(Received for publication, April 3, 1997, and in revised form, May 30, 1997)
From the University of Memphis, Department of
Microbiology and Molecular Cell Sciences, Memphis, Tennessee 38152,
University of Wisconsin Medical School, Milwaukee Clinical
Campus, Department of Medicine, Sinai Samaritan Medical Center,
Milwaukee, Wisconsin 53233, Departments of ** Chemistry and
Pathology and Laboratory Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599, and
§ University of Tennessee-Campbell Clinic, Department of
Orthopaedic Surgery, Memphis, Tennessee 38163
In this study, endothelial cell-mediated clot
retraction was supported by fibrin generated from several purified
fractions of plasma fibrinogen, purified proteolytic fragments of
plasma fibrinogen, recombinant normal fibrinogen, and recombinant
variant fibrinogen. These results were surprising because some of these fibrinogens lack domains that are known binding sites for the integrin
receptors that support clot retraction. Specifically, fibrinogens
lacking A-chain RGD residues at 572-574 or lacking the
-chain
residues AGDV 408-411 supported endothelial cell-mediated clot
retraction as well as intact fibrinogen. Thus, clot retraction mediated
by endothelial cells is not dependent on either of these sites. A
variety of monoclonal antibodies against the integrin
v
3 partially inhibited the endothelial
cell-mediated retraction of clots formed from plasma fibrinogen. As
expected, an antibody to the platelet integrin
IIb
3 did not inhibit endothelial
cell-mediated clot retraction. These results indicate that this
retraction is mediated at least in part by
v
3. These results support the conclusion that (a) neither of the two fibrinogen cell binding sites
described above is required to support clot retraction or that
(b) either site alone or in conjunction with other
fibrin(ogen) region(s) can support clot retraction. Thus, endothelial
cell-mediated clot retraction appears to be dependent on fibrinogen
cell binding sites other than those required to support adhesion of
resting platelets to immobilized fibrinogen and platelet
aggregation.
This study was undertaken to evaluate the role in clot retraction
mediated by endothelial cells of presumptive endothelial cell and
platelet binding sites on fibrinogen. Platelets, fibroblasts, melanoma
cells, and endothelial cells are known to support clot retraction
(1-4). However, it is not known whether clot retraction mediated by
endothelial cells is dependent on either the presumptive endothelial
cell fibrinogen A-chain binding site, the 572-574 RGD residues (5),
or the fibrinogen
A-chain carboxyl-terminal AGDV sequence as is
resting platelet adhesion to immobilized fibrinogen (6, 7) and platelet
aggregation (8, 9).
Clot retraction is dependent on fibrin binding to activated
IIb
3 in platelets (10, 11) or to the
homologous integrin
v
3 (12, 13) in
nucleated cells (3, 4). Katagiri et al. (3) used monoclonal
antibodies and immunoelectron microscopy to show that clot retraction
mediated by melanoma cells is dependent on fibrin binding to
unstimulated
v
3. In their study, clot
retraction mediated by melanoma cells was blocked by RGD-containing
peptides and anti-
3 as well as
anti-
v
3
mAbs1 but not by an
IIb
3-specific inhibitor. The conclusion
that
v
3 can support clot retraction
mediated by nucleated cells was confirmed by Chen et al.
(4). Alemany et al. (14) provided evidence that a fibrinogen
A-chain binding region of the platelet integrin
IIb
3 is on its
3 subunit
and that ligand binding to this site is independent of platelet
activation. Their results support the possibility that the
v
3 integrin of endothelial cells, like
the homologous
IIb
3 on platelets, may be
able to bind fibrinogen via the
A-chain carboxyl termini and that
this hypothetical binding may play a role in endothelial-mediated clot retraction.
Fibrinogen platelet binding sites have been identified using a variety
of experimental systems. Farrell et al. (8) used recombinant
forms of fibrinogen to show that platelet aggregation appears to be
dependent on residues within the sequence 408-411 of the fibrinogen
A-chain. This was shown more directly by Rooney et al.
(9). These observations confirmed and extended earlier results obtained
using aggregated genetically modified
-chains (15). Under static
conditions, the adhesion of both resting and stimulated platelets to
immobilized fibrinogen appears to be dependent on the fibrinogen
A-chain carboxyl-terminal platelet binding sites (6, 7, 16). The
activation-independent adhesion of platelets to fibrinogen also appears
to be dependent on the fibrinogen
A-chain carboxyl-terminal platelet
binding sites under flow conditions (17). Rooney et al. (9),
using a recombinant form of fibrinogen (without the
A-chain terminal
sequence AGDV on either
A-chain), tested
A-chain involvement in
platelet aggregation and clot retraction. The recombinant fibrinogen
did not support platelet aggregation in response to ADP but did support
clot retraction. Therefore, the ligand sites on fibrinogen that support
platelet aggregation may be different than the sites on fibrin that
support clot retraction. These latter studies did not exclude the
possibility that normal fibrinogen secreted from the platelet
granules may have provided functional fibrinogen
A-chains to support
the clot retraction. However, a recent study by Holmback et
al. (18) confirms the conclusion of Rooney et al. (9)
by demonstrating that mice which have only fibrinogen lacking the QAGDV
sequence of both
-chains can support clot retraction even though the
altered fibrinogen cannot support platelet aggregation. These results raise the interesting possibility that non-
-chain platelet binding sites on fibrinogen can support platelet-mediated clot retraction (9,
18).
Others have reported the blocking of clot retraction by certain
RGD-containing peptides (presumably by binding to the receptors that
mediate retraction). Unfortunately this inhibition did not identify the
receptor binding site(s) on the ligand (10, 11). Likewise, the role of
the A-chain carboxyl-terminal platelet binding regions is not clear.
Despite the fact that the
A-chain carboxyl-terminal peptide mimetics
LGGAKQAGDV (L10) and HHLGGAKQAGDV (H12) have been shown to inhibit
platelet-mediated clot retraction (10), the dependence of clot
retraction on the corresponding platelet binding sites on fibrinogen
has not been shown. In fact, as described above, recent evidence
demonstrates that clot retraction mediated by human and mouse platelets
is not dependent on the AGDV sequence of the fibrinogen
A-chain (9,
18). Thus, care must be taken in interpreting the results of peptide
inhibition studies (19).
In the experiments described here, human umbilical vein endothelial
cells (HUVECs) were tested in clot retraction assays because, unlike
platelets, they do not secrete fibrinogen in response to treatment with
thrombin. Thus, various forms of exogenous fibrinogen in conjunction
with HUVECs would be useful to try to identify the cell binding site(s)
on fibrinogen that is required to support clot retraction. Peak 1 fibrinogen (A
A), fibrinogen fraction I-9 (a fibrinogen fragment
that lacks about 100 carboxyl-terminal residues from each A
-chain
including the 572-574 RGD sequence and that is bivalent with respect
to the
A-chain binding sites (20)), peak 2 fibrinogen fraction I-9
(a fragment of peak 2 fibrinogen that has one platelet reactive
A-chain and one nonplatelet reactive
-chain and the same
A
-chain composition as fibrinogen fraction I-9), recombinant normal
human fibrinogen (9), recombinant fibrinogen
407, which lacks
residues 408-411 in both
A-chains (9), and the mAb 4A5 (a mAb
specific for the carboxyl-terminal region of the
A-chain) (21)
were used to evaluate the role of presumptive fibrinogen cell binding
sites in clot retraction mediated by HUVECs.
HUVECs and endothelial cell growth medium (containing 10 ng/ml human recombinant epidermal growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, 3 mg/ml bovine brain extract, 2% fetal bovine serum) were purchased from Clonetics Corp. (San Diego, CA). The cells were grown to 80-95% confluence, and then a 0.025% trypsin, 0.01% EDTA solution (Clonetics) was used to release the cells from the surface of the flask, after which trypsin neutralization solution (Clonetics) was added. The cell suspension was centrifuged at 220 × g for 5 min, the supernatant liquid was decanted, and the cells were suspended in fresh endothelial cell growth medium. The suspended cells were allowed to recover from trypsinization by incubation for 30 min at 37 °C before use in the clot retraction assay.
Clot RetractionThe clot retraction assay was a modification of published methods (3, 4, 10). The cell suspension was centrifuged, the liquid was decanted, and the cells were washed three times with a Tyrodes Hepes solution (150 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2, 5.0 mM Hepes, pH adjusted to 7.35) containing 1 mg/ml glucose and 3.5% bovine serum albumin. The washed cells were added to the incubation solution (wash solution containing 2 mM CaCl, 3 µg/ml aprotinin, and 250 µg/ml fibrinogen or fragment) and allowed to incubate for 5 min at 37 °C. Cell numbers were estimated using a hemocytometer. ~2 × 105 cells were suspended in 0.5 ml of clotting medium (250 µg/ml fibrinogen or fragment in Tyrodes Hepes solution containing glucose, bovine serum albumin, CaCl2, and aprotinin) in a sylanized (using trimethylsylyl) aggregometer cuvette. mAbs that bind integrins were added to cells in incubation buffer before addition to clotting medium. mAbs that bind fibrinogens or their fragments were added to the clotting medium before the addition of the cells. Thrombin (Sigma) (1 unit/ml) was added to inititate fibrin formation. The retracting clot was photographed at several time intervals, and the photos were traced on a back-lit digitizing pad (Ortho-Graphics, Inc.) providing automated data entry into a computer. The longitudinal cross-sectional area not occupied by the clot was calculated and expressed as a percentage of the total area. The results are reported as percent clot retraction where 0% is no retraction and 100% would be complete retraction, which is undefined. Each clot retraction experiment was repeated at least once. Some experiments were repeated only once due to the large amount of antibodies and recombinant fibrinogen required. Although data are presented as quantitative, these studies are not meant to be a stringent quantitative analysis; however, the experiments provide unequivocal data as to the support or inhibition of HUVEC-mediated clot retraction by the forms of fibrinogen, antibodies, and peptides used in the experiments described above.
AntibodiesmAbs used were LM609 (50 µg/ml), which binds
to the v
3 receptor (22); CLB-706 (50 µg/ml), which binds to
v (LM609 and CLB-706 were from
Chemicon International, Inc.); 7E3 (50 µg/ml), which binds to
IIb
3 (23) and
v
3 (24) (courtesy of Dr. Barry Coller);
A2A9 (50 µg/ml), which binds to
IIb
3
(25) (courtesy of Dr. J.S. Bennett); AP3 (100 µg/ml), which binds the
3 subunit (26) (courtesy of Dr. Peter Newman); 4A5 (21)
50 µg/ml (courtesy of Dr. Gary Matsueda), which binds the fibrinogen
A-chain carboxyl terminus and prevents platelet adhesion; and JB1a
(1:100 dilution from ascites fluid), an anti-
1 antibody
(27). Mouse IgG (mIgG) (Sigma) (50 µg/ml) was used as control
IgG.
The following fibrinogens were used in this
study to identify the binding sites utilized by endothelial cells to
support clot retraction: peak 1 fibrinogen (A
A) (6), peak 1 fibrinogen fraction I-9 (20), peak 2 fibrinogen fraction I-9 (6), and fibrinogen 325, which lacks the first 42 amino acids (amino terminus) of the B
-chains (28, 29). The fibrinogens used and the important binding sites present or absent on each form are listed in Table I.
Peak 1 (
A
A) and peak 2 (
A
) fibrinogens were prepared as
described by Mosesson and Finlayson (30) from fraction I-2 fibrinogen
(31) containing >80% intact A
-chains (20) or from fraction I-9
fibrinogen (20). Fraction I-9 fibrinogen is devoid of intact
A
-chains and contains instead A
-chain derivatives of the size of
B
-chains (54 kDa) or smaller (20) that lack carboxyl-terminal
segments. Its composition was verified by SDS-polyacrylamide gel
electrophoresis. Chromatographic separation into peak 1 and peak 2 subfractions was verified by DEAE-cellulose ion exchange chromatography
using the gradient elution system described by Siebenlist et
al. (32). Fibrinogen 325 (des-B
1-42 fibrinogen) that lacks the
first 42 amino acids of B
-chains (33) was produced from fraction I-2
fibrinogen as described by Pandya et al. (28) and Pandya and
Budzynski (29). Its structure was verified by SDS-polyacrylamide gel
electrophoresis.
|
Normal recombinant fibrinogen and recombinant fibrinogen 407 (which
lacks residues 408-411 on both
A-chains) were synthesized by
transfected Chinese hamster ovary cells, and purification was monitored
as described (9). Briefly, samples were run on SDS-polyacrylamide gel
electrophoresis under reduced conditions according to the method of
Laemmli (34) and appeared as three bands corresponding to the A
-,
B
-, and
-chains. Western blot analysis was performed as described
(35) using 4A5. Normal recombinant and plasma fibrinogen developed
bands corresponding to the
-chain; however, the
-chain from
407 fibrinogen was undetectable, consistent with previous results
(9).
Peptide inhibitors of fibrinogen binding to
platelets were tested as inhibitors of endothelial cell-mediated clot
retraction. The peptide LGGAKQAGDV, a A-chain carboxyl-terminal
fibrinogen peptide mimetic, GRGDSP, and a control scrambled version of
GRGDSP, PGRSGD, were tested in the clot retraction assay at different concentrations. Quantity and sequences were verified by St. Jude Children's Research Hospital Biotechnology Center laboratories. The
methods used for the synthesis, purification, and characterization of
these peptides have been described (19).
Endothelial cells retracted clots formed from thrombin-treated
peak 1 fibrinogen A
A, peak 1 fibrinogen fraction I-9, and peak 2 fibrinogen fraction I-9 (Fig. 1). The
rates of clot retraction mediated by the HUVECs were approximately the
same for all three types of fibrinogen used in these experiments (Fig.
2). The ability of peak 1 fibrinogen
fraction I-9 to support clot retraction mediated by the endothelial
cells means that the A
-chain RGD (572-574) sequence was not
required for retraction. Furthermore, the fact that clot retraction was
also supported by peak 2 fibrinogen fraction I-9 demonstrates that if
the carboxyl terminus of the
A-chain is required to support clot
retraction, a single
-chain is sufficient.
The mAb 4A5 was used to determine if endothelial cell-mediated clot
retraction is dependent on the carboxyl terminus of the fibrinogen
A-chain. This mAb antibody was used because it binds to the carboxyl
terminus of the fibrinogen
A-chain (19) and thereby inhibits
cross-linking of fibrinogen by Factor XIIIa (19) and the adhesion of
platelets to immobilized fibrinogen (6). As shown in Fig. 3, the mAb
4A5 inhibited the retraction of clots formed from both peak 1 fibrinogen and fibrinogen fraction I-9 compared with mIgG controls.
This inhibition of endothelial cell-mediated clot retraction by the mAb
4A5 indicated either a requirement for the
A-chain carboxyl terminus
or an indirect inhibitory effect on clot retraction by the antibody. To
distinguish between these alternatives, recombinant fibrinogen
containing normal A
- and B
-chains, but lacking residues
A408-411 (9) was used in the clot retraction assay. Recombinant
normal fibrinogen was used as a control. The recombinant normal
fibrinogen supported the adhesion of normal and resting platelets (data
not shown) and clot retraction in HUVECs (Fig. 1). Likewise,
recombinant fibrinogen
407, though it did not support the adhesion
of resting platelets (data not shown), supported clot retraction
mediated by HUVECs without any apparent impairment of function (Figs. 1
and 4).
Fibrinogen 325, which lacks the first 42 amino acid residues of the
B-chains was also used in the clot retraction assay. It has been
shown that fibrin prepared from fibrinogen molecules lacking residues
1-42 of the B
-chains failed to support endothelial cell spreading
(36, 37). Fibrinogen 325 in this study supported HUVEC clot retraction
(data not shown).
Two peptides that inhibit platelet-mediated clot retraction were tested
in the HUVEC system (10). Although A-chain carboxyl-terminal mimetic
peptide L10 (LGGAKQAGDV) inhibited platelet-mediated clot retraction
(data not shown), it did not inhibit retraction in the HUVEC system at
the concentrations up to 6 mM (Fig.
5). In contrast, the RGD peptide GRGDSP,
but not a scrambled control peptide (PGRSGD), inhibited clot retraction
mediated by platelets (10) (data not shown) and endothelial cells (Fig.
5).
Anti-integrin mAbs also provided useful information concerning the
details of endothelial cell-mediated clot retraction. First, the
v
3-specific mAb LM609 partially inhibited
clot retraction (Fig. 6A). The
v-specific mAb CLB-706 also inhibited clot retraction (Fig. 6A). These results confirm observations made by others
(3, 4) indicating a role for the
v
3
integrin in clot retraction mediated by a variety of nucleated cells.
Similarly, the mAb 7E3, but not A2A9, inhibited clot retraction in
HUVECs (Fig. 6B). In contrast, both of those mAbs inhibited
platelet-mediated clot retraction, presumably reflecting a role for
IIb
3 in the process (Fig. 6B).
These data demonstrate that clot retraction mediated by HUVECs is
v
3-dependent since LM609,
CLB-706, and 7E3 but not A2A9 or JB1a (which blocks the function of
1 integrin subunits) can inhibit
v
3-dependent functions.
Finally, the AP3 mAb, which binds to the
3 integrin
subunit and can inhibit platelet-mediated clot retraction (10, 28),
also partially inhibited (at a concentration of 100 µg/ml) clot
retraction in the HUVEC system (Fig. 6A). The
IIb
3-specific mAb Tab, which also
inhibits platelet-mediated clot retraction (10), did not inhibit
endothelial cell-mediated clot retraction (50 µg/ml) compared with a
mouse IgG control (data not shown).
Studies by Cheresh et al. (5) and others (16) provide
evidence implicating the fibrinogen A-chain RGD 572-574 sequence as
a putative endothelial cell binding site recognized by
v
3 (22). These studies also demonstrated
that the fibrinogen A
-chain 95-97 RGD residues do not play a
significant role in HUVEC adhesion to fibrinogen (5, 16). mAb data
indicated that endothelial cell adhesion to this region of immobilized
fibrinogen was mediated by the integrin
v
3 (5, 22). Consequently, the possibility was tested that the fibrinogen A
-chain 572-574 sequence is required to support
v
3-mediated clot retraction by
HUVECs. This possibility was tested by using fibrinogen fragment
fraction I-9 in an endothelial cell-supported clot retraction assay.
Surprisingly, the fibrinogen fraction I-9 (Table
I) supported apparently normal clot
retraction. These results mean that the A
-chain RGD (572-574)
sequence is not required for clot retraction mediated by HUVECs.
The finding that HUVECs-mediated clot retraction is not dependent on
the fibrinogen A-chain RGD 572-574 sequence resulted in the
evaluation of the role of the fibrinogen
A-chain carboxyl-terminal sequences in the endothelial cell clot retraction system. Four approaches were used for this aspect of the study. Proteolytic fragments of fibrinogen, an anti-fibrinogen mAb, recombinant
fibrinogens, and peptides were used in these studies. Peak 2 fibrinogen
fraction I-9 (Table I) was found to support HUVEC-mediated clot
retraction normally (Fig. 1), demonstrating that if a fibrinogen
-chain carboxyl terminus is required, a single functional
A-chain
carboxyl terminus is sufficient even in the absence of the A
-chain
572-574 RGD sequences. The mAb 4A5 results reflected either a
requirement for the
A-chain carboxyl-terminal platelet binding site
or indirect inhibitory effects caused by 4A5 (Fig. 3). Use of
recombinant human fibrinogen, which does not support platelet
aggregation (9) or the adhesion of resting platelets (data not shown), supported normal clot retraction in the endothelial cell clot retraction system (Fig. 4). These results demonstrate that the inhibition of clot retraction by mAb 4A5 apparently resulted from indirect effects (steric hindrance or the induction of an incompatible conformational change of the fibrinogen), since clot retraction was not
dependent on the presence of the AGDV sequence of the fibrinogen
A-chain. The
A-chain carboxyl-terminal mimetic peptide L10 has
been shown to inhibit platelet-supported clot retraction (10) but did
not inhibit clot retraction at concentrations up to 6 mM
mediated by endothelial cells in our assays (Fig. 5). The data from the
proteolytic fragments of fibrinogen, an anti-fibrinogen mAb,
recombinant fibrinogens, and peptides demonstrate that HUVEC-mediated clot retraction is not dependent on the 408-411 AGDV sequence of the
fibrinogen
A-chain. Thus, the fibrinogen cell binding sites required
to support endothelial cell-mediated clot retraction are different than
those required to support platelet aggregation (8, 9) and adhesion (6,
7, 16, 17).
The amino-terminal region of the fibrinogen B-chain is a presumptive
endothelial cell interaction site. Since this region of the B
-chain
appears to affect the interaction of HUVECs with fibrin, a
protease-treated form of fibrinogen that lacks the first 42 amino acid
residues of the B
-chains (fibrinogen 325) (28, 29) was tested and
shown to support clot retraction (data not shown). Even though this
region has been shown to interact with HUVECs, it is not necessary to
support HUVECs clot retraction. The amino-terminal 42 residues of the
B
-chains are therefore not required to support clot retraction
mediated by HUVECs.
The results of this study reveal that HUVECs can mediate clot
retraction in a manner that is not dependent on the A-chain 572-574
RGD sequences, the 408-411 AGDV sequence of the
A-chain carboxyl-terminal platelet binding sites, or residues 1-42 of the
fibrinogen B
-chains. In summary, the data mean that (a)
either of these fibrinogen cell binding sites may be sufficient to
support clot retraction mediated by HUVECs or that (b)
another region(s) of fibrinogen alone or in conjunction with either of
the above mentioned fibrinogen platelet or nucleated cell binding sites can be utilized by endothelial cells to support clot retraction.
The data presented here for HUVECs agree with those reported by others
for the role of the fibrinogen A-chain in clot retraction mediated
by platelets (9, 18). Rooney et al. (9) report that the
IIb
3 binding sites on fibrinogen that
mediate platelet aggregation appear to differ from the
IIb
3 binding site(s) on fibrin that are
used during clot retraction (9). Their conclusion that the
A-chain
408-411 AGDV sequence is not required for clot retraction was
supported by the results of Holmback et al. (18) obtained
using homozygous mutant mice. The results presented here extend the
observation that the AGDV sequence is not required to support clot
retraction to a new system, the endothelial cell clot retraction
system. Additionally, these results demonstrate that clot retraction in
this system is not dependent on either the A
-chain 572-574 RGD
sequence or the B
-chain 1-42 residues.
A role for the v
3 integrin in endothelial
cell-mediated clot retraction is supported by the facts that the
v
3-specific mAb LM609, the
v-specific mAb CLB-706, and the mAb 7E3, which blocks
both
v
3 and
IIb
3, inhibited endothelial cell-mediated clot retraction, whereas the
IIb
3-specific A2A9 and the
anti-
1 function blocking mAb, JB1a, did not inhibit clot
retraction in this system. The fact that the mAb AP3 partially
inhibited clot retraction may indicate a role for residues within the
3 348-426 sequence, which encompass the AP3 binding
site, in clot retraction mediated by endothelial cells as well as
platelets, since AP3 has also been shown to inhibit platelet-mediated
clot retraction (10, 38). Alternatively, the inhibitory effect of AP3
may be indirect.
Finally, the observations that sites other than the A-chain 408-411
AGDV sequences and the A
-chain 572-574 sequences appear to be
required for both platelet (fibrinogen fraction I-9 supports platelet-mediated clot retraction, data not shown) and HUVEC-mediated clot retraction may mean that receptor sites other than those used for
platelet aggregation and platelet and endothelial cell adhesion are
used to bind fibrin during clot retraction mediated by either platelets
or HUVECs.
We thank Jason Holcomb, Stephanie Long, Tursha Hamilton, and Robert Loudon for their invaluable technical assistance with this project.