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The Contribution of the Three Hypothesized Integrin-Binding Sites in Fibrinogen
to Platelet-Mediated Clot Retraction
By Michael M. Rooney, David H. Farrell, Bettien M. van Hemel, Philip G. de Groot, and Susan T. Lord
Fibrinogen is a plasma protein that interacts with integrin
aIIbb3 to mediate a variety of platelet responses including
adhesion, aggregation, and clot retraction. Three sites on
fibrinogen have been hypothesized to be critical for these
interactions: the Ala-Gly-Asp-Val (AGDV) sequence at the
C-terminus of the g chain and two Arg-Gly-Asp (RGD)
sequences in the Aa chain. Recent data showed that AGDV is
critical for platelet adhesion and aggregation, but not retraction, suggesting that either one or both of the RGD sequences are involved in clot retraction. Here we provide
evidence, using engineered recombinant fibrinogen, that no
one of these sites is critical for clot retraction; fibrinogen
lacking all three sites still sustains a relatively normal, albeit
delayed, retraction response. Three fibrinogen variants with
the following mutations were examined: a substitution of
RGE for RGD at position Aa 95-97, a substitution of RGE for
RGD at position Aa 572-574, and a triple substitution of RGE
for RGD at both Aa positions and deletion of AGDV from the
g chain. Retraction rates and final clot sizes after a 20-minute
incubation were indistinguishable when comparing the Aa
D97E fibrinogen or Aa D574E fibrinogen with normal recombinant fibrinogen. However, with the triple mutant fibrinogen, clot retraction was delayed compared with normal
recombinant fibrinogen. Nevertheless, the final clot size
measured after 20 minutes was the same size as a clot
formed with normal recombinant fibrinogen. Similar results
were observed using platelets isolated from an afibrinogenemic patient, eliminating the possibility that the retraction
was dependent on secretion of plasma fibrinogen from
platelet a-granules. These findings indicate that clot retraction is a two-step process, such that one or more of the three
putative platelet binding sites are important for an initial
step in clot retraction, but not for a subsequent step. With
the triple mutant fibrinogen, the second step of clot retraction, possibly the development of clot tension, proceeds
with a rate similar to that observed with normal recombinant fibrinogen. These results are consistent with a mechanism where a novel site on fibrin is involved in the second
step of clot retraction.
r 1998 by The American Society of Hematology.
C
copies per cell.1 This receptor binds ligand after platelet
activation by an agonist. The receptor is a member of the
integrin superfamily of receptors and is composed of an a and a
b subunit, specifically aIIbb3.2 aIIbb3 is a receptor found on
platelets and their parental cells, megakaryocytes. Previous
experiments using antibodies directed against either aIIb, b3, or
the receptor complex have shown that this receptor is required
for platelet adhesion, platelet aggregation, and plateletmediated clot retraction.3-5 These results were confirmed using
platelets isolated from patients diagnosed with Glanzmann’s
thrombasthenia, a condition in which functional aIIbb3 is not
expressed on the surface of platelets. These platelets do not
adhere to immobilized fibrinogen, do not aggregate in the
presence of soluble fibrinogen and agonist, and do not support
clot retraction.6-8
Three sites on fibrinogen have been hypothesized to bind to
aIIbb3 during interactions with platelets: two Arg-Gly-Asp
(RGD) sites and the C-terminal portion of the g chain. RGD is a
consensus binding sequence for integrins and was identified
using synthetic peptides9; the two RGD sequences in fibrinogen
are both located in the Aa chain at residues 95-97 and
572-574.10 The third site is specific for fibrinogen and is
composed of the 12 C-terminal residues of the g chain (H12:
HHLGGAKQAGDV).11,12 The role of each of these sites in the
interactions of fibrinogen with platelets has been probed in a
variety of ways. Initially, these sites were identified because
peptides containing the RGD or H12 sequence inhibit fibrinogen binding to platelets, a prerequisite for aggregation.12,13
Subsequent experiments using antibodies directed against either
H12 or the Aa RGD at position 95-97 implicated both sites as
players in fibrinogen binding to aIIbb3 and in platelet aggregation.14 In addition, experiments using an RGDS-containing
peptide, the sequence corresponding to the Aa 572-575 putative
binding site, suggested that this C-terminal site in the Aa chain
interacts with the fibrinogen receptor on activated platelets.15
Direct observation of aIIbb3-fibrinogen complexes by electron
LOT RETRACTION IS believed to be a primary step in
the clearance of a thrombus, which initiates wound
healing. The process of clot retraction is modeled in vitro using
three blood components: fibrinogen, platelets, and thrombin.
Fibrinogen is a large (340 kD) glycoprotein composed of six
polypeptide chains: two each of Aa, Bb, and g. Platelets are
small, anuclear cells that contain a variety of inflammatory
agents and are responsible for formation of an initial hemostatic
plug at sites of injury. Thrombin, a serine protease, converts
soluble fibrinogen to fibrin monomers that spontaneously
polymerize in an ordered fashion to form fibrin fibers. These
fibers serve as the lattice of a clot and function as the ligand for
platelets during clot retraction. Thrombin, in addition, is a
platelet agonist that activates platelets by cleaving a specific
receptor.
Platelets express a specific receptor for fibrinogen and fibrin
on their surface in a high-copy number, approximately 45,000
From the Departments of Chemistry and Pathology and Laboratory
Medicine, University of North Carolina at Chapel Hill, Chapel Hill,
NC; the Department of Biochemistry and Molecular Biology, Pennsylvania State University, College of Medicine, Hershey, PA; and the
Department of Haematology, University Hospital Utrecht, Utrecht, The
Netherlands.
Submitted March 31, 1998; accepted May 28, 1998.
Supported by the National Institutes of Health Grants No. HL 31048
and HL 45100 (S.T.L.) and a predoctoral fellowship from the American
Heart Association, NC Affıliate (M.M.R.).
Address reprint requests to Susan T. Lord, PhD, University of North
Carolina, Department of Pathology and Laboratory Medicine, CB#
7525, 605 Brinkhous-Bullitt Bldg, Chapel Hill, NC 27599.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9207-0027$3.00/0
2374
Blood, Vol 92, No 7 (October 1), 1998: pp 2374-2381
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FIBRINOGEN SITES FOR CLOT RETRACTION
microscopy supported the conclusion that the C-terminal g
chain domain is the primary interaction site.16 Genetically
engineered fibrinogen variants have since shown that the Aa
chain RGD sequences are not required for binding of aIIbb3 to
soluble fibrinogen or for platelet aggregation. Instead, the g
chain dodecapeptide, specifically residues Ala-Gly-Asp-Val
(AGDV), seems to be the important sequence on fibrinogen for
aIIbb3 binding and platelet aggregation.17-19
In contrast, the g chain binding site is not required for clot
retraction in either human or mouse systems.19,20 Therefore, it
has been suggested that the RGD sites in the Aa chain mediate
clot retraction. We tested this hypothesis using three variant
fibrinogens, two with substitutions of RGE for RGD at either
Aa 95-97 or Aa 572-574, and a third variant with both RGD
sites changed to RGE and the AGDV residues of the g chain
H12 sequence deleted. Our results indicate that loss of all three
hypothesized platelet binding sites on fibrinogen only influence
the initial phase of clot retraction and are consistent with the
presence of an additional, unknown site on fibrin that is
involved in clot retraction.
MATERIALS AND METHODS
Materials. The Chinese hamster ovary (CHO) cells; culture media;
and vectors pMLP-Aa, pMLP-Bb, and pMLP-g; encoding for the three
fibrinogen cDNAs; have been described.21 Restriction enzymes were
purchased from New England Biolabs (Beverly, MA). Monoclonal
antibody IF-1 was kindly provided by Dr Michio Matsuda (Institute of
Hematology, Jichi Medical School, Tochigi-Ken, Japan). Human
a-thrombin was a generous gift of Dr Frank Church (University of
North Carolina at Chapel Hill).
Construction of mutant expression vectors. A mammalian vector
encoding for the deletion of residues AGDV from the C-terminus of the
g chain (pMLP-g 407) has been described.22 Likewise, baby hamster
kidney (BHK) cells expressing recombinant fibrinogen with RGE
substituted for RGD at either position Aa 95-97 or Aa 572-574 were
previously constructed and cells grown as described.17 The proteins
expressed by these cells are referred to as Aa D97E and Aa D574E,
respectively, and were purified as outlined below.
A plasmid encoding for RGD to RGE mutations at both sites in the
Aa chain (pMLP-Aa DE2) was constructed using plasmid pMLP-Aa
and the Clontech Transformer Site Directed Mutagenesis kit (Palo Alto,
CA), as described below. The mutagenic primers have the following
sequences: Aa D97E-TTG AGA GGA# GAG* TTT TCC TCA GC and
Aa D574E-AGA GGA GAG* TCA° ACG° TTT GAA AGC, where *
indicates a base substitution resulting in a D to E mutation, # indicates
the silent introduction of a BseRI site, and ° marks the base changes
required to introduce a silent Psp1406I site. The selection primer has the
sequence TCT AGG GCC CAG GCT TGT TTC C, and encodes for the
deletion of a unique HindIII site located in the vector. The parent
plasmid was heated to 100°C in the presence of 58-phosphorylated
selection and mutagenic oligonucleotides and allowed to anneal on ice.
T4 DNA polymerase and T4 DNA ligase were added and the mutant
DNA strand was synthesized at 37°C. The parental plasmid DNA was
linearized with HindIII and the plasmid mixture used to transform BMH
71-18 mut S bacteria (Clontech) by electroporation using a Gene Pulser
(Bio-rad, Hercules, CA). Bacteria were grown in culture overnight and
the plasmid pool was isolated using a Qiagen Plasmid Kit (Chatsworth,
CA). Purified plasmid pools were linearized with HindIII and the
products were used to transform DH5aF8 bacteria by electroporation
using a Gene Pulser (Bio-rad). The bacteria were selected by growth on
ampicillin-containing media and plasmids were isolated from resistant
colonies using the Qiagen Plasmid Kit. The introduction of both RGD to
2375
RGE mutations was confirmed by digestion with BseRI and Psp1406I
followed by sequencing the entire fibrinogen Aa chain cDNA using an
Applied Biosystems automated sequencer (Foster City, CA). The
positively identified plasmid is referred to as pMLP-Aa DE2.
Synthesis of recombinant fibrinogens. The preparation of a CHO
line expressing recombinant fibrinogen with the desired alterations was
performed essentially as described.21 Briefly, plasmids pMLP-Aa DE2,
pMLP-g 407, and pMSVhis were used to transfect CHO cells containing pMLP-Bb and pRSVneo. Colonies that grew in the presence of
histidinol (Sigma Chemical Co, St Louis, MO) and G418 (Life
Technologies, Inc, Grand Island, NY) were selected and screened for
fibrinogen expression by enzyme-linked immunoassay (ELISA). The
clone with the highest expression level was used to seed roller bottles.
Each week all 200 mL of serum-free media from roller bottles
containing CHO cells was collected and pooled, and phenylmethylsulfonyl fluoride was added to 150 µmol/L, and stored at 270°C until the
fibrinogen was purified. Fresh media were added to the cultures and
fibrinogen production continued for 3 months. The fibrinogen purified
from the media obtained from these clones is designated Aa DE2/g 407.
Normal recombinant fibrinogen was grown in roller bottles and media
collected as described.20
Purification and characterization of recombinant fibrinogens. All
of the recombinant fibrinogens were purified from pooled serum-free
media collected from CHO or BHK cells as described.23 Briefly,
fibrinogen was precipitated from the media by adding ammonium
sulfate to 40% final concentration. The precipitate was collected by
centrifugation at 16,000g at 4°C and resuspended in Tris-buffered
saline, pH 7.4 (TBS; 20 mmol/L Tris-HCl and 150 mmol/L NaCl) with 1
mmol/L CaCl2. The resuspended precipitate was clarified by centrifuging at 27,000g before loading onto an immunoaffinity column equilibrated in the same buffer. The column was washed consecutively with
high salt (1 mol/L NaCl) and low pH (6.0) buffers followed by TBS with
10 mmol/L EDTA. The antibody (IF-1) is calcium dependent, binding
fibrinogen in the presence of calcium and failing to bind fibrinogen in
the absence of calcium.24 Therefore, fibrinogen was eluted with
EDTA-containing buffer. The protein was then dialyzed for 3 hours at
4°C against 1 L TBS with 1 mmol/L added calcium followed by
extensive dialysis against TBS without added calcium. The dialyzed
fibrinogen was aliquoted and stored at 270°C until it was used. Except
for the dialysis steps, every step of the purification procedure was
performed in the presence of a cocktail of protease inhibitors, as
previously described.23 The purity of the recombinant fibrinogens was
monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to the method of Laemmli.25
Preparation of platelets. Platelets were prepared from a normal
donor’s blood as described.19 Platelets prepared from a patient diagnosed with afibrinogenemia were purified with the following modifications, as described.26 Human blood (60 mL) from the patient, who had
abstained from aspirin, was collected in tubes containing one-tenth
volume of 3.4% acid/citrate/dextrose (ACD) anticoagulant. Plateletrich plasma was obtained by centrifugation at 150g for 10 minutes at
room temperature. ACD was added to 10% final concentration before
applying the platelet-rich plasma to a Sepharose CL-2B column
(Sigma) equilibrated with Tyrodes buffer, pH 7.2 (10 mmol/L Hepes,
135 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L NaHCO3, 5.5 mmol/L
glucose, 2% bovine serum albumin [Fr V, pH 7.0; Bayer Corp,
Kankakee, IL]). The platelets were eluted with Tyrodes and ACD added
to 10% final concentration before centrifuging the platelet suspension at
700g for 10 minutes at ambient temperature. The platelet pellet was
resuspended in Tyrodes buffer, counted with a Cell-Dyn 1600 (Abbott
Diagnostics, Abbott Park, IL) and adjusted to a final concentration of
4 3 108 platelets/mL with 1 mmol/L calcium and 2 mmol/L magnesium.
Platelet aggregation. Aggregation experiments with gel-filtered
platelets were performed as described.19 Fibrinogen was added to
platelets to give final concentrations of 250 nmol/L and 2 3 108
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2376
ROONEY ET AL
platelets/mL, respectively. The sample was placed in an aggregometer
(Chrono-Log Corp, Haverton, PA) with stirring at 37°C, using Tyrodes
buffer as the reference solution. Adenosine diphosphate (ADP; ChronoLog Corp) was added to a final concentration of 10 µmol/L and the
change in light transmission was recorded versus time.
Clot retraction. Platelet-mediated clot retraction experiments were
performed similarly to Tuszynski et al27 and as described.19 Briefly,
fibrinogen (final concentration 300 nmol/L) was preincubated with
platelets (final concentration 2 3 108 platelets/mL) at 37°C for 10
minutes before adding human a-thrombin to a final concentration of 0.5
U/mL. The tubes were inverted several times and allowed to incubate at
37°C for 20 minutes. During this period the length and width of the clots
were measured with a ruler and were used to calculate clot area. For the
experiment involving platelets isolated from an afibrinogenemic patient, clot area was calculated by measuring the length and width of
computer-generated images of the clot at 2-minute intervals. The clot
areas were then used to calculate percent of retraction using the
following equation:
% Retraction 5 51 2 [Clot Area (t)/Clot Area (t 5 0)]6 3 100%
After the appropriate incubation time, the tubes were placed on ice until
a photograph was taken.
RESULTS
Production and characterization of recombinant fibrinogens.
Oligonucleotide-directed mutagenesis was used to introduce
RGD to RGE mutations at both positions 95-97 and 572-574 in
the fibrinogen Aa chain. The mutations made in the expression
vector pMLP-Aa were confirmed by restriction digests with
BseRI and Psp1406I and by sequence analysis of the complete
cDNA (data not shown). The sequence data ensured that there
were no unanticipated codon changes. The altered expression
plasmid was called pMLP-Aa DE2. After cotransformation of
pMLP-Aa DE2 and pMLP-g 407 into CHO cells containing
pMLP-Bb, the media were screened for fibrinogen by ELISA.
Of 17 colonies tested, 4 were positive. The clone with the
highest expression level (0.6 µg/mL) was expanded and grown
in roller bottles for 3 months, essentially as previously described.21 Likewise, two previously described BHK clones
expressing fibrinogen with either Aa D97E or Aa D574E
mutations were used to synthesize these two variant proteins.17
The different variant fibrinogens were all purified as described.23 When analyzed by SDS-PAGE under reduced conditions, the different fibrinogen variants all showed three major
bands with molecular weights corresponding to the a, b, and g
chains (Fig 1).
Aggregation of ADP-stimulated platelets. We tested the
variants in aggregation experiments using ADP-stimulated
platelets. Platelet aggregation was measured by monitoring the
change in light transmission versus time.28 As shown in Fig 2,
all four aggregation curves had an initial decrease in light
transmission after ADP addition (indicated by arrows), as
expected for agonist-induced platelet activation.29 In addition,
the aggregation curves generated with recombinant fibrinogens
containing intact g chains (normal recombinant fibrinogen: Fig
2A, curve 1; Aa D97E fibrinogen: Fig 2A, curve 2; and Aa
D574E fibrinogen: Fig 2B, curve 2) showed a rapid increase in
light transmission characteristic of platelet aggregation. In
contrast, in the presence of fibrinogen Aa DE2/g 407 no change
in light transmission was observed (Fig 2B, curve 1). This
indicates that the platelets did not aggregate in the presence of
fibrinogen Aa DE2/g 407, which is consistent with previous
results showing that residues AGDV of the g chain are required
for platelet aggregation.19
Platelet-mediated clot retraction. The fibrinogen variants
were tested for their ability to retract a clot formed by adding
Fig 1. SDS-PAGE analysis of
purified recombinant fibrinogens. Recombinant fibrinogens
in Laemmli25 sample buffer with
SDS were separated on an 8%
gel run under reduced conditions and stained with Coomassie Brilliant Blue R-250.
Lanes: 1, plasma fibrinogen; 2,
normal recombinant fibrinogen;
3, recombinant Aa D97E fibrinogen; 4, recombinant Aa D574E
fibrinogen; 5, recombinant Aa
DE2/g 407 fibrinogen; 6, recombinant g 407 fibrinogen.
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FIBRINOGEN SITES FOR CLOT RETRACTION
2377
genemic patient were able to retract clots formed with both
normal recombinant and Aa DE2/g 407 fibrinogen. As with
experiments using platelets isolated from normal donors, clots
formed with added Aa DE2/g 407 fibrinogen showed delayed
retraction when compared with clots formed with normal
recombinant fibrinogen (Fig 5). In addition, the retraction of
clots formed with normal recombinant fibrinogen and platelets
from the afibrinogenemic patients was delayed relative to
retraction using platelets from normal donors (compare Figs 3
and 5). The retraction rates were different even though the
ADP-induced aggregations were comparable (data not shown).
DISCUSSION
Fig 2. ADP-induced platelet aggregation. Platelets (2 3 108 mL21
final concentration) were preincubated at 37°C with 250 nmol/L final
concentration of the indicated fibrinogen before adding ADP to 10
mmol/L final concentration (indicated by arrows). The increase in
light transmission is plotted versus time. Shown are representative
curves, with each experiment performed at least four times. (A) Curve
1, normal recombinant fibrinogen; curve 2, recombinant Aa D97E
fibrinogen. (B) Curve 1, recombinant Aa DE2/g 407 fibrinogen; curve
2, recombinant Aa D574E fibrinogen.
thrombin to platelets in the presence of fibrinogen. Figure 3A
shows the average (n 5 4) change in clot area with time for the
normal recombinant and three variant fibrinogens. Both fibrinogen Aa D97E and fibrinogen Aa D574E showed a slightly
lower retraction rate relative to normal recombinant fibrinogen;
however, this difference was within the large systematic error
inherent in this semiquantitative method of calculating clot area
and therefore was not statistically significant. On the other
hand, Aa DE2/g 407 fibrinogen showed a marked delay in
retraction. After the delay, the retraction rate of Aa DE2/g407
fibrinogen seemed similar to normal recombinant fibrinogen.
When the data were displayed as shown in Fig 3B the rate of Aa
DE2/g407 fibrinogen retraction approximated the rate of retraction of normal recombinant fibrinogen, as indicated by the
similarities in the slopes of the curves. The final size of all four
clots was indistinguishable as shown in the representative
photograph taken after a 20-minute incubation at 37°C (Fig 4).
Two control experiments were performed to ensure that clot
retraction was dependent on both platelets and exogenously
added fibrinogen. Fibrinogen, in the absence of platelets,
formed a clot that failed to retract, whereas platelets treated with
thrombin in the absence of added fibrinogen failed to form a clot
(data not shown).
To exclude the possible involvement of fibrinogen from
platelet a-granules, clot retraction experiments were performed
in an identical manner with platelets isolated from an afibrinogenemic patient. These platelets contain minute levels of
fibrinogen in their a-granules (10 µg/109 platelets) as determined by ELISA with a peroxidase-conjugated rabbit antihuman fibrinogen antibody.30 Platelets isolated from an afibrino-
The use of recombinant technology has contributed to our
understanding of the role of the two RGD sequences in both
platelet adhesion and aggregation. Farrell et al17,18 found that
neither RGD site is required for platelet aggregation or adhesion
of stimulated or unstimulated platelets. Furthermore, recombinant fibrinogens with alterations to the H12 sequence were
found to have a reduced ability to support platelet aggregation
and adhesion when compared with normal recombinant fibrinogen.17-19 Similarly, recombinant technology has clarified the
role of the three putative binding sites on fibrinogen during
interactions with endothelial cells. The importance of the
C-terminal RGD sequence in the Aa chain of fibrinogen in
endothelial adhesion by integrin avb3 was identified by replacement of Asp with Glu at residue 574; this mutation substantially
reduced endothelial cell adhesion to fibrinogen via integrin
avb3.31 In addition, g407 fibrinogen, with deletion of AGDV
from the g chain, and proteolytic fragments of plasma fibrinogen were used in a study of endothelial cell-mediated clot
retraction. These experiments showed that neither the RGD at
residues 572-574 of the Aa chain nor the AGDV residues of the
g chain were required for endothelial cell avb3-mediated clot
retraction.32 However, the role of these sites in plateletmediated clot retraction was not addressed.
This communication reports on the results of a study of
platelet-mediated clot retraction using similar recombinant
technology. As previously shown, both Aa D97E fibrinogen and
Aa D574E fibrinogen could support platelet aggregation to the
same extent as normal recombinant fibrinogen.17 Moreover, the
results of this study indicate that both the rate of clot retraction
and the final size of the clots were indistinguishable when using
normal recombinant fibrinogen, Aa D97E fibrinogen, or Aa
D574E fibrinogen (Figs 3 and 4). Combining these results with
previous data indicating that the g chain AGDV residues are not
required for clot retraction19 shows that no one of the three
proposed aIIbb3 binding sites on fibrin is critical for plateletmediated clot retraction.
It remained possible that each of the platelet binding sites
could support clot retraction independently of one another such
that elimination of a single site would show no adverse effects,
as one or both of the other two sites would compensate for the
loss. To test this possibility we constructed a third variant
fibrinogen, called Aa DE2/g 407, where both RGD sequences in
the Aa chain were changed to RGE and the AGDV residues of
the g chain were deleted. Although this variant did not support
platelet aggregation (Fig 2), it did support clot retraction.
However, the initiation of retraction was delayed when com-
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2378
ROONEY ET AL
Fig 3. Clot retraction kinetics. Platelets (2 3 108
mL21 final concentration) were preincubated for 10
minutes at 37°C with 300 nmol/L final concentration
of the indicated fibrinogen before adding human
a-thrombin to 0.5 U/mL final concentration. The
length and width of the clot were measured with a
ruler every 2 minutes and used to calculate clot area,
which was then used to calculate % retraction. (A)
Average % retraction, expressed as mean 6 standard error from four experiments, is plotted versus
time as a bar graph for the various recombinant
fibrinogens, which are indicated in the legend. (N),
Normal recombinant; (h), Aa D97E fibrinogen; (°),
Aa D574E fibrinogen; (j), Aa DE2/g 407 fibrinogen.
(B) Curves generated by plotting average % retraction versus time for normal recombinant and Aa
DE2/g407. (j), Normal recombinant; (d), Aa DE2/g
407 fibrinogen.
pared with normal recombinant fibrinogen (Fig 3A). This result
suggests that clot retraction occurs in two distinct phases. The
first phase would involve one or more of the putative fibrinogen
sites, whereas the second phase would be independent from
these sites. Several mechanisms are consistent with such a
two-step process. We favor a model where in the first phase
platelets are recruited to the clot, either by interactions between
the platelets and fibrinogen or between the platelets and
polymerizing fibrin. After recruitment of the platelets to the
clot, the second phase of clot retraction involves the transmission of the contractile force from the platelets to the fibrin
strands. The result of this second phase is the collapse of the
fibrin clot. This two-step mechanism would predict that platelets cannot interact with fully polymerized fibrin, as has been
observed.33,34
In our experiments with Aa DE2/g407 fibrinogen, loss of all
three sites affected the initial phase of clot retraction, suggesting
that one or more of the three putative binding sites on fibrinogen
or polymerizing fibrin play a role in the initial recruitment of
platelets to the clot. In contrast to Aa DE2/g407 fibrinogen,
retraction with three fibrinogens that lack only one of the
binding sites was indistinguishable from normal recombinant
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FIBRINOGEN SITES FOR CLOT RETRACTION
2379
Fig 4. Photograph of clots after 20 minutes. Clot retraction experiments were performed as described in Fig 3A and a photograph of the
tubes containing the clots was taken after 20 minutes. The photograph is representative of the results observed in all four experiments
performed. Tubes: 1, normal recombinant fibrinogen; 2, recombinant
Aa D97E fibrinogen; 3, recombinant Aa D574E fibrinogen; 4, recombinant Aa DE2/g 407 fibrinogen.
recombinant fibrinogen was similar to that observed using
platelets from donors with normal fibrinogen levels, even
though the afibrinogenemic platelets have a-granule fibrinogen
levels of less than 10 µg/109 platelets.30 This is in contrast to the
normal fibrinogen levels in platelet a-granules, reported to be
between 63 to 140 µg/109 platelets.36-38 In the experiments
described here, the exogenous fibrinogen concentration (51 µg)
is approximately 63 times greater than the fibrinogen released
by the afibrinogenemic platelets (0.8 µg from 500 µL of
1.6 3 108 platelets/mL). Therefore, these results suggest that
either a-granule fibrinogen is not required for retraction of clots
formed from Aa DE2/g407 fibrinogen or minute quantities are
sufficient. These results do not eliminate the possibility that
other a-granule proteins, most notably von Willebrand factor,
vitronectin, fibronectin, and thrombospondin, may bind to fibrin
and participate in clot retraction when fibrinogen lacking all
three binding sites is used to form the clot.
We observed a difference in the retraction rates of clots
formed with platelets from normal donors versus platelets from
the afibrinogenemic patient using both normal recombinant
fibrinogen and Aa DE2/g 407 fibrinogen. The difference may
result from a variety of factors, including the normal donor-todonor variation in platelet responsiveness and the differences in
the platelet purification procedures for the two experiments. In
addition, whereas the normal platelet data is the average from
four experiments, the afibrinogenemic experiment was performed only once because of the limited availability of these
platelets. Because of these differences, our data are not sufficient to establish whether platelet a-granule fibrinogen participates in clot retraction. However, we can conclude that platelet
fibrinogen (Fig 3A).19 This suggests that the loss of one site can
be compensated by either one or both of the other two sites.
Once a sufficient number of platelets are incorporated into the
clot formed with the Aa DE2/g407 fibrinogen, retraction
proceeded to completion at a normal rate, as shown by the
similarities in the retraction rate curves for Aa DE2/g407 and
normal recombinant fibrinogen (Fig 3B). This finding indicates
that the loss of the three hypothesized binding sites has no
bearing on the force generation phase of retraction. Therefore,
the second phase of clot retraction seems to involve a novel site
on fibrin. This introduces the possibility that distinct platelet
receptors are involved in the two phases of clot retraction. This
possibility is consistent with the finding of Cohen et al35 that the
receptor necessary for clot tension may be another glycoprotein
and/or domain of aIIbb3 not involved in fibrinogen binding.
Perhaps aIIbb3 mediates the interactions between platelets and
fibrinogen or polymerizing fibrin during the recruitment phase
and a second, unidentified receptor and/or domain of aIIbb3
mediates the contractile phase.
Alternatively, platelet a-granule fibrinogen, which contains
all three binding sites, may be required for platelet adhesion and
subsequent clot retraction using Aa DE2/g 407 fibrinogen. The
observed delay would then arise from the time required for
a-granule release and the incorporation of the platelet fibrinogen into the fibrin fibers. This possibility prompted us to
examine clot retraction using platelets isolated from an afibrinogenemic patient. In this experiment, the delay in retraction of
clots formed with Aa DE2/g 407 compared with normal
Fig 5. Clot retraction kinetics using platelets from an afibrinogenemic patient. Platelets (1.6 3 108 mL21 final concentration) were
preincubated for 10 minutes at 37°C with 300 nmol/L final concentration of the indicated fibrinogen before adding human a-thrombin to
0.5 U/mL final concentration. The retraction was filmed on videotape,
which was used to generate computer images of the clots at
2-minute intervals. The length and width of the computer images
were measured with a ruler and used to calculate clot area, which
was then used to calculate % retraction. Percent retraction is plotted
versus time for the various recombinant fibrinogens, which are
indicated in the legend (N 5 1). (h), Normal recombinant; (j), Aa
DE2/g407 fibrinogen.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2380
ROONEY ET AL
fibrinogen does not have a critical role in retraction of clots
formed with Aa DE2/g407, as the relative delay of Aa
DE2/g407 fibrinogen versus normal recombinant fibrinogen
with afibrinogenemic platelets is similar to the relative delay
with normal platelets (Figs 3 and 5).
Recombinant technology has allowed us to eliminate a
critical role in clot retraction for any one of the hypothesized
platelet binding sites on fibrin. However, our results do indicate
that the three putative binding sites on fibrinogen are involved
in the initial recruitment of platelets to the fibrin clot. Because
Aa DE2/g407 fibrinogen does support clot retraction, we
conclude that at least one of the RGE sites and/or an unidentified site on fibrin interacts with platelets during the second, clot
tension phase of retraction. Likely candidates for the novel
fibrin site include other regions on fibrinogen found to impair
platelet aggregation, another aIIbb3-driven process. These include two regions on the g chain identified in the dysfibrinogens
Vlissingen and Frankfurt VII, which have a deletion of g
319-320 and a substitution of Thr for Met at g 310, respectively.
These two variants from heterozygous patients supported
platelet aggregation less than 50% of that observed with normal
fibrinogen, indicating these residues can directly or indirectly
influence binding to aIIbb3.39 Moreover, as stated previously, the
second phase may involve a second receptor to mediate the
development of clot tension. Therefore, the presumptive novel
fibrin site may be one of several sites on fibrinogen or fibrin that
interact with other integrin and nonintegrin receptors. These
include g 190-201, which was found to interact with MAC-1 on
leukocytes, g 117-133, which is found to inhibit binding of
fibrinogen to endothelial cells or B lymphoblastoid Daudi cells
expressing intracellular adhesion molecule 1 (ICAM-I), and b
15-42, which is hypothesized to interact with a 130-kD
glycoprotein during endothelial cell spreading on fibrin.40-43
Finally, the possibility exists that the site on fibrin required for
clot retraction is buried in fibrinogen and only becomes
available after conformational changes as a result of the
thrombin-catalyzed transition from fibrinogen to fibrin. For
example, neoantigenic sequences on fibrin, such as a 148-160,
may only be available for interaction with aIIbb3 after conversion of fibrinogen to fibrin or after subsequent polymerization
of fibrin.44 This and other fibrin-exposed sequences are leading
candidates for a novel binding site important in clot retraction.
ACKNOWLEDGMENT
We thank Li Fang Ping for her tissue culture work and Dr Leslie V.
Parise for critical review of this manuscript.
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1998 92: 2374-2381
The Contribution of the Three Hypothesized Integrin-Binding Sites in
Fibrinogen to Platelet-Mediated Clot Retraction
Michael M. Rooney, David H. Farrell, Bettien M. van Hemel, Philip G. de Groot and Susan T. Lord
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