (5, 6)
and are thought to act on macrophages, which orchestrate the cascade of
reactions involved in tissue repair (2).
The IGF-I and IGFBP-3 in platelets are important not only as early
determinants of wound healing, but could be involved in other platelet
functions, such as the maintenance of vascular integrity and
atherogenesis (7). Therefore, the ability to selectively modulate the
platelet -granule content of IGF-I and IGFBP-3 may assume
considerable importance. To accomplish this, the mechanism by which
IGF-I and IGFBP-3 appear in the -granules has to be established.
-Granule factors, such as epidermal growth factor, transforming
growth factor-ß, and platelet-derived growth factor, are synthesized
by megakaryocytes (8, 9, 10), whereas albumin, fibrinogen, and Igs are
endocytosed by megakaryocytes and packaged into platelet -granules.
Therefore, we asked which of these mechanisms was responsible for IGF-I
and IGFBP-3 appearance in the -granules of platelets and whether we
could modulate their amounts in platelets.
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Materials and Methods |
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Animals
Timed pregnant and nonpregnant Long-Evans rats (Simonsen
Laboratories, Gilroy, CA) and hypophysectomized Fisher rats (Hilltop
Lab Animals, Scottsdale, PA), weighing 200–250 g, were housed under a
12-h dark, 12-h light cycle and fed Purina rat chow (Ralston-Purina,
St. Louis, MO) and water ad libitum.
Hypophysectomy was verified by significantly low plasma IGF-I levels, failure to gain weight, and significantly reduced levels of IGFBP-3 on a ligand blot. All laboratory procedures on animals were carried out in accordance with the NIH guidelines and the approval of the California Pacific Medical Center animal care committee (San Francisco, CA). Animals were anesthetized by inhalation of Metofane (Pitman-Moore, Mundelein, IL).
Isolation and purification of megakaryocytes
Rat megakaryocytes were isolated by a two-step procedure of
Percoll density gradient centrifugation, followed by velocity
sedimentation of the cells on a Ficoll medium (11). Marrow cells were
flushed from femurs of 250-g Long-Evans rats with CATCH medium (12) (1
mM adenosine, 2 mM theophyline, 0.76 g/liter
sodium citrate, and 3.5% bovine fraction V, dissolved in Hanks’
medium, pH 7.15). The cells were filtered through a nylon mesh, then
layered on top of a Percoll step gradient (5%:10%:20%:30%) in CATCH
medium and centrifuged at 1000 x g for 10 min at room
temperature. The top megakaryocyte-containing layer was placed on top
of a 2–4% continuous gradient of Ficoll in CATCH medium containing
5% FCS and centrifuged at 100 x g for 5 min. The
bottom 0.1 vol was removed and spun briefly to collect the
megakaryocytes. A yield of 103-104
megakaryocytes/rat was obtained, with up to 90% purity as assessed by
microscopic characteristics.
Isolation and purification of platelets
Eight milliliters of rat blood were drawn from the inferior vena
cava and collected in 50 U heparin and 1% acid citrate-dextrose
solution. The blood was layered gently on top of 1-Step-Platelet medium
at a ratio of 1:1 (vol/vol) and centrifuged at 350 x g
for 20 min at room temperature. The top platelet-rich layer was
removed, diluted 3-fold with Tyrode buffer (50 mM HEPES, 30
mM dextrose, 4 mM KCl, 140 mM NaCl,
0.5 mM EDTA, and 0.35% bovine fraction V, pH 7.3), and
centrifuged at 800 x g for 20 min at room temperature.
The pellet was washed in Tyrode buffer three times and used for RNA
processing or thrombin activation. The platelets were counted and
assessed for purity on a Coulter STKS-2A (Coulter Corp., Hialeah,
FL).
Purification of messenger RNA (mRNA)
The mRNA from megakaryocytes, platelets, or rat embryo
fibroblasts (purchased from the Tissue Culture Facility, University of
California-San Francisco) was isolated using the Ultraspec RNA reagent
according to the manufacturer’s recommendations. Megakaryocytes
(1 x 104), platelets (1 x 109), or
fibroblasts (1 x 106; grown to confluence in DMEM in
the presence of 5% FCS) were homogenized in 1 ml Ultraspec RNA
reagent, extracted with 0.2 ml chloroform, then precipitated with
isopropanol at a 1:2 ratio (vol/vol) and pelleted. After an ethanol
wash, the final mRNA pellet was dried and dissolved in 25 µl
ribonuclease-free water.
RT-PCR amplification
RT was performed using the Superscript preamplification kit. The
reaction was carried out in the presence of 10 U of Superscript II
reverse transcriptase and 1–5 µl mRNA sample [primed with
oligo(deoxythymidine)] in a buffer of 0.5 mM NTP mix, 0.04
M dithiothreitol, 20 mM Tris-HCl (pH 8.4), 50
mM KCl, 2.5 mM MgCl2, and 0.1
µg/µl BSA for 50 min at 45 C. Ribonuclease H was added to the
20-µl reaction mixture for 20 min at 37 C before termination by
heating. PCR amplification was performed, using RT mixture (1–2 µl),
with sequence-specific primers against rat IGF-I
(5'-ATCTCTTCTACCTGGCACTCTG-3'/5'-GAAGCAACACTCATCCACAAT-3'), IGFBP-3
(5'-ACATCGCGTGACTGATTCC-3'/5'-CAGATGATTCAGTGTGTCCTCC-3'), and platelet
factor-4 (PF-4; 5'-TGTGGTTGCTGTCACCAG-3'/5'-CTCCAGGAGTTTCTTGATTATT-3').
The oligonucleotide pairs were designed to amplify a region that
spanned an intron to remove the possibility of DNA contamination. PCR
was carried out for 30–50 cycles using TwinBlockCycler (Ericomp, San
Diego, CA). Each cycle consisted of denaturation at 98 C (30 sec),
annealing at 52–55 C (1 min), and extension at 72 C (1 min). Twenty
microliters of a 50-µl PCR mixture were electrophoresed on a 5%
acrylamide gel and stained in ethidium bromide, and amplified products
were visualized by UV illumination. Molecular sizes were estimated
using a 100-bp DNA ladder.
Lysis of platelet -granules with thrombin
Purified platelets (1 x 109) in Tyrode buffer
were spun down and resuspended in 200 µl Dulbecco’s PBS (pH 7.4)
containing 0.1 g/liter CaCl2 and 0.1 g/liter
MgCl2. One unit of thrombin was added to the platelet
suspension and incubated at 37 C for 5 min. The activated platelet
suspension was spun down, and supernatant containing -granule
lysate was analyzed by Western ligand blotting or IGF-I RIA.
Western ligand blot analysis of IGFBPs
Western ligand blot analysis for IGFBPs was carried out using
the method of Hossenlopp et al. (13) with significant
modification. Three parts of sample were mixed with one part sample
buffer and electophoresed under nonreducing conditions on a 12.5%
SDS-polyacrylamide gel in Tris-HCl, pH 8.3. The separated proteins were
blotted onto a nitrocellulose membrane, washed in 3% Nonidet P-40
Tris-buffered saline (TBS), then blocked in 1% bovine fraction V in
TBS for 1 h. The nitrocellulose membrane was enclosed in a
Seal-O-Meal bag, and 1 x 106 cpm
[125I]IGF-I in 10 ml blocking buffer was added. The bag
was incubated on a rotary shaker for 2 h at room temperature. The
membrane was subjected to three washes of TBS containing 0.1%
Tween-20, followed by three washes in TBS alone. After drying, the
membrane was exposed to Kodak Biomax MS film for 1–3 days at -80 C
before development of the film.
Systemic administration of the human IGF-I:IGFBP-3 complex into a
hypophysectomized rat
A 0.1-µmol 1:1 complex of recombinant human IGF-I and 28.7-kDa
nonglycosylated recombinant human IGFBP-3 in 1.0 ml phosphate buffer
saline and 0.1% rat serum albumin was injected over 2 min by a 1-ml
syringe into the jugular vein of an anesthetized hypophysectomized rat.
Two hundred microliters of heparinized blood were taken at 1 h,
and plasma was saved. At 24 h, the animal was exsanguinated, and
blood was drawn from the inferior vena cava; 200 µl were collected
for plasma, and the rest was used for platelet processing.
RIA of IGF-I
IGF-I was separated from IGFBPs before RIA by the formic
acid-acetone extraction method (14). The method was validated by
obtaining the same results as those with chromatography on a 1 x
110-cm Sephadex G-50 column equilibrated with 1 N acetic
acid (the gold standard). One hundred microliters of platelet
-granule lysate or plasma were mixed with 50 µl 8 N
formic acid-0.05% Tween solution. After 5 min at room temperature, 350
µl acetone were added to the mixture, which was placed at -20 C
overnight to precipitate the IGFBPs. After centrifugation, the plasma
supernatant was analyzed for IGF-I by RIA. The platelet lysate
supernatant was dried completely by Speed-Vac (Savant Instruments,
Farmingdale, NY), then reconstituted in assay buffer (50 mM
basic sodium phosphate, 0.1% NaCl, 0.1% EDTA, 0.1% sodium azide,
0.02% protamine sulfate, and 0.05% Tween-20 adjusted to pH 7.5 with
NaOH) before RIA. Antirat IGF-I antiserum was used at a final
concentration of 1:60,000 in assay buffer. The RIA was performed at 4 C
for 16–20 h in polypropylene tubes, which contained 200 µl standard
(human IGF-I) or diluted sample in assay buffer, 200 µl first
antibody, and 200 µl trace containing 10,000 cpm
[125I]IGF-I. After incubation, 100 µl of a 1:15
dilution of goat antirabbit 
-globulin, 100 µl of a 1:200 dilution
of rabbit -globulin, and 0.7 ml 9.14% polyethylene glycol 8000
(Sigma Chemical Co., St. Louis, MO) (pH 7.3) were added and mixed.
After 15 min, the samples were centrifuged at 3000 x g
for 30 min at 4 C, and radioactive pellets were counted on a Berthold
Gamma Counter LB 2104 (Wallac, Inc., Gaithersburg, MD). The nonspecific
binding was less than 1%, and the specific binding of tracer was
43–50%. The interassay coefficient of variation was 2.6%.
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236 bp (Fig 1A
). The PF-4 PCR product was confirmed
as corresponding to PF-4 complementary DNA (cDNA) by restriction
endonuclease digestion with BamHI, resulting in fragments of
the predicted sizes, 154 and 82 bp (Fig. 2A). As expected, PF-4 transcripts were
also present in platelets (Fig. 1A).
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198 bp, whereas IGFBP-3 primers
yielded a PCR product of 202 bp (Fig. 1B
). Both amplified cDNAs were
validated by restriction enzyme analysis and corresponded to specific
cDNAs of IGF-I and IGFBP-3 (data not shown). RT-PCR of rat
megakaryocyte mRNA using IGF-I-specific primers resulted in a predicted
IGF-I PCR product of 198 bp. As megakaryocytes could be contaminated
with leukocytes, platelet mRNA was analyzed and found to contain IGF-I
transcripts (Fig. 1C). The platelet IGF-I product was validated as
corresponding to IGF-I cDNA by EaeI digestion, which
produced the predicted DNA fragments of 155 and 43 bp (Fig. 2B). No
IGFBP-3 transcript was detected in either megakaryocytes or platelets
by the RT-PCR (Fig. 1D).
Changes in platelet IGFBP-3 parallel serum changes in pregnant
rats
Although IGF-I in -granules could theoretically be explained by
megakaryocyte synthesis, the presence of IGFBP-3 cannot. Therefore,
the uptake of extracellular IGFBP-3 by megakaryocytes in the bone
marrow was indirectly demonstrated by measuring IGFBP-3 incorporation
into platelet -granules. It is well established that serum IGFBP-3
decreases during late pregnancy in the rat due to a protease specific
for the IGFBP-3 (15). If IGFBP-3 were incorporated from extracellular
fluid, the IGFBP-3 levels in platelets would be expected to mirror the
changes in levels found in both serum and marrow fluid.
The Western ligand analysis of serum IGFBPs levels in rats during days
14, 15, 17, and 20 of pregnancy and days 1 and 3 postpartum was
characterized by a major decrease in the 40- to 45-kDa IGFBP-3 starting
on day 15 of pregnancy (Fig. 3A). From
day 17 of pregnancy until delivery, the 40- to 45-kDa IGFBP-3 remained
virtually undetectable in serum. On day 1 postpartum, a rapid recovery
of the serum IGFBP-3 levels occurred (Fig. 3A). Marrow fluid showed a
similar IGFBP-3 profile during gestation and postpartum (Fig. 3B). When
the IGFBP-3 contents of -granule of rat platelets were analyzed,
their levels paralleled the changes found in both the serum and marrow
fluid (Fig. 3C). The pattern of platelet IGFBP-3 was characterized by a
decrease in the amount on day 17 of pregnancy followed by its return to
nonpregnancy levels on day 3 postpartum. The slight increase in IGFBP-3
in platelet lysate from days 14 to 15 of gestation was not reproduced
in a repeat experiment; thus, we speculate that it might be caused by a
minor variation in our platelet preparations. This particular ligand
blot was presented because we felt it best reflected our overall
results. Other IGFBPs of 24 and 30 kDa were also noted in serum,
marrow fluid, and platelet lysate. Interestingly, an increase in the
24- and 30-kDa IGFBPs in platelet lysate was observed at about
the same time as IGFBP-3 declined in serum during late pregnancy.
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Changes in platelet IGF-I in pregnant rats
Comparison of plasma IGF-I levels of nonpregnant rats with plasma
IGF-I levels of day 20 pregnant and day 3 postpartum rats revealed a
reduction in the IGF-I levels on day 20 in pregnant rats compared with
the nonpregnant rat levels. On day 3 postpartum, the plasma IGF-I
concentrations were restored to 85% of the nonpregnancy levels (Fig. 4A). The IGF-I levels in the platelet
lysate mirrored the changes in plasma IGF-I levels characterized by a
lower amount of IGF-I on day 20 of pregnancy followed by their
restoration to 80% of the nonpregnant levels on day 3 postpartum (Fig. 4B). These changes are consistent with the previous finding of a
decline in serum IGF-I during late gestation (15).
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-granules of rat platelets-granules. The
IGF-I:IGFBP-3 complex was injected into the animals, and plasma levels
were monitored at 1 and 24 h by Western ligand analysis (Fig. 5, A–C). The analysis revealed a
prominent band characteristic of 28.7-kDa rhIGFBP-3 in the plasma
1 h after the injection (lane B) and minor bands before infusion
of the complex (lane A) corresponding to rat IGFBPs. After 24 h,
only 5% of the original 28.7-kDa rhIGFBP-3 remained in plasma (lane
C). Western ligand analysis of platelet lysate of a rat that received
the iv complex showed a new prominent band of 28.7 kDa (Fig. 5D),
whereas none of the 28.7-kDa protein was seen in platelet lysate of the
control rat injected with saline. The results were additionally
validated by immunoblotting with antihuman IGFBP-3 antiserum (data not
shown). Other forms of IGFBPs were also present in the platelet lysate
of hypophysectomized rats, albeit in reduced amounts. They were not
visible on the Western ligand blot due to the much shorter film
exposure time used to demonstrate the dominant 28.7-kDa IGFBP-3. The
wash of platelets collected before their activation with thrombin
contained only trace amounts of the 28.7-kDa IGFBP-3 (Fig. 5E).
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). One hour postinfusion, plasma
IGF-I levels increased 4-fold and reverted to preinfusion levels at
24 h. At 24 h, 4-fold higher IGF-I levels were detected in
the platelet -granule lysate of animals administered the iv
complex.
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Discussion |
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-granules of platelets. Our data show that
IGFBP-3 in the -granules is derived from the extracellular fluid in
the bone marrow by megakaryocytic endocytosis. The IGF-I in
-granules may also be incorporated with the IGFBP-3 as a complex. As
IGF-I transcripts were expressed in megakaryocytes, endogenous
synthesis could also contribute to the IGF-I in platelet -granules.
In addition, this study demonstrates that the platelet -granule
contents of both IGF-I and IGFBP-3 can be modulated by altering their
plasma levels. mRNA phenotyping detected no IGFBP-3 transcripts in megakaryocytes; however, IGF-I transcripts were demonstrated. Although our megakaryocyte preparations could contain up to 10% leukocytes, these results were shown to reflect megakaryocyte functions by an analysis of platelet mRNA. Platelets can be purified essentially free of other blood cells and contain an appreciable amount of mRNA derived only from megakaryocytes. RT-PCR of platelet mRNA for the presence of transcripts of PF-4, a megakaryocyte-specific factor, and IGF-I gave the same results as those using megakaryocyte mRNA. No IGFBP-3 transcripts were found in platelets underscoring its absence in megakaryocytes.
As our data showed that megakaryocytes did not express IGFBP-3 mRNA,
the IGFBP-3 present in the -granules of platelets (6) had to be
derived from another source. Direct uptake by platelets was first
excluded by in vitro studies, leaving the most likely
mechanism to be endocytosis of IGFBP-3 by megakaryocytes. It was
previously shown that albumin, fibrinogen, and Igs were incorporated
into the megakaryocytes from plasma via marrow fluid in this fashion
(16). If this mechanism pertains to IGFBP-3, then the platelet
-granule IGFBP-3 content should parallel the changes in serum
IGFBP-3 levels. This was demonstrated by Western ligand analysis in
pregnant rats, which shows a major decline in serum IGFBP-3 during late
pregnancy due to an extracellular IGFBP-3-specific protease (15).
Indeed, the dramatic decrease in the 40- to 45-kDa IGFBP-3 occurring in
the serum and marrow fluid of late pregnant rats was also reflected in
the platelet -granule IGFBP-3 content. In addition, the decline in
IGFBP-3 levels in platelet -granules was delayed during pregnancy in
the rat. This is consistent with the time required for endocytosis of
IGFBP-3 into megakaryocytes, packaging into -granules, and platelet
maturation. For the same reasons, a delay in the reappearance of
IGFBP-3 levels in -granules of platelets in postpartum rats was also
observed on Western ligand analysis, as the half-life of platelets in
rats is approximately 24 h. The changes in -granule IGFBP-3
content are probably not caused by intracellular protease digestion, as
the pregnancy-associated IGFBP-3 protease activity is generally
accepted to be an extracellular phenomenon (17). However, it is
theoretically possible that the protease could be endocytosed and thus
act intracellularly. This possibility will be explored in future
studies. The increase in the 24-kDa IGFBP (presumably IGFBP-4) and
30-kDa IGFBPs evident during late pregnancy in rats may be explained
by decreased competition from IGFBP-3, possibly for a common carrier,
or increased endocytosis to compensate for reduced levels of IGFBP-3
in the -granules. However, we have not tested this concept. As
other IGFBPs on a Western ligand blot of platelet lysate of pregnant
rats also mirrored the forms of IGFBPs found in corresponding serum
samples, the incorporation process may be common to other forms of
IGFBPs.
To further test the endocytotic mechanism of megakaryocytes, a foreign
IGFBP-3, rhIGFBP-3, complexed to human IGF-I was injected iv into
hypophysectomized rats. The appearance of this human 28.7-kDa IGFBP-3
in rat -granules 24 h later further established the endocytotic
mechanism. The recombinant human IGFBP-3 in the platelet -granule
lysate was not due to carry-over of plasma recombinant human IGFBP-3,
as the wash of the purified platelets contained only trace amounts of
the IGFBP-3, most likely due to mechanical lysis. Hypophysectomized
rats were used because their platelets contained very low IGF-I and
IGFBP-3 levels, minimizing any possible competition for the endocytosis
with the injected IGF-I:IGFBP-3 complex.
The iv injection of IGF-I:IGFBP-3 complex also caused higher
amounts of IGF-I to appear in rat platelets. If the complex was
endocytosed, this would explain the increase in IGF-I. The finding is
consistent with the previous demonstration of parallel changes between
platelet IGF-I levels and serum IGF-I levels during pregnancy and
postpartum in rats. To what extent endogenous synthesis by
megakaryocytes contributes to the -granule content of IGF-I has yet
to be shown. Although it is unlikely, it is theoretically possible that
the endocytosed IGFBP-3 could regulate the megakaryocyte synthesis of
IGF-I. However, a previous finding of equimolar amounts of IGF-I and
IGFBP-3 in -granules of platelets (6) strengthens the hypothesis
that -granule IGF-I and IGFBP-3 were probably derived from
extracellular fluid and housed in the -granules as a complex.
IGF-I and IGFBP-3 are probably endocytosed into megakaryocytes as a complex. If they were endocytosed separately, the mode of transport of each would involve either pinocytosis or receptor-mediated uptake. Neither mechanism offers sufficient explanation for IGF-I, because pinocytosis of small polypeptides is unusual, and cell surface receptors target small polypeptides for lysosomal degradation. However, when one considers the IGF-I:IGFBP-3 complex, both mechanisms are possible.
IGFBPs are known to associate with cell membranes (18), and IGFBP-3 has
been found to react strongly with a receptor-like protein in Hs578T
human breast cancer cells (19). Thus, we speculate that the endocytosis
of the IGF-I:IGFBP-3 complex is probably mediated by a IGFBP-3 carrier
on the cell membrane. In fact, Li et al. (20) have recently
reported the internalization and nuclear translocation of both IGF-I
and IGFBP-3 in proliferating opossum kidney cells, suggesting that an
endocytotic mechanism may operate in other cell systems. However, as
the function and fate of the endocytosed IGF-I and IGFBP-3 in
megakaryocytes are vastly different compared with those in other
tissues, unique mechanisms may be involved in their uptake and
packaging into -granules. We have further demonstrated that the
IGFBP-3 incorporated and stored in the platelet -granules is
functional.
The ability to modulate the concentrations of IGF-I and IGFBP-3
in -granules of platelets has many therapeutic potentials. First, in
wound healing, the IGF-I and IGFBP-3 levels in platelets are important
because platelet contents are released immediately after trauma
directly into the tissue, where additive and synergistic actions with
other growth factors in -granules, e.g. platelet-derived
growth factor, epidermal growth factor, and transforming growth
factor-ß, can promote tissue repair (21). As there is a significant
association of low plasma IGF-I and impaired wound healing, the list of
conditions in which wound healing might benefit from increasing the
-granule content of IGF-I and IGFBP is impressive: malnutrition,
glucocorticoid therapy, catabolic states, aging, chronic disease, GH
deficiencies, and trauma (2). Second, boosting the levels of IGF-I and
IGFBP-3 in platelets before blood donation may help prolong the
activity of platelets that is IGF dependent (see above). Third,
platelets are important for preserving vascular integrity. As
functional IGF-I receptors are present on endothelial cells (22),
increasing IGF-I levels in -granules may improve vascular integrity
and decrease the risk of bleeding in thrombocytopenia. Conversely,
there may be benefits from lowering the -granule content of
IGF-I:IGFBP-3. Shepard has postulated that the atheromatous process is
stimulated by deposition of IGF-I from plasma into the lesion (7).
Possibly a more significant source could be from the release of
-granule contents of IGF-I and IGFBP-3 (plus other growth factors)
during platelet activation. Lowering -granule IGF-I and IGFBP-3
contents could also be beneficial if IGF-I and IGFBP-3 were involved in
the smooth muscle hypertrophy postangioplasty (23) (probably by acting
synergistically with other growth factors). In the above-mentioned
conditions, the role of IGFBP-3 may not be passive because of its
ability to potentiate IGF-I actions in certain situations.
In summary, our findings demonstrate that megakaryocytes are
capable of endocytosing both IGFBP-3 and IGF-I from the extracellular
fluid and subsequently package them into platelet -granules. This
study also shows that platelet -granule levels of IGF-I and IGFBP-3
are modulated by their plasma concentrations. As the IGF-I, but not the
IGFBP-3, gene is expressed by megakaryocytes, there may be more than
one mechanism for directing IGF-I into the -granules of platelets.
The IGF-I synthesized by the megakaryocytes may also be exported or act
in a paracrine fashion. Further understanding of the underlying
mechanisms involved in the endocytosis and incorporation of IGF-I and
IGFBP-3 into platelet -granule should permit various IGFs-dependent
platelet functions to be influenced by manipulation of their
-granule contents.
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Footnotes |
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Received July 9, 1997.
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