(Received for publication, July 22, 1994; and in revised form, November 28, 1994)
From the
-Macroglobulin (
M) is a
potentially important regulator of platelet-derived growth factor-BB
(PDGF-BB)-stimulated cell growth due to our previous observation that
PDGF-BB binds to
M noncovalently (Bonner, J. C.,
Goodell, A. L., Lasky, J. A., and Hoffman, M. R.(1992) J. Biol.
Chem. 267, 12837-12844). We examined the in vitro effect of native and plasmin-activated (receptor-recognized)
M on the PDGF-BB-induced proliferation of mouse Swiss
3T3 and rat lung fibroblasts. Nondenaturing polyacrylamide gel
electrophoresis showed that plasmin converted
M to its
electrophoretically ``fast'' form at a 2:1 molar ratio and
that
I-PDGF-BB bound both
M and
M-plasmin. PDGF-BB-induced growth was not affected by
native
M (0.3 µM) or plasmin (0.6
µM). The combination of plasmin and
M
(2:1 molar ratio) inhibited PDGF-BB-induced cell proliferation
80-90%. Complexes of PDGF-BB
M purified by
gel filtration chromatography retained growth promoting activity, but
the PDGF-BB
M-plasmin complex did not.
Preincubation of fibroblasts (37 °C for 24 h) with
M-plasmin did not change
I-PDGF-BB
binding or affect gene expression of the 6.5-kilobase PDGF-
receptor or 5.2-kilobase PDGF-
receptor mRNA. However,
preincubation with
M-plasmin (0-4 °C for 4
h) increased
I-PDGF-BB binding 2-fold, and this increase
was blocked by a receptor-associated protein antagonist of the
M-receptor/low density lipoprotein receptor-related
protein. The receptor-associated protein antagonist blocked
I-
M-methylamine binding, inhibited
PDGF-BB-
M-plasmin uptake from fibroblast-cultured
supernatants, and abolished the inhibitory effect of
M-plasmin on PDGF-stimulated growth. These data
suggest that inhibition of PDGF-stimulated proliferation by
M-plasmin is mediated in part by clearance of
PDGF-BB-
M-plasmin through the lipoprotein
receptor-related protein.
Platelet-derived growth factor (PDGF) ()and
homologues of PDGF secreted by macrophages(1, 2) ,
endothelial cells(3) , smooth muscle cells(4) , and
fibroblasts (5) are mesenchymal cell mitogens and
chemoattractants that operate in the normal processes of development,
wound healing, and tissue remodeling(6) . The aberrant
expression of PDGF has been postulated as a common feature in the
progression of fibroproliferative diseases such as atherosclerosis (7) and pulmonary fibrosis(8, 9) . PDGF is a
disulfide-linked dimer of two polypeptide chains termed A or B that
give rise to functional PDGF-AA, PDGF-AB, or PDGF-BB
isoforms(10) . Two subtypes of PDGF cell-surface receptors,
termed PDGF-R
and PDGF-R
are present on cells of mesenchymal
origin(5, 11) , and two related but distinct cDNAs
encoding
and
PDGF receptors have been
cloned(12, 13) . The PDGF-A chain binds only the
PDGF-R
subtype, whereas the PDGF-B chain binds both PDGF-R
and PDGF-R
(11, 14) . Apparently, PDGF receptor
dimerization, mediated by ligand binding, is required for signal
transduction(15) . The precise role for each of the different
PDGF isoforms and receptor subtypes is not well understood, but it has
been suggested that the different subtypes of PDGF and receptors could
allow for fine tuning of cellular responses, due to observations that
different cell types can vary greatly in the ratio of PDGF isoforms
secreted and in the receptor subtype composition that the responding
target cell possesses(11) .
The biological activity of PDGF
is also regulated by -macroglobulin
(
M)(4, 16, 17, 18, 19) .
The function of
M as a proteinase inhibitor has been
well described and the mechanism whereby native or electrophoretically
``slow''
M covalently entraps serine,
aspartic, cysteine, and metalloproteinases has been extensively
studied(20, 21, 22, 23, 24) .
Proteinases ``activate''
M through cleavage
at a specific ``bait region'' which inititates a series of
conformational changes in the molecule that entraps the
proteinase(20) . The conformational change reveals latent
receptor recognition sites on the molecule and also makes the
M more compact; conferring ``fast'' mobility
when subjected to nondenaturing gel electrophoresis as compared to the
native or slow form of
M. The irreversible triggering
of the proteinase trap is mimicked by primary amines(25) , and
fast
M-proteinase or
M-amine complex,
but not native
M, bind high affinity receptors on
fibroblasts(26, 27) , hepatocytes(28) , and
macrophages(29, 30) . This receptor, termed the
M receptor/low density lipoprotein receptor-related
protein (LRP), binds
-migrating very low density lipoproteins
activated with apolipoprotein E as well as fast
Ms(31, 32) . It is synthesized as a
600-kDa precursor protein which undergoes post-translational processing
into a 515-kDa ligand-binding subunit and an 85-kDa transmembrane
subunit(33) . A 39-kDa protein that has been copurified with
LRP, termed receptor-associated protein (RAP), can reversibly bind to
the 515-kDa subunit and inhibit binding and uptake of ligands which
interact with the LRP(34) .
PDGFM
complexes have been isolated from plasma (17) and from
macrophage supernatants(18) . PDGF binds both native and
proteinase- or amine-activated forms of
M(35) , and PDGF-stimulated fibroblast
proliferation (19) and chemotaxis (36) are inhibited by
native
M at concentrations above 0.3 µM.
Below this concentration, the native form has no significant effect on
the biological properties of PDGF.
M activated with
methylamine synergistically enhances the growth promoting activity of
PDGF purified from human platelets (19) .
M
also binds and modulates the biological activities of several other
growth factors, including transforming growth factor-
(TGF-
)(37) , tumor necrosis factor-
(TNF-
) (38) , basic fibroblast growth factor(39) ,
interleukin-1
(IL-1
) (40) , interleukin-6
(IL-6)(41) , nerve growth factor (NGF)(42) , and
vascular endothelial growth factor(43) . The action of native
and activated forms of
M in regulating this wide
spectrum of growth factors has been reviewed(44) . However, the
precise role of
M in regulating growth factor activity
is poorly understood.
The potential modulatory activity of
proteinase-activated M on PDGF-stimulated mitogenesis
has not yet been investigated. Because methylamine-modified and
proteinase-modified fast
Ms are both receptor
recognized by fibroblasts, we hypothesized that both would possess
similar biological activities with regard to modulation of
PDGF-stimulated cell growth. On the contrary, we report that
plasmin-activated fast
M inhibited the proliferation
of Swiss mouse 3T3 fibroblasts and rat lung fibroblasts induced by
PDGF-BB, and this inhibitory effect was blocked by the RAP antagonist
of LRP. We hypothesize that, in extravascular tissues, native
(nonreceptor recognized)
M serves as a latent
reservoir for PDGF-BB, whereas proteinase-activated (receptor
recognized)
M serves to clear PDGF-BB through the LRP.
Figure 1:
[I]PDGF-BB
binds to native and plasmin-activated
M. Panel
A, Native
M (10 µg) was treated with plasmin
for 24 h at 37 °C prior to loading onto a nondenaturing 6%
Tris-borate-EDTA gel as described under ``Materials and
Methods.'' Increasing plasmin concentrations resulted in a
progressive increase in receptor-recognized, electrophoretically fast (F)
M with complete conversion occurring at a
2:1 molar ratio of plasmin to
M. Panel B,
autoradiography of TBE gel demonstrating that
M and
M-plasmin bound nearly equivalent amounts of
I-PDGF-BB. Native and plasmin-activated
M (10 µg) were incubated with
I-PDGF-BB (2 ng) at 37 °C for 24 h and
electrophoresis was performed as described
above.
Figure 2:
PDGF-BB-induced fibroblast proliferation
is inhibited by a combination of M and plasmin. The
proliferation of rat lung fibroblasts (panel A) and Swiss 3T3
fibroblasts (panel B) was measured as described under
``Materials and Methods.'' Subconfluent fibroblast cultures
were treated with increasing concentration of PDGF-BB in the absence or
presence of
M (0.3 µM), plasmin (0.6
µM), or a combination of
M and plasmin
(1:2 molar ratio) in serum-free defined medium. After 3 days in culture
the cells were removed from the plates by trypsin treatment and
enumerated with an electronic particle counter. Native
M (closed circles) or plasmin (open
triangles) had no significant affect on PDGF-stimulated cell
growth as compared to PDGF-BB alone (open circles, dashed
line), while the combination of
M and plasmin (closed triangles) inhibited PDGF-BB-stimulated proliferation.
Results are the mean of six separate experiments (S.E. < 5% of the
mean). Each assay was performed in triplicate
wells.
Figure 3:
The
PDGF-BBM complex retains mitogenic activity,
while the PDGF-BB
M-plasmin complex does not. One
mg of
M or
M-plasmin was incubated
with 200 ng of nonradioactive PDGF-BB for 24 h at 37 °C prior to
loading onto a Superose 6 FPLC column and elution in PBS, pH 7.4.
Column fractions were diluted 1:1 with serum-free defined medium and
tested for mitogenic activity on lung fibroblasts in a 3-day
proliferation assay as described under ``Materials and
Methods.'' The equivalent binding of
I-PDGF-BB (2 ng) to either
M or
M-plasmin (100 µg) was determined on Superose 6
FPLC in an identical manner as described above, and the column
fractions were
-counted. A, PDGF-BB bound
M-plasmin (open triangles, dashed
line) but the PDGF-BB/
M-plasmin did not elicit
mitogenesis of rat lung fibroblasts (solid line, closed
circles). A peak of mitogenic activity was apparent at the
approximate molecular mass of uncomplexed PDGF-BB. B, the
PDGF-BB
M complex stimulated fibroblast
proliferation by as much as 40% above control cells maintained in SFDM
alone. Arrows indicate the elution of molecular weight mass
markers: 1, blue dextran (V), 2,
thyroglobulin (669 kDa, also marks the elution of
M as
determined by ELISA), 3, apoferritin (440 kDa), 4,
-amylase (200 kDa), 5, BSA (66 kDa), 6, carbonic
anhydrase (29 kDa), and 7, cytochrome (12.4 kDa). The data are
typical of four separate experiments.
Figure 4:
M-plasmin and a 39-kDa
RAP antagonist compete for the specific binding of
I-
M-methylamine to fibroblasts.
Confluent quiescent cultures of rat lung fibroblasts (panel A)
or Swiss 3T3 fibroblasts (panel B) were chilled to 0-4
°C for 30 min and assayed for specific binding of
I-
M-methylamine as described under
``Materials and Methods.'' Increasing concentration
of
M-plasmin (open circles) and the 39-kDa
RAP antagonist (closed triangles) were added immediately prior
to the addition of 0.1 nM
I-
M-methylamine. A 200-fold molar
excess of native
M did not inhibit the specific
binding of
I-
M-methylamine (closed
squares). The RAP antagonist inhibited specific binding (IC
= 0.1-0.2 nM) with about a 5-fold lesser
potency than that of
M-plasmin (IC
= 0.5-1.0 nM). The data are representative
of four experiments each performed in
triplicate.
Figure 5:
The 39- kDa M RAP
antagonist does not compete for
I-PDGF-BB binding to
fibroblasts. Increasing concentrations of PDGF-BB or the RAP
M-receptor antagonist were added with 1 ng/ml
I-PDGF-BB to confluent rat lung fibroblasts rendered
quiescent in SFDM for 24 h. Receptor binding was assayed as described
under ``Materials and Methods.'' These data show
that RAP is a specific antagonist for the LRP and does not directly
interfere with PDGF-BB binding to its cell-surface receptor. The data
are representative of four experiments each performed in
triplicate.
Figure 6:
Preincubation of fibroblasts with
M-plasmin at 0-4 °C increases the specific
binding of
I-PDGF-BB. Confluent, quiescent rat lung
fibroblasts were incubated with
M or
M-plasmin, with or without the RAP antagonist, for 4 h
on ice prior to washing the cells three times with ice-cold binding
buffer and performing the radioligand binding assay for
I-PDGF-BB (20 ng/ml) as described under ``Materials
and Methods.''
M-plasmin, but not native
M, caused a 2-fold increase in the specific binding of
I-PDGF-BB that was significant (
p < 0.01 paired Student's t test), but only at
radioligand concentrations >10 ng/ml. The increase in
I-PDGF-BB binding was blocked by co-incubation with a
100-fold excess of the RAP antagonist. These data indicate that
I-PDGF-BB binds to surface-bound
M-plasmin, but only when the cell-surface PDGF
receptors are saturated. Data are the mean ± S.E. of four
separate experiments each performed in
triplicate.
Figure 7:
The uptake of I
PDGF-BB
M-plasmin complex by
fibroblasts in culture is inhibited by RAP. Confluent cultures of rat
lung fibroblasts grown in 10% FBS-DMEM in 150-cm
flasks
were rendered quiescent with SFDM for 4-6 h at 37 °C and then
treated with BSA-free SFDM alone or supplemented with 100 nM RAP for an additional 1 h prior to adding 1 nM
I
PDGF-BB
M or
I
PDGF-BB
M-plasmin complex.
These complexes were prepared by incubating 20 µg of
M or
M-plasmin with 0.3 ng of
I-PDGF-BB in a final volume of 100 µl for 4 h at 37
°C prior to diluting in 10 ml BSA-free SFDM and adding to 10 ml of
pre-existing medium (with or without RAP) on the cultures. The final
concentration of
M was 1 nM. At various time
points the media was removed, concentrated with a 100-kDa cutoff
filter, lyophilized, reconstituted to 50 µl, and electrophoresed on
a nondenaturing gel as described under ``Materials and
Methods.'' Following electrophoresis, the gels were dried
and stained with Coomassie Blue to visualize
M slow (S) and fast (F) forms (panel A). The same
gel was exposed to autoradiographic film to visualize
I-PDGF-BB bound to S and F forms of
M (panel B). The
I
PDGF-BB
M-plasmin complex,
but not the native complex, was taken up by the fibroblasts, and this
uptake was blocked the RAP antagonist.
Figure 8:
The RAP antagonist blocks inhibition of
PDGF-BB-induced growth by M-plasmin. Subconfluent
cultures of rat lung fibroblasts in 10% FBS-DMEM were washed three
times with SFDM and treated with PDGF-BB (10 ng/ml) in the absence or
presence of
M (0.3 µM), plasmin (0.6
µM), or a combination of both (2:1 molar ratio of plasmin
to
M) as described in Fig. 2. A 100-fold molar
excess of RAP antagonist significantly blocked the growth inhibitory
effect of the
M-plasmin mixture on PDGF-BB-stimulated
growth (**p < 0.01 paired Student's t test).
Data are expressed as the percent increase in cell number above that
obtained in SFDM without PDGF-BB and are the mean ± S.E. from
six separate experiments each performed in triplicate. All treatments
shown contain 10 ng/ml PDGF-BB.
M has been described as a potential
modulator of several growth factors and cytokines. Its precise function
in regulating growth factor activity appears to vary greatly depending
on the specific growth factor, the conformation of
M
to which the growth factor is bound, and the biological function
studied. In general,
M could serve several regulatory
roles, including 1), inactivation and clearance of growth
factors, 2) potentiation of growth factor activity,
3) protection of growth factors against proteolytic
inactivation, and 4) as a latent extracellular reservoir of
growth factors. In this study we report that
M
activated by plasmin is a negative growth regulator of PDGF-stimulated
fibroblast proliferation in vitro, and this appears to be
related to the clearance of PDGF-BB
M-plasmin
complex through an LRP-dependent mechanism. In contrast, the
PDGF-BB
M complex, which does not bind LRP or
PDGF receptors(18, 19) , retained growth-promoting
activity. This is likely explained by our previous observation that
PDGF is bound to
M in a reversible, noncovalent
manner, and PDGF-BB dissociates from
M with a
half-time of about 2 h(35) . PDGF-BB bound to
M does not bind PDGF-R
or PDGF-R
, apparently
because the receptor-recognized sites on the PDGF molecule are masked
by the
M molecule (18) . Thus, PDGF-BB was
probably released from the PDGF-BB
M complex
during the course of the 3-day growth assay to bind cell-surface PDGF
receptors (Fig. 3). The relative low affinity binding of PDGF-BB
to
M in the micromolar range (47) as compared
to PDGF-BB binding either the PDGF-R
or PDGF-R
in the
nanomolar range (Fig. 5) supports our hypothesis that native
M serves as a reservoir of latent PDGF-BB that can be
released to bind high affinity PDGF cell-surface receptors on
mesenchymal cells.
Our data suggest that the
M
plasmin complex does not suppress PDGF action
by down-regulation of the PDGF receptor, since preincubation of
fibroblasts with
M-plasmin did not alter PDGF-R
or PDGF-R
gene expression. Furthermore,
I-PDGF-BB
binding to fibroblasts was not affected by
M-plasmin
preincubation for 24 h at 37 °C. However, preincubation of
fibroblasts with
M-plasmin at 0-4 °C for 4
h, which prevents internalization of
M-plasmin
LRP complex, increased the specific
binding of
I-PDGF-BB 2-fold. This increase in specific
binding was observed only at concentrations of
I-PDGF-BB
above receptor saturation (0.3 nM
I-PDGF-BB) (Fig. 6). Recently, Crookston and co-workers (47) reported a K
of
0.3 µM for PDGF-BB binding to
M , indicating that
PDGF-BB binds to
M with an affinity 1000-fold less
than PDGF-BB binding to its own receptor. In general, this is in
agreement with our observation that
I-PDGF-BB will bind
to LRP-bound
M-plasmin only when cell-surface PDGF
receptors are occupied (i.e. at concentrations of
I-PDGF-BB above 10 ng/ml). We observed that the increase
in
I-PDGF-BB binding following
M-plasmin
preincubation on ice was blocked by the RAP antagonist of the LRP (Fig. 6). This proved that PDGF-BB bound to LRP-bound
M-plasmin at the cell surface, since excess
concentrations of RAP did not inhibit the binding of
I-PDGF-BB binding to its own receptor on fibroblasts (Fig. 5). The RAP antagonist inhibited
I-
M-methylamine binding to fibroblasts (Fig. 4), inhibited uptake of
I
PDGF-BB
M complex by
fibroblasts (Fig. 7), and abolished the inhibitory effect of
M-plasmin on PDGF-induced proliferation (Fig. 8). Thus, we concluded that the inhibitory effect of
M-plasmin on PDGF-BB-stimulated fibroblast growth is
mediated at least in part by uptake and degradation of
PDGF-BB
M-plasmin complex through the LRP.
Our
findings are novel in that we have demonstrated that fibroblasts can
mediate growth factor clearance via their cell-surface LRP, and this
results in a mitoinhibitory effect. The concentration of
M used in this study (300 nM) is much lower
than that found in plasma (2-4 µM) (23, 24) and could be similar to concentrations found
in extravascular spaces. Activated macrophages produce
M(18) , and we have observed that human lung
fibroblasts in culture spontaneously secrete
0.5 ng
M/million cells. (
)Thus, in vivo fibroblasts could have nanomolar levels of
M in
their immediate environment. Several other investigators have reported
that activated
Ms mediate the plasma clearance and
inactivation of growth factors and cytokines, including
PDGF-BB(48) , TGF-
1(48, 49) , and
TNF-
(38) . These studies are particularly relevant to
circumstances of vascular injury, where platelets degranulate and
release growth factors such as PDGF and TGF-
. Crookston and
co-workers showed that, while PDGF-BB and TGF-
1 bound both native
and methylamine-activated
M, only the
I
PDGF-BB
M-methylamine and
I
TGF-
1
M-methylamine
complexes were cleared from the circulation(48) . These
investigators suggested that native, non-receptor-recognized
M is the major carrier of TGF-
1 and PDGF-BB in
the blood, whereas activated
Ms mediate the rapid
clearance of these growth factors through the liver. This is a
reasonable assessment, since it has been established that TGF-
1
and PDGF-BB bind to slow and fast
Ms and because
M-proteinase complexes are cleared through the liver
with a half-time of minutes(28) . Our findings herein suggest
that
M and
M-proteinase complexes
could be critical mediators of growth factor clearance in extravascular
compartments, particularly during inflammation and tissue repair where
activated macrophages have accumulated and release numerous growth
factors (e.g. PDGF (1) , TGF-
(50) ,
IL-1
(51) ),
M(18) ,and a wide
spectrum of proteolytic enzymes(52) . We reported earlier that
the majority of macrophage-derived PDGF in culture supernatants is
complexed to macrophage-derived homologues of plasma
M
and that the macrophage-derived PDGF
M complex
does not bind to PDGF receptors, nor is it recognized by antibodies
raised against PDGF(18) . However, these complexes retain
PDGF-like activity for fibroblasts. Like fibroblasts, macrophages also
possess LRP (29) and potentially could reclaim secreted
PDGF
M complex in the presence of proteinases
through an LRP autocrine loop. This hypothetical situation would be
further complicated by inflammatory mediators (e.g. colony
stimulating factor, interferon-
, and lipopolysaccharide) which
have been shown to either up-regulate or down-regulate the macrophage
LRP(53, 54) .
We previously reported that
M-methylamine enhanced the growth promoting activity
of PDGF for fibroblasts(19) . A similar effect of
M-methylamine was reported for TGF-
1-induced
mitogenesis of smooth muscle cells(55) . TGF-
1 induces a
growth response in smooth muscle cells via a PDGF-AA autocrine loop (56) and
M-MA was recently shown to
synergistically enhance the TGF-
1-stimulated proliferation of
these cells(55) . On the other hand,
M blocks
the mitoinhibitory effect of TGF-
1 for mink lung epithelial
cells(57) . We postulated that the opposing effects of
M-plasmin and
M-methylamine on
PDGF-stimulated growth could be due to a differences in the fate of the
complexes once endocytosed, i.e. whether the
PDGF
M-methylamine or
PDGF
M-plasmin complexes follow the same pathways
of degradation or recycling to the cell surface. However, no
differences were observed in the fate of
I-PDGF-BB that
was endocytosed by fibroblasts in the absence or presence of
M-plasmin or
M-methylamine. In all
instances, the endocytosed radioactivity was released from fibroblasts
in a time-dependent manner that was maximal at 2 h and all of the
radioactivity possessed a molecular mass less than 30 kDa (i.e. degraded PDGF). Thus, the enhancement of PDGF-stimulated growth by
M-methylamine was not due to recycling of bioactive
PDGF through the LRP. Stouffer and co-workers reported that
M-methylamine, but not native
M,
stimulated mitogenesis of smooth muscle cells. Thus, it appears that
M-methylamine can directly induce mitogenesis, and
this finding is consistent with the discovery of a second
G-protein-coupled
M-receptor by Misra and co-workers (58) that possesses signaling properties. While it is clear
that
M-methylamine and
M-proteinase
complex bind and are cleared through the LRP, possible differences in
the interaction of
M-methylamine and
M-proteinase complex have not been investigated with
regard to the G-protein-coupled
M receptor. In the
present study, we observed that
M-methylamine, but not
M-plasmin, up-regulated the PDGF-
receptor
subtype (Table 1). We previously reported that up-regulation of
this receptor increases the mitogenic response of fibroblasts to PDGF (59) in a manner reminiscent of that observed with the
combination of
M-methylamine and PDGF(19) ; i.e. in both cases the growth response is increased
2-3-fold above the maximal response obtained with PDGF alone.
Thus, these data taken together suggest that
M-methylamine, but not
M-plasmin,
enhance PDGF-stimulated growth through up-regulation of the PDGF-
receptor subtype which then increases the mitogenic activity of PDGF
isoforms. It is important to note that proteinase-activated forms of
M, but not
M-methylamine, occur in vivo. Thus, an understanding of how
M-proteinase complexes modulate growth factor activity
should be more relevant to tissue repair and disease.
In light of
our findings, it seems reasonable to speculate that native and
proteinase-activated M differentially regulate PDGF-BB
activity in extravascular tissues, i.e. native
PDGF
M complexes purfied by Superose 6 FPLC
chromatography retained growth-promoting activity, while
PDGF
M-plasmin complexes did not (Fig. 3).
Other studies of growth factor/
M binding and clearance
have been related to the plasma(38, 48, 49) .
However, it is probable that
M regulates growth factor
activity in the extravascular tissues as well. Macrophages and
fibroblasts both produce
M and possess
LRP(60) . Thus, we postulate that native
M
serves as a reservoir of secreted PDGF in tissues, where it can be
released under conditions such as decreased pH(19) .
Conversely, proteinases secreted by inflammatory cells such as
macrophages and neutrophils mediate the slow to fast conformational
change in
M and could trigger the clearance of growth
factors via the LRP on macrophages and fibroblasts.
In summary, we
have shown that M-plasmin, but not
M
or plasmin alone, inhibits PDGF-BB-stimulated proliferation of rat lung
fibroblasts and Swiss mouse 3T3 fibroblasts. Preincubation of
M-plasmin complexes with these fibroblasts does not
alter PDGF-R
or PDGF-R
gene expression. The inhibitory effect
of
M-plasmin on PDGF-induced growth is blocked by the
RAP antagonist of the LRP (i.e. the
M
receptor). We hypothesize that
M fast forms activated
by proteinases function in vivo as a vehicle for PDGF
clearance and inactivation via the LRP, while native, slow
M serves as an extracellular reservoir for PDGF where
it could be released in the vicinity of responding target cells.