(Received for publication, December 13, 1995; and in revised form, February 16, 1996)
From the
Fibrinogen mediates the processes of platelet aggregation and
clot retraction. Previous studies have demonstrated that fibrinogen
binding to the platelet receptor requires the C-terminal residues of the fibrinogen
chain.
We made a recombinant human fibrinogen that lacks the
chain
C-terminal four residues (AGDV). As expected this fibrinogen did not
support platelet aggregation. Unexpectedly, this variant did support
clot retraction that was indistinguishable from retraction with normal
recombinant or plasma fibrinogen. These results suggest that the site
on fibrinogen that is required for platelet aggregation differs from
the site on fibrin that is required for clot retraction.
The processes of platelet aggregation and clot retraction are
mediated by fibrinogen in vitro and play a role in maintaining
hemostasis in vivo. Fibrinogen is a large (340 kDa) plasma
protein consisting of two sets of A, B
, and
chains. The
protein is arranged in a symmetrical fashion with two lateral D domains
and a central E domain(1) . Previous work employing electron
microscopy(2) , synthetic peptides(3) , and recombinant
proteins (4, 5) has implicated the D domain, in
particular residues
408-411 (AGDV), as the site on
fibrinogen that interacts with platelets. This interaction involves the
platelet integrin
, a heterodimeric
transmembrane receptor. Antibodies and peptides that prevent fibrinogen
binding to
also prevent platelet
aggregation and clot retraction(6, 7, 8) .
Together, these findings predict that the
recognition site on fibrinogen (i.e. residues
408-411) must be present and
accessible for aggregation or clot retraction to occur. Two studies
support this prediction with respect to platelet
aggregation(4, 5) , but both have limitations. In
one(4) , recombinant
chains lacking residues
408-411 were unable to support platelet aggregation; however,
this variant
chain was studied outside the context of the entire
molecule. In the other(5) , recombinant fibrinogen with a
20-amino acid insert in place of these four residues was unable to
support platelet aggregation. However, the lack of aggregation cannot
be directly attributed to the loss of the AGDV residues, as the
additional 20 amino acids may sterically impair aggregation. Moreover,
neither study explored the effects that these alterations may have had
on clot retraction.
We report that an intact recombinant fibrinogen
lacking only residues 408-411 does not support aggregation,
consistent with previous work (4, 5) , but
unexpectedly supports clot retraction to the same extent as normal
recombinant and plasma fibrinogen. These findings suggest that other
domains in fibrinogen participate in clot retraction.
We characterized the purified recombinant proteins by
SDS-polyacrylamide gel electrophoresis and Western blots.
SDS-polyacrylamide gel electrophoresis run under reducing conditions (Fig. 1) demonstrated that all three chains were present in both
the normal recombinant fibrinogen and the 407 variant.
Electrophoresis under non-reducing conditions showed that the chains
assembled into a protein with a molecular mass of 340 kDa (data not
shown). Western blot analysis with a polyclonal antibody (Fig. 2A) confirmed the presence of all three chains in
both recombinant proteins. A duplicate blot developed with monoclonal
antibody 4A5 (Fig. 2B), a monoclonal antibody specific
for the C terminus of the
chain, showed immunoreactivity with
both plasma and normal recombinant fibrinogen but not with the
407
variant. Thus, the C-terminal epitope for 4A5 has been perturbed in the
407 variant. This result is consistent with the lack of
chain residues 408-411, as predicted from the DNA sequence data
of the
chain expression plasmid.
Figure 1:
Polyacrylamide gel
electrophoresis on 8% gels run under reduced conditions according to
the method of Laemmli (11) and developed with Coomassie
Brilliant Blue R-250. Lanes: 1, plasma fibrinogen; 2, normal recombinant fibrinogen; 3, recombinant
407 fibrinogen.
Figure 2:
Western blot analysis of recombinant
fibrinogen under reduced conditions. An 8% polyacrylamide gel, run as
in Fig. 1, was blotted onto a nitrocellulose membrane and
developed as in Binnie et al.(9) . Lanes: 1, plasma fibrinogen; 2, normal recombinant
fibrinogen; 3, recombinant 407 fibrinogen. Primary
antibodies were: A, rabbit polyclonal antiserum reactive with
all three fibrinogen chains; B, 4A5, a monoclonal antibody
specific for the C terminus of the
chain(10) .
We tested the recombinant
proteins in platelet aggregation assays (Fig. 3). Platelets were
activated upon addition of 10 µM ADP, as indicated by the
initial decrease in light transmission due to platelet shape
change(13) . Following activation, platelet samples in the
presence of normal recombinant fibrinogen exhibited a dramatic increase
in light transmission due to the formation of aggregates(14) .
In contrast, platelets in the presence of 407 changed shape but
did not aggregate. Similar results were obtained with the addition of
ADP to platelets in the absence of added fibrinogen (data not shown).
Figure 3:
ADP-induced aggregation of platelets.
Platelets (2 10
platelets/ml) were preincubated at
37 °C with 250 nM fibrinogen prior to activation with 10
µM ADP (indicated by arrows). Aggregation of
stirred solutions was measured as the increase in light transmission. Curve A is for normal recombinant; curve B is for
recombinant
407 fibrinogen.
We examined clot retraction to see if the region on fibrinogen
required for aggregation was also required for this seemingly related
process. The clot formed in the presence of 407 was
indistinguishable from the clots formed with plasma or normal
recombinant fibrinogen. Clot retraction was followed for 20 min at 37
°C. Retraction rates were similar for both normal recombinant and
407 fibrinogen (Fig. 4). The final contracted clots formed
with normal recombinant,
407, or plasma fibrinogen (Fig. 5)
were also indistinguishable. When thrombin was added to platelet
suspensions without added fibrinogen, there was no clot formation (Fig. 5). When thrombin was added to solutions of recombinant or
plasma fibrinogen, fibrin clots formed but did not retract (data not
shown). Thus, the residues AGDV on the
chain of fibrinogen are
apparently not required for clot retraction.
Figure 4:
Time course for clot retraction. Platelets
(2 10
platelets/ml) were preincubated with 300
nM fibrinogen (to give a final volume of 0.5 ml) at 37 °C
for 10 min prior to the addition of 0.5 unit/ml human
-thrombin.
The clots were incubated at 37 °C, and length and width were
measured in mm at the indicated times. The figure presents the average
clot area (n = 2). White bars represent normal
recombinant fibrinogen, and black bars represent
407
fibrinogen.
Figure 5:
Photographs of the final retracted clots.
Platelets (2 10
platelets/ml) were preincubated
with 300 nM fibrinogen (to give a final volume of 0.5 ml) at
37 °C for 10 min prior to the addition of 0.5 unit/ml human
-thrombin. The clots were incubated at 37 °C for 15 min and
transferred to ice. The photograph was taken after 1 h. Tubes: 1, normal recombinant fibrinogen; 2, recombinant
407 fibrinogen; 3, plasma fibrinogen; 4, no
added fibrinogen.
Because activation of
platelets by thrombin results in the release of fibrinogen from the
-granules(15) , it is possible that the platelet
fibrinogen may contribute to the retraction of the clots. Therefore, we
plan to perform clot retraction experiments using platelets from an
afibrinogenemic patient, which will serve as a definitive control.
In the experiments discussed here, we used genetic engineering to
produce a variant fibrinogen molecule to probe two seemingly related
events. Consistent with previous results, removal of the C-terminal
AGDV residues from the chain of fibrinogen abolished the ability
of the protein to support platelet aggregation. Unexpectedly, deletion
of these residues did not impair clot retraction. This finding
apparently contradicts previous findings that implicate AGDV in binding
to
during clot retraction. We
hypothesize that
interacts with
residues AGDV during aggregation and interacts with one or both of the
consensus binding sequences for integrins (RGD) in the A
chain
during clot retraction. It has been shown that the peptide RGDS can
retard clot retraction(16) . Using monoclonal antibodies, other
investigators have shown that inaccessible sites on fibrinogen become
accessible upon conversion to fibrin(17) . Perhaps the RGD
sites are not accessible in fibrinogen but are exposed in the fibrin
molecule. These regions on the A
chain may then interact with
and mediate fibrin-platelet
interactions, including clot retraction. Thus, the ligand site on
fibrinogen that mediates aggregation may differ from the site on fibrin
that mediates clot retraction. Yet, the platelet receptor for both
processes is
(7, 8, 18) .
We plan to test the hypothesized role of RGD in clot retraction by
analysis of additional variant fibrinogens.
In summary, we find that
the chain C-terminal residues AGDV are critical for platelet
aggregation but not for clot retraction. These findings suggest a
separate role for the consensus binding sequence RGD at either position
95-97 or 572-574 on the A
chain of fibrinogen.
Finally, the
407 variant may also provide a means of
differentiating between platelet signaling events involved in
aggregation versus clot retraction.