(Received for publication, August 17, 1995)
From the
We have investigated how modulation of integrin
function influences the mechanisms
that initiate platelet thrombus formation onto surface-bound fibrinogen
and isolated fibrinogen domains. Under stationary conditions and with
full activation of platelets blocked by prostaglandin E
,
the carboxyl-terminal
sequence is
necessary for establishing initial contact with the immobilized
substrate. Molecules containing a single copy of this sequence, like
the plasmin-generated fibrinogen fragment D, support platelet
spreading, but the resulting attachment to the surface is loose and
disrupted by minimal peeling force. In contrast, platelets adhere
firmly to intact fibrinogen under the same conditions, suggesting that
recognition of contact sites outside a single D domain can secure the
firm interaction not supported by a single
sequence. If platelets are activated, the
sequence is no longer necessary to initiate the adhesion process
but becomes sufficient, even as a single copy, to mediate stable
surface attachment in the absence of shear stress. Under conditions of
flow, however, intact fibrinogen but not fragment D can support
adhesion, regardless of whether platelets have the potential to become
activated or not. These results indicate the functional relevance of
multiple fibrinogen domains during the initial stages of the platelet
adhesion process.
Fibrinogen is required for normal hemostasis not only as the
precursor of fibrin but also to mediate platelet thrombus formation (1) . With respect to the latter role, fibrinogen can support
both platelet-surface and platelet-platelet interactions, i.e. platelet adhesion and aggregation,
respectively(2, 3) , by binding to the glycoprotein
IIb-IIIa receptor (integrin
)(4, 5, 6) .
These two functions occur in sequence at the onset of hemostasis and,
when deranged in pathological conditions, may cause vascular occlusion.
Platelet adhesion and aggregation are influenced by changes in the
recognition specificity of
, which,
as present on the membrane of nonactivated platelets, serves as a
specific receptor for surface-bound fibrinogen (7, 8) but, after activation, acquires the ability to
interact with other immobilized adhesive proteins, particularly von
Willebrand factor(8) . Moreover,
activation is required for the binding of soluble fibrinogen and
von Willebrand factor (9, 10) leading to
aggregation(3, 11) .
Fibrinogen contains three
putative platelet interaction sites, namely the sequence
Arg-Gly-Asp-Phe (RGDF) at A, the sequence
Arg-Gly-Asp-Ser (RGDS) at A
, and the
dodecapeptide sequence His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val
(HHLGGAKQAGDV) at
(12, 13) . Small
synthetic peptides reproducing each of the three sequences have been
shown to bind to
regardless of its
state of activation(14, 15) . It is not yet known,
however, how these different sites, and possibly others(16) ,
contribute to the complex adhesive functions of the intact fibrinogen
macromolecule. Evidence obtained with recombinant mutants indicates a
predominant role for the carboxyl-terminal
chain dodecapeptide in
the binding of soluble fibrinogen to activated platelets and thus in
mediating aggregation(17, 18) . Moreover, it has been
postulated that the presence of two
chain carboxyl-terminal
domains in the dimeric fibrinogen molecule may influence the adhesion
of nonstimulated platelets when the ligand is immobilized onto a
surface(19) . To elucidate mechanisms important in the
initiation of platelet response to vascular injury, we have evaluated
the ability of intact fibrinogen and isolated fibrinogen fragments to
interact with platelets and support their attachment to a surface. The
results obtained indicate that multiple sites are responsible for the
adhesive potential of fibrinogen depending on the state of
activation. Modulation of the
interaction with distinct domains in an appropriate substrate may be an
example applicable to the activity of different integrins involved with
the adhesive functions of vascular cells exposed to flowing blood.
Platelets interacting with the surface-bound substrate were visualized by scanning electron microscopy in two ways. For the study of loosely attached platelets, wells were subjected to a single gentle washing by adding 100 µl of PBS-7.4 followed by mild aspiration of the fluid. Attached platelets were immediately fixed with modified Karnovsky fixative (1.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4; 50 µl/well) for 60 min at 4 °C. Samples were dehydrated using graded ethanol and freon 113, critical point-dried in freon 13, and sputter-coated using a carbon target prior to analysis. For the study of irreversibly attached platelets, washing was repeated four times before fixing.
Figure 1:
Morphologic evaluation of platelets
attached to surface-bound fibrinogen and fibrinogen fragments under
stationary conditions. Platelets in plasma, isolated from freshly drawn
blood containing PPACK, were allowed to attach to polystyrene wells
coated with fibrinogen (A), fragment D (B), or
fragment E (C) in the absence (upper row) or in the
presence (lower row) of PGE. After 60 min at room
temperature, nonadherent platelets were removed by four aspiration and
washing steps, and attached platelets were immediately fixed and
processed for analysis by scanning electron microscopy. Note the
equivalent extensive platelet spreading and aggregate formation on
fibrinogen and fragment D as compared with the absence of platelets on
fragment E when platelet activation is not inhibited (upper
row) and the absence of platelets on fragments D and E when
platelet activation is inhibited with PGE
(lower
row). Bar, 5 µm.
These findings indicate that the carboxyl terminus of the
chain in fragment D can support stable adhesion mediated by
only when platelets have the
potential to become activated but not when they are kept in the
``resting'' state and that the RGDF sequence in fragment E is
not active under the same conditions. Thus, isolated fragments D and E (Fig. 1), as well as a combination of the two immobilized
together on a surface (not shown here), cannot function like intact
fibrinogen in supporting adhesion. This suggests that additional
sequences not present in these two fragments are involved in the
process and/or that only the native molecule can present multiple
adhesive sites in the appropriate conformation for interacting with
platelets. As shown below, however, this does not rule out the
occurrence of specific interactions with single domains resulting in
loose and/or transient attachment that cannot be detected in standard
assays because it is disrupted during the washing steps performed to
reduce ``nonspecific'' background.
These interpretations
rely on the notion that platelets maintained in their plasma
environment with intact divalent cation concentrations should be
minimally altered by uncontrolled stimulation. Other investigators,
however, have found that they behave like washed platelets stimulated
by the combination of epinephrine and ADP, whereas nonstimulated washed
platelets behave like PGE-treated platelets in
plasma(19) . It is debatable whether any ex vivo study
is compatible with the absence of stimulation, because the unavoidable
manipulations necessary to remove platelets from the circulation may be
sufficient to cause functional perturbation. Moreover, the definition
of whether circulating platelets are truly resting is essentially
arbitrary. For example, platelets in plasma never exposed to exogenous
agonists may act like stimulated washed platelets because of the
presence of small quantities of activating substances, like ADP,
released from erythrocytes or platelets themselves. Alternatively,
nonstimulated washed platelets may appear to function like
PGE
-treated platelets because they are rendered refractory
to weak stimuli by activation during isolation procedures. In spite of
these problems, our results are in agreement with the previous
study(19) , demonstrating that platelets can adhere firmly to
fragment D, containing a single
chain carboxyl-terminal domain,
only when they become activated and that nonstimulated platelets adhere
only to substrates, like intact fibrinogen, with two such domains. In
view of these findings, additional experiments were designed to
evaluate whether the
chain carboxyl-terminal sequences are
necessary and sufficient to support the adhesive functions of
fibrinogen and whether platelet activation can influence their
interaction with
.
Figure 2:
Time course of adhesion of untreated
platelets to fibrinogen and fragment D: effect of a monoclonal antibody
against the chain carboxyl terminus. Platelets in plasma were
allowed to incubate in polystyrene wells coated with fibrinogen (upper panel) or fragment D (lower panel) for the
times indicated. Before adding platelets, the immobilized ligands were
treated with the monoclonal antibody LJ-Z69/8 (100 µg/ml) directed
against the fibrinogen
chain carboxyl terminus, including
residues
, or with control buffer
(PBS-7.4). After 60 min of incubation, unbound antibody was removed by
two washing steps before adding the platelets. In another set of wells,
platelets were treated with the anti-
monoclonal antibody LJ-CP8 (100 µg/ml) for 30 min before
incubation with the immobilized ligands. At the end of the incubation
period, nonadherent platelets were removed by two consecutive
aspiration and washing steps with PBS-7.4. Adherent platelets were
detected by adding the nonfunction blocking
anti-
monoclonal antibody, LJ-P4,
labeled with
I. After 30 min at room temperature,
residual unbound antibody was removed by two consecutive aspiration and
washing steps. The contents of the wells were solubilized with 2%
sodium dodecyl sulfate, and the
I-associated
radioactivity was measured to obtain a relative estimate of the number
of adherent platelets. The results represent the means ± S.E.
for nine separate experiments, each performed in duplicate. Statistical
evaluation confirmed that the inhibitory effect of the antibody
LJ-Z69/8 was progressively less significant with time in the case of
adhesion to fibrinogen (Student's t distribution of
control versus treated groups: p < 0.001 at 40
min; p < 0.1 at 60 min) but remained highly significant in
the case of adhesion to fragment D (p < 0.001 at 60
min).
Platelets treated with PGE to inhibit the response to
activating stimuli still adhered well to intact fibrinogen but, unlike
untreated platelets, their interaction was completely inhibited by the
anti-
chain dodecapeptide antibody, as well as by the
anti-
antibody, regardless of the
length of incubation (Fig. 3). Moreover, PGE
-treated
platelets could not attach firmly to fragment D (Fig. 3).
Figure 3:
Time
course of adhesion of PGE-treated platelets to fibrinogen
and fragment D: effect of a monoclonal antibody against the
chain
carboxyl terminus. The experiment was performed as described in the
legend to Fig. 2with the only exception being that platelets in
plasma supplemented with PGE
were used instead of untreated
platelets. The results represent the means ± S.E. for nine
separate experiments, each performed in
duplicate.
In
marked contrast to these results, platelets in plasma stimulated by the
addition of exogenous epinephrine attached firmly to fragment D even
after the first time interval of 20 min; the interaction was completely
inhibited by the monoclonal antibodies LJ-Z69/8 against the chain
carboxyl terminus and LJ-CP8 against
(Fig. 4). The same platelets adhered well to fibrinogen,
but in this case the interaction was still completely inhibited by
LJ-CP8 but only minimally affected by LJ-Z69/8 (Fig. 4).
Figure 4: Time course of adhesion of epinephrine-treated platelets to fibrinogen and fragment D. The experiment was performed as described in the legend to Fig. 2with the only exception being that platelets in plasma were treated with epinephrine (final concentration, 20 µM) for 10 min before addition to the substrate-coated wells. The results represent the means ± S.E. for nine separate experiments, each performed in duplicate. Statistical evaluation confirmed that the inhibitory effect of the antibody LJ-Z69/8 was not significant at later time points in the case of adhesion to fibrinogen (Student's t distribution of control versus treated groups: p < 0.025 at 20 min; p > 0.5 at 60 min) but remained highly significant in the case of adhesion to fragment D (p < 0.001 at all time points tested).
These findings support the concept that the chain carboxyl
terminus is the only
interactive
site in fragment D but cannot mediate irreversible attachment unless
platelets are activated. The
sequence is
clearly necessary for initiating platelet adhesion to fibrinogen when
full activation is blocked and under these conditions two copies of it,
as opposed to the single one in fragment D, may support irreversible
attachment, as previously suggested(19) . Alternatively, the
distinct properties of intact fibrinogen may indicate functional
co-operation between this sequence and one or more additional sites
present in fibrinogen but not in fragment D. When platelets can become
activated, the
chain carboxyl-terminal sequence is no longer
strictly required to initiate or mediate adhesion to intact fibrinogen,
although it retains its essential role in fragment D. This is in
agreement with the results of previous studies demonstrating that
several domains in fibrinogen can interact with activated
(7) .
Our findings show
that untreated platelets (not inhibited by PGE) exposed for
a sufficiently long time to immobilized fibrinogen or fragment D in the
absence of any added stimulus, exhibit adhesive properties similar to
those of platelets activated by exogenous epinephrine. Indeed, in
assays like the one described here performed in the absence of flow,
membrane contacts developing when platelets sediment onto the surface
over time can induce the release reaction(27) , leading to the
local availability of agonists like ADP and thromboxane
A
(28) , as well as favor guanylate cyclase
activation and signal transduction (29) ; thus platelets become
activated. Of note, in experiments not reported here, we found that
agitation preventing platelet sedimentation during the assay
effectively inhibited adhesion to fragment D but not to fibrinogen, in
agreement with all the other results indicating that activation is
essential for stable attachment to the former but not the latter. It
appears, therefore, that the relatively slow time course of adhesion to
fragment D exhibited by untreated platelets is a reflection of the slow
process of activation upon sedimentation, not the consequence of other
undefined properties of this substrate. In agreement with this
hypothesis, platelet attachment to fragment D occurred more rapidly
after activation with exogenous epinephrine. In the latter situation,
as well as in the case of adhesion to fibrinogen, the time course of
the process may reflect the rate at which platelets sediment and can
interact with the surface more than the generation of stimuli deriving
from close contact. Along similar lines, the fact that the anti-
chain antibody had no effect on adhesion to fibrinogen of platelets
stimulated with epinephrine (Fig. 4), whereas it inhibited
partially but significantly that of untreated platelets (Fig. 2), also seems to indicate that activation upon
sedimentation is a slow process involving platelets in a nonsynchronous
manner; thus only platelets that are not yet activated in the well
would fail to adhere to fibrinogen in the presence of the antibody, as
shown in Fig. 3for those treated with PGE
.
Figure 5:
Effect of activation with the monoclonal antibody, AP5, on platelet
adhesion to fibrinogen and fragment D. The experiment was performed as
described in the legend to Fig. 2, except that platelets in
plasma with or without the addition of PGE
were treated
with the activating monoclonal antibody AP5 (Fab; final
concentration, 50 µg/ml) for 20 min before addition to the
substrate-coated wells for 60 min.
Because all the experiments reported to this
point were performed in the presence of plasma proteins, it is possible
that unidentified molecules interacting differently with immobilized
fragment D or fibrinogen modify the surface to which platelets are
exposed and influenced the process of adhesion. To rule this out, we
performed additional studies, to be described in detail elsewhere, ()using heterologous cells expressing recombinant
. In this case, in the absence of
any plasma protein, the monoclonal antibody AP5 in the presence of
Mn
could induce the cells to mimic the function of
``stimulated'' platelets (adhesion to both fibrinogen and
fragment D), whereas cells treated with the antibody but without
Mn
behaved like resting platelets (more prominent
adhesion to fibrinogen), demonstrating that specific differences in the
receptor-substrate interaction are responsible for the findings
observed.
Figure 6:
Interaction of nonactivated
PGE-treated platelets with fragment D. Platelets in plasma
supplemented with PGE
were allowed to attach to immobilized
fibrinogen or fibrinogen fragments under the conditions described in
the legend to Fig. 2. However, in order to determine the
morphology of any loosely attached platelets that would otherwise be
detached by the four washing steps described for the experiment
presented in Fig. 2, at the end of the 60-min incubation,
surface-associated platelets were fixed and processed for scanning
electron microscopy after a single washing step. Upper row,
high magnification; bar, 5 µm. Lower row, low
magnification; bar, 30 µm. Note extensive platelet
spreading on fibrinogen (A) and fragment D (B) but
not on fragment E (C) where residual platelets retain a
nonspread morphology. Compare the results shown here for fragment D
with those shown in Fig. 2(lower row) performed under
identical conditions except for the number of washing
steps.
If
platelets can be activated normally and function can be modulated, platelet adhesion to fibrinogen may
proceed along different pathways, because the
chain carboxyl
terminus is no longer strictly required to initiate or mediate the
process (Fig. 4) but, even as a single copy, becomes sufficient
to support stable adhesion (Fig. 2). Thus, other fibrinogen
domains in addition to and distinct from the dodecapeptide
can interact with activated
. In a static adhesion assay this
occurs whenever platelet activation is not impaired, most likely
through mechanisms requiring close platelet contact; it can also occur
after platelet stimulation with an exogenous agonist (Fig. 4).
The difference between the former and the latter is probably one of
extent of activation. In the case of a weak stimulus, like that
developed when platelets sediment onto a surface, the synergistic
effect of the initial contact with an appropriate fibrinogen domain,
like the
sequence, is still required for
adhesion to occur; therefore, nonstimulated but metabolically active
platelets adhere selectively to fibrinogen and fragment D, as shown
here, but not to other potential adhesive substrates like fibronectin
or vitronectin(8) . In the case of a stronger stimulus, for
example the combination of ADP plus epinephrine (7) , activated
becomes less selective and
mediates adhesion to other fibrinogen domains, like fragment E, and to
other substrates(7) . The latter processes take place under the
same conditions required for soluble ligand binding to
, indicating that they depend on
full activation of the receptor. Clearly, functional modulation of
has a profound impact on the
mechanism of platelet adhesion to surface-bound fibrinogen.
Figure 7:
Direct real-time observation of platelet
interaction with surface-bound fibrinogen and fibrinogen fragments
under flow. Substrates were coated onto glass coverslips that were used
in a parallel plate flow chamber. Blood containing PPACK as
anticoagulant and treated with the fluorescent dye mepacrine (final
concentration, 10 µM) was perfused through the flow
chamber in the direction indicated by the arrow at 37 °C.
A computerized epifluorescence video microscopy system was used for
real-time visualization of platelet interaction with surface-bound
fibrinogen and fibrinogen fragments. Fluorescent images after 5 min of
perfusion at a wall shear rate of 50 s were captured
from video tapes; single frames are shown
here.