© 1997 American Heart Association, Inc.
Articles |
IIbß3 Redistribution Triggered by Receptor Cross-Linking
From the Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wis.
Correspondence to Ralph M. Albrecht, PhD, Department of Animal Health and Biomedical Sciences, 1655 Linden Drive, Madison, WI 53706.
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Abstract |
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Abstract Fibrinogen binding to
IIbß3 on adherent, spread
platelets triggers active, cytoskeletally-directed redistribution
of fibrinogen/IIbß3 complexes on the platelet surface.
Gold-conjugated fibrinogen, unlabeled, soluble fibrinogen, and
individual fibrinogen molecules have been demonstrated to trigger
receptor redistribution. Here we examine the respective roles of
receptor cross-linking and ligand occupancy of receptors in initiating
this movement. Monovalent, IIbß3-binding fibrinogen fragments RGDS
and HHLGGAKQAGDV did not trigger receptor redistribution, suggesting
that ligand binding to a single receptor is an insufficient stimulus.
Binding of monoclonal antibodies 10E5, AP2, and AP3 to the receptor did
not trigger receptor movement. However, cross-linking these
receptor-bound monoclonal antibodies by polyclonal anti-mouse IgG or by
conjugation of the anti-receptor antibody to large colloidal gold
particles triggered receptor redistribution identical in rate, pattern,
and final distribution to that previously seen with fibrinogen binding.
We conclude that receptor cross-linking provides the signal for
initiation of fibrinogen/IIbß3 complex redistribution on
platelet surfaces.
Key Words: platelet aggregation • fibrinogen • integrin • IIbß3 • signal transduction
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Introduction |
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Besides providing attachment points for extracellular matrix proteins, cell surface adhesion receptors in the integrin family play significant roles in signal transduction, control of cytoskeletal organization, and translocation of bound ligand over the surface of the plasma membrane.1–3 In recent years it has become clear that integrins are capable of two-way signal transduction.4,5 The specificity and affinity of the extracellular portion of the receptors is regulated from within the cell. Ligand binding, receptor clustering, and receptor cross-linking in turn provide signals that are transmitted via the integrin receptor, providing modulation of cellular behavior by extracellular matrix or intercellular interactions. Signal transduction by integrins appears to depend on the ability of the receptors to interact with and organize the actin cytoskeleton and assemble a cytoskeleton-based, multimeric signal transduction complex of enzymes, linker proteins, transmembrane receptors, and structural proteins.4–6 Ligand occupancy of integrin receptors and receptor cross-linking have both been implicated in initiation of integrin-transmitted signals.1,7,8 Receptors may be linked by a single multivalent ligand, by immobilized ligand, or by a nonligand structure such as a latex sphere that binds and cross-links proteins nonspecifically.9 In some cases triggering of integrin activity requires a combination of ligand occupancy and receptor cross-linking.8
The integrin IIbß3 (glycoprotein IIbIIIa) serves as
the receptor for fibrinogen and other RGD-containing adhesive proteins
on surfaces of activated platelets. Binding of fibrinogen
to this receptor is required for formation of platelet aggregates
and normal blood clotting. Ligand occupancy, by either whole fibrinogen
or peptide fragments containing the RGD or 
400–411 (HHLGGAKQAGDV)
receptor-binding portions of the molecule, induces conformational
changes in IIbß310–13 and triggers
formation of small clusters of receptors on the activated
platelet surface.14,15 Occupancy of receptors
by whole fibrinogen, but not monovalent RGD or 400–411 fibrinogen
fragments, also has been shown to alter the linkage of the receptors to
the actin cytoskeleton,16–18 induce kinase
association with the membrane skeleton, and increase tyrosine
phosphorylation on receptor- and
cytoskeleton-associated proteins.18–23 In
platelets, cross-linking of fibrinogen receptors either by antibody
(primary monoclonal anti-receptor followed by polyclonal goat
anti-mouse IgG)24 or by the dimeric,
RGD-containing disintegrin contortrostatin25 has
been shown to trigger increased tyrosine
phosphorylation. Receptor occupancy, via the
ligand-induced conformational change in IIbß3, thus may provide
the signal that initiates receptor clustering. Some further signal,
also provided by binding of whole fibrinogen to IIbß3, possibly
via receptor cross-linking, is required to stimulate the increase in
receptor/actin linkage and tyrosine phosphorylation
seen in response to fibrinogen binding.
In addition to the receptor clustering induced by binding of fibrinogen
or fibrinogen fragments, binding of soluble or gold-conjugated whole
fibrinogen to IIbß3 triggers longer range movement of the
receptor/ligand complexes across the platelet
surface.3,26–28 This is in contrast to von
Willebrand factor binding to glycoprotein Ib on
adherent, spread platelets. von Willebrand factor remains
dispersed on the platelet surface unless subsequently cross-linked
by addition of an antibody specific to von Willebrand
factor.29 Fibrinogen receptors on spread
platelets initially are distributed over much of the platelet
surface, somewhat more concentrated near the periphery. Fibrinogen
binding triggers cytoskeletally directed, centripetal movement of
involved receptors. Fibrinogen-bound receptors move in the plane of the
membrane toward the granulomere, forming a band of receptor/ligand
complexes on the platelet surface overlying the inner filamentous
zone of the subjacent cytoskeletal matrix surrounding the
granulomere.27 Unbound fibrinogen receptors
remain dispersed until they, too, are bound.3
This centripetal movement of receptors over the spread platelet
surface requires the presence of the intact filamentous actin
cytoskeleton30,31 and can be inhibited by agents
that block myosin phosphorylation by myosin light chain
kinase.32 In this study we show that binding of
non-cross-linking fibrinogen fragments does not trigger this long range
receptor redistribution. Using colloidal gold labeling in conjunction
with whole IgG or Fab fragments of antibody against IIbß3, we
examine the role of receptor cross-linking in triggering
receptor/ligand redistribution.
The fibrinogen molecule is dimeric, consisting of three pairs of
nonidentical polypeptide chains, A, Bß, and
chains.33 Several receptor-recognition sequences
have been identified within the molecule. The fibrinogen chains
each contain two RGD sequences. The position of the chains in the
molecule in its solution conformation is
uncertain.34–37 In terms of supporting
platelet aggregation, the principal platelet receptor
recognition domains are the carboxy-terminal sequences of the
chains, 406–411, which are located near the extreme ends of the
molecule.38–42 Seen by electron microscopy, the
fibrinogen molecule appears roughly rod-shaped, 6 nm in diameter by 45
nm in length.43 Electron microscopic examination
of the interaction of isolated IIbß3 with fibrinogen revealed that
85% of the isolated receptors bound specifically to the distal
domains of the fibrinogen molecule.40 The
remaining 15% bound, presumably to the chain RGD sequences, at a
variety of positions on the molecule. Many of the fibrinogen molecules
were bound to two receptor complexes, illustrating the capacity of the
fibrinogen molecule to link two receptors separated by a distance of up
to 45 nm. The roles of the RGD sequences on the fibrinogen chain
and the relationship between the RGD and HHLGGAKQAGDV binding sites on
IIbß3 remain obscure. While the short linear sequences RGD and
KQAGDV are essential for receptor-specific binding, it is likely that
other, yet unidentified, ligand regions may also play roles in
ligand/receptor interaction. For example, the whole fibrinogen
D-domain, which contains the chain binding site, is 70-fold more
active than the isolated HHLGGAKQAGDV peptide in inhibiting
fibrinogen-mediated platelet aggregation.44
Thus, interaction of other regions on the D-domain with the receptor is
required for high-affinity binding. Furthermore, conformational changes
in fibrinogen elicited by its interaction with IIbß3 have been
shown to expose neoepitopes within both the and
chains.45,46 Exposure of these epitopes may
create additional sites for interaction with the receptor or may create
self-adhesive sites within the fibrinogen molecule. In a recent
scanning electron microscopic study of binding of unlabeled fibrinogen
to adherent platelets, fibrinogen displayed an increased tendency
to undergo nonfibrillar self-adhesive interactions after binding to
platelet surface receptors.47 Consequently,
groups of fibrinogen/receptor complexes are cross-linked via
fibrinogen-fibrinogen cohesion on the platelet surface.
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Methods |
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Fibrinogen was a gift from Dr. Michael Mosesson, Sinai Samaritan Medical Center, Milwaukee, Wis. GRGDS was purchased from Novabiochem. Fibrinogen
chain dodecapeptide HHLGGAKQAGDV (H12) was
a gift from Dr. E.F Plow, Center for Thrombosis and Vascular Biology,
Cleveland Clinic Foundation, Cleveland, OH. Anti-mouse IgG, either
whole molecule or F(ab')2, affinity isolated,
goat polyclonal IgG, was purchased from Sigma Chemical Co. Monoclonal
antibody 10E5 was a gift from Dr. Barry Coller, Mount Sinai Medical
Center, New York, NY. Monoclonal antibodies AP2 and AP3 were gifts from
Dr. Thomas Kunicki, Research Institute of Scripps Clinic, La Jolla,
CA. Fab fragments of IgG were prepared by papain (immobilized papain, Pierce) digestion. IgG was digested at 37°C for 5 hours in phosphate buffer containing 10 mmol/L EDTA and 20 mmol/L cysteine HCl at pH 7.0. Digested IgG was separated from the immobilized papain by centrifugation. Fab fragments were separated from Fc fragments by passage through an immobilized protein G column (Pierce).
Five-and 15-nm colloidal gold particles were prepared by the reduction of a dilute solution of HAuCl4 by a mixture of sodium citrate and tannic acid.48 The gold colloids were stored at 4°C until used.
The pH of gold sols used in the preparation of protein-gold conjugates was adjusted with 0.2 N K2CO3 to pH 6.9 for fibrinogen and pH 7.3 for antibody. The amount of protein necessary to stabilize the gold was determined by adsorption isotherms. Increasing concentrations of proteins were mixed with 1.0 mL of gold, 0.1 mL of saturated aqueous NaCl was added, and the lowest protein concentration that prevented flocculation of the gold on addition of the NaCl was used for conjugation. All gold-protein conjugates were concentrated and separated from unbound protein by centrifugation, adjusted to between 5x1012 and 5x1013 particles/mL, and suspended in modified Tyrode's buffer.
Human platelets were separated from citrated blood by passage through a Sepharose CL-4B column in calcium-free Tyrode's buffer, pH 7.3 (136 mmol/L NaCl, 2.7 mmol/L KCl, 3.3 mmol/L NaH2PO4, 10.5 mmol/L HEPES salt, 4.45 mmol/L HEPES free acid, 2 mmol/L MgCl2, 1 g/L of dextrose, and 2 g/L of albumin). A drop (10 µL) of the platelet suspension was deposited on a Formvar-coated Ni electron microscope grid, and platelets were allowed to settle and adhere at 37°C in a moist chamber for 15 minutes. Specimens were incubated for 5 minutes in ligand or antibody dissolved in Tyrode's buffer, and the same buffer was used for all washes. Where light fixation was needed to prevent receptor rearrangement between labeling steps, platelets were fixed for 5 minutes in 0.05% glutaraldehyde in albumin-free Tyrode's buffer, and free aldehyde groups were quenched for 10 minutes with 0.05 mol/L glycine in Tyrode's buffer.
To determine whether binding of non-cross-linking fragments of fibrinogen was sufficient to trigger long-range receptor redistribution, GRGDS or H12 was added, individually or together, at a concentration of 1.0 mg/mL for 10 minutes. Some specimens were then labeled with fibrinogen-gold (whole molecule) to evaluate the extent of receptor occupancy by the peptides. To determine the distribution of receptors after peptide binding, specimens were incubated with AP3, a monoclonal antibody that binds to the receptor in the presence or absence of fibrinogen.49 After rinsing, the platelets were lightly fixed in 0.05% glutaraldehyde to prevent further receptor movement, and AP3-bound receptors were identified by addition of goat anti-mouse IgG gold label. As a control, some specimens were treated only with whole fibrinogen followed by AP3 and anti-mouse IgG gold.
For examination of cross-linking of receptors with the primary
antibodies (whole molecule), spread platelets were incubated with
AP2, AP3, or 10E5 at a concentration of 0.1 mg/mL for 5 minutes.
Unbound antibody was rinsed away, the platelets were incubated in
buffer for an additional 5 minutes to allow time for receptor
redistribution and fixed for 5 minutes in 0.05%
glutaraldehyde, and the bound antibody was detected
with gold-conjugated goat anti-mouse IgG. For examination of secondary
antibody cross-linking of receptors, glutaraldehyde
fixation after treatment with primary antibody was omitted, and
receptor/antibody complexes were cross-linked by anti-mouse IgG added
as soluble antibody or as gold conjugate. After treatment with soluble
primary (mouse monoclonal anti-IIbß3) and soluble secondary
(polyclonal goat anti-mouse) antibodies, platelets were lightly
fixed with 0.05% glutaraldehyde to prevent further
receptor movement, then labeled with gold-conjugated anti-goat IgG to
determine the final distribution of receptors. Primary antibody was
also cross-linked by conjugation to colloidal gold.
Five-nanometer-diameter gold particles are smaller than an IgG
molecule, and only one IgG molecule can adsorb to each 5-nm gold label.
Fifteen-nanometer-diameter particles have room for several
antibody molecules to attach.50,51 Thus, in 5-nm
gold-IgG conjugates the IgG remains as individual molecules, while in
15-nm gold-IgG conjugates IgG molecules are "linked" by virtue of
several being bound to each gold particle. Gold-conjugated 10E5 was
prepared with 5-and 15-nm colloidal gold. Spread, adherent
platelets were labeled for 5 minutes with the two sizes of
10E5-gold together or separately. Excess gold conjugate was rinsed
away, and the platelets were incubated in buffer for an additional
5 minutes before fixation and preparation for electron microscopy. The
larger, 15-nm-diameter, gold-10E5 was also used to follow receptor
movement in the light microscope via the inflated diffraction images of
the 15-nm gold particles.
Antibody cross-linking of receptors on live platelets was observed
by time-lapse, video enhanced, differential interference contrast light
microscopy.52 Column-washed platelets were
allowed to contact-activate and spread for 5 to 20 minutes on
Formvar-coated grids affixed in a chamber open at two ends to enable
exchange of media for labeling, washing, and fixation of specimens on
the light microscope stage. The spread platelets were incubated
with antibody to IIbß3 (10E5, AP2, or AP3), rinsed with Tyrode's
buffer, and labeled with 15-nm gold-conjugated goat anti-mouse IgG, or
were labeled with 10E5 conjugated directly to 15-nm colloidal gold.
Unbound label was rinsed away. The specimens were fixed with
glutaraldehyde while still under observation in the
light microscope and then removed and prepared for electron microscopy.
The entire course of light microscopic experiments was recorded on
a high-resolution videotape (Panasonic NV 9240 XD). This permits
repeated examination of the experiment to enable the investigator to
follow the binding and movement of multiple gold particles. Single
frames were then collected at time points throughout the experiment.
Frames presented here are selected to experiments.
To address the concern that the platelet Fc receptor may be
involved in movement of antibody-cross-linked IIbß3, Fab fragments
of 10E5 were prepared. Fab 10E5 at a concentration of 0.1 mg/mL
was incubated with spread, adherent platelets for 5 minutes.
Unbound Fab 10E5 was rinsed away and bound Fab 10E5 was cross-linked by
addition of F(ab')2 goat anti-mouse IgG, either
soluble or conjugated to 5-nm gold particles. In experiments where Fab
10E5 was cross-linked by soluble F(ab')2 goat
anti-mouse IgG, unbound F(ab')2 was rinsed away,
and the platelets were incubated in buffer for an additional 5
minutes to allow time for receptor redistribution, fixed for 5 minutes
in 0.05% glutaraldehyde to prevent further receptor
movement, and then labeled with gold-conjugated anti-goat IgG to
determine the final distribution of cross-linked receptors. To ensure
that the receptor movement seen after binding of Fab 10E5 and
F(ab')2 goat anti-mouse IgG resulted from
receptor cross-linking rather than from conformational changes caused
by the binding of the goat anti-mouse IgG to the Fab 10E5, Fab
fragments of goat anti-mouse IgG were prepared and substituted for
F(ab')2 in parallel trials.
After labeling, platelets were washed with protein-free Tyrode's buffer and prepared for electron microscopy. All samples were fixed in 0.1 mol/L HEPES-buffered 1% glutaraldehyde containing 0.5% tannic acid at pH 7.2 for 30 minutes at room temperature. Specimens were postfixed for 15 minutes in HEPES-buffered 0.05% OsO4 and for 15 minutes in aqueous 1% uranyl acetate before dehydration through a graded alcohol series to absolute ethanol. Samples were dried by the critical point method with molecular sieve-dried CO2 as the transitional fluid. Specimens were examined on a Hitachi S-900 scanning electron microscope at 4.5 to 10 kV in back-scattered electron imaging mode. All experiments were repeated at least three times. Micrographs presented here represent the typical appearance of platelets after each treatment.
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Results |
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Whole fibrinogen molecules, whether soluble or conjugated to large or small gold particles, bound to receptors on adherent, spread platelet surfaces and triggered centripetal redistribution of receptor/ligand complexes (Fig 1
), as
previously described.3,26–28 Fibrinogen
receptors on spread platelets initially are distributed over much
of the platelet surface. On interaction with whole fibrinogen
molecules, ligand-bound receptors move in the plane of the membrane
over the surface toward the granulomere to create a band of bound
fibrinogen on the platelet surface surrounding the granulomere. To
determine whether fibrinogen binding could trigger movement of
IIbß3 by ligand occupation of the receptor without receptor
cross-linking, fibrinogen receptor-binding fragments GRGDS or H12 were
added to adherent, spread platelets. The platelets were
incubated with the fragments for sufficient time to allow peptide
binding and possible receptor redistribution. The final locations of
the receptors were identified by AP3, an antibody to
glycoprotein IIIa that binds to the receptor in the
presence or absence of fibrinogen. To verify that ligand-bound
receptors could be identified by AP3 in this system, platelets were
treated with fibrinogen, fixed, treated with AP3, and labeled with goat
anti-mouse IgG gold. AP3 bound to surface receptors on
fibrinogen-treated platelets in the same centralized pattern as
previously seen with fibrinogen or fibrinogen-gold conjugates (Fig 2). To verify that the fibrinogen
fragments were binding to the platelet surface receptors,
peptide-treated platelets were labeled with gold-conjugated
fibrinogen. Treatment of platelets with GRGDS or H12 completely
blocked binding of gold-conjugated fibrinogen, demonstrating that every
available receptor site on the peptide-treated platelet surface was
occupied by a fibrinogen fragment (Fig 3).
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On platelets that had been treated with GRGDS or H12 alone or in
combination, then treated with AP3, fixed with
glutaraldehyde to prevent further receptor movement,
and labeled with gold-conjugated goat anti-mouse IgG, the gold-labeled
receptors were distributed in a diffuse pattern across the platelet
surface (Fig 4). Thus, binding of the
ligand fragments was not sufficient to trigger long-range receptor
redistribution. However, if the fixation before addition of goat
anti-mouse IgG gold (whole molecule) was omitted, cross-linking of
AP3-bound, peptide-occupied receptors by the goat anti-mouse IgG gold
triggered centralization of bound receptors on the live platelet
surface, showing that the presence of receptor-bound ligand fragments
did not inhibit receptor movement triggered by receptor
cross-linking.
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Treatment of platelets with antibodies to IIbß3, whether as
whole molecule or Fab fragments, did not trigger receptor
redistribution (Fig 5). However,
cross-linking of the primary antibody, AP2, AP3, or 10E5, by addition
of a second antibody, polyclonal goat anti-mouse IgG, whole molecule,
did trigger receptor movement whether the second antibody was soluble
or gold-conjugated (Fig 6). This was true
also when 10E5 was prepared as Fab and the second, cross-linking,
antibody was prepared as F(ab')2, eliminating the
concern that the platelet Fc receptor was involved in the reaction
(Fig 7). Addition of the secondary
antibody in non-cross-linking, monovalent, Fab form did not trigger
receptor redistribution.
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A second approach to cross-linking of the receptor by anti-IIbß3
was through direct conjugation of the antibody to 15-nm-diameter
colloidal gold particles. Gold particles in this size range will adsorb
approximately nine whole antibody molecules per particle when prepared
with the minimum stabilizing protein concentration, as was used
here.51,53 Fifteen-nanometer gold-10E5
bound to receptors on the platelet surface and triggered movement
of bound receptors. Smaller, 5-nm-diameter, colloidal gold particles
have room for only one antibody molecule to adsorb to each gold
label.50 Five-nanometer gold-10E5 (whole
molecule) also bound to IIbß3, but did not trigger receptor
movement, remaining in a diffuse pattern on the spread platelet
surface. When 5-nm gold-10E5 and 15-nm gold-10E5 were added
simultaneously, the larger gold particles centralized on
the platelet surface, but the smaller particles remained dispersed,
illustrating that only cross-linked receptors moved, while receptors
that were antibody-bound but not cross-linked remained in place (Fig 8).
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Binding of gold-labeled ligand or antibody to live platelets was
observed by video-enhanced, differential interference contrast light
microscopy. Individual 15- to 20-nm gold particles can be detected and
their movement followed over time via their inflated diffraction images
in interference-based light microscopy. Previously we have shown by
this technique that fibrinogen or anti-IIbß3 conjugated to 18-nm
gold will bind to receptors diffusely distributed over the spread
platelet surface and trigger centripetal movement of bound
receptors. The gold-labeled ligand/receptor and gold-labeled
antibody/receptor complexes move in a directed fashion toward the
granulomere, slowing and collecting in a band overlying the inner
filamentous zone of the subjacent cytoskeletal matrix surrounding the
granulomere. Unbound receptors remain dispersed, but can be triggered
to undergo centralization by subsequent binding of ligand-gold or
antibody-gold label.3,28,31,32,52 In this study,
whole molecule goat anti-mouse IgG conjugated to 15-nm gold was added
to platelets pretreated with whole molecule AP3. The goat
anti-mouse IgG-gold initially bound in a dispersed pattern,
demonstrating, as described above, that binding of whole molecule AP3
anti-IIbß3 to the receptor is insufficient to initiate receptor
redistribution (Fig 9b). However, after
binding, anti-mouse IgG-gold labels moved toward the platelet
granulomere. Binding of the gold-conjugated goat anti-mouse IgG to the
receptor-bound anti-IIbß3 triggered receptor redistribution
identical to that previously seen with fibrinogen-gold or 15-nm
anti-IIbß3-gold. Each bound gold label moved centripetally across
the platelet surface, collecting in a band of bound label on the
platelet surface surrounding the granulomere (Fig 9c through 9f).
Location and identity of gold labels followed in the light microscope
were subsequently confirmed by electron microscopy (Fig 10). AP3-bound receptors remained
dispersed until cross-linked by the goat anti-mouse IgG gold conjugate,
then underwent centripetal redistribution identical in rate, direction,
and final location to that previously described in binding of
fibrinogen-gold.32,52
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Discussion |
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Previously we have shown that fibrinogen binding to receptors on surface-adherent, spread platelets triggers redistribution of receptor-ligand complexes. This redistribution can be initiated by binding of whole fibrinogen, whether gold-conjugated or in native, unlabeled form.47 Individual fibrinogen molecules directly conjugated to small gold particles trigger receptor movement.47 In this study we demonstrate first that non-cross-linking, monovalent fibrinogen fragments do not trigger receptor movement. This indicates that ligand occupancy of individual receptors may provide insufficient stimulus to trigger receptor movement. However, neither HHLGGAKQAGDV nor GRGDS provides a perfect model for monovalent fibrinogen binding. Although each of these short peptides can bind to
IIbß3 and some of the platelet responses
to whole fibrinogen binding are seen also with peptide binding, the
respective roles of these sequences in binding of whole fibrinogen to
platelet receptors, particularly regarding ligand-induced changes
in receptor conformation, remain unclear. The RGD sequences in soluble
fibrinogen do not appear to support fibrinogen binding or platelet
aggregation, although they may play a role in IIbß3 binding to
fibrinogen, which is immobilized on a surface or converted
to fibrin.41,54 The chain terminal
dodecapeptides are the primary receptor-binding sequences in soluble
fibrinogen. However, once the molecule binds to IIbß3, other
portions of the fibrinogen molecule also interact with the
receptor.44,45 Hence, the role of receptor
occupancy by ligand in triggering receptor/ligand complex
redistribution remains in question.
In contrast with fibrinogen fragment binding, antibody cross-linking of
IIbß3 triggered receptor redistribution identical to that seen in
response to fibrinogen binding. This was true whether or not the
receptor was occupied by ligand fragments. Binding of individual,
soluble, or gold-conjugated antibody molecules, whole molecule, or Fab
fragments to IIbß3 did not trigger movement. Triggering of
receptor movement required that the primary antibody be cross-linked by
addition of a second antibody, whole molecule, or
F(ab')2, or by conjugation of the primary
antibody to large gold particles, where multiple IgG molecules adsorb
to each particle. Two of the primary antibodies tested, 10E5 and AP2,
bind at or near the fibrinogen-binding site on the
receptor.55,56 The third, AP3, does not bind
directly to the fibrinogen-binding site.49
Initiation of receptor complex redistribution by cross-linking of
AP3-bound receptors with goat anti-mouse IgG gold thus provides an
additional indication that receptor cross-linking can trigger receptor
movement in the absence of fibrinogen-binding site occupancy.
Failure of individual primary antibody molecules to trigger receptor
movement may indicate that more than two receptors must be cross-linked
or that an individual IgG molecule is too small to bridge the gap
between two receptors. The extracellular portion of IIbß3 in its
functional, heterodimeric form is 12 nm in
diameter.40 The maximum spread of the two Fab
arms of an IgG molecule appears to be around 15 nm. Cross-linking of
receptors by addition of Fab fragments of antibody (10E5, which binds
at or near the fibrinogen binding site) against the fibrinogen receptor
followed by second antibody either as whole IgG or as
F(ab')2 fragments did trigger receptor movement.
The length of an individual Fab fragment of IgG is 4 to 6 nm. Adding
this length to the 15-nm spread of an IgG molecule suggests that the
point to point distance between the fibrinogen binding sites on
adjacent cross-linked receptors is at most 27 nm. Although this figure
is, at best, an approximation, it does show that the individual
fibrinogen molecule could span the distance between two receptors on
the platelet surface.
Receptor cross-linking in vivo could be accomplished by a single
fibrinogen molecule binding to two or more receptors or through
adhesive interactions between fibrinogen molecules, each bound to
IIbß3. Individual gold-labeled, bivalent fibrinogen molecules can
be shown to move across the platelet
surface.47 The fibrinogen molecule is >40 nm in
length, with potential receptor-binding sequences in both and
chains.33,40,43 In electron microscopic
examination of isolated IIbß3 bound to fibrinogen, single
fibrinogen molecules were seen to bind to two receptor complexes,
primarily via the chain sequence at the ends of the fibrinogen
molecule.40 Thus, an individual fibrinogen
molecule will trigger receptor movement, is large enough to bridge the
space between receptors, and is capable of binding to and cross-linking
adjacent receptors. In addition, fibrinogen displays an increased
tendency to undergo self-adhesive interactions when bound to
platelet surface receptors.47 Receptor-bound
fibrinogen on adherent platelets forms small, branched, and
globular aggregates, each of which represents a cluster of
receptors whose external domains are linked by fibrinogen. These
aggregates undergo redistribution on the platelet surface identical
in rate, pattern, and final destination to that previously described in
studies using gold-conjugated fibrinogen or soluble
fibrinogen.47
While there is abundant evidence that platelet aggregation is
dependent on fibrinogen-IIbß3 interaction, the precise mechanism
by which fibrinogen links platelets remains unclear. Most models to
date have assumed that an individual fibrinogen molecule binds to two
receptors, one on each of two apposed platelets. There have been
numerous electron microscopic examinations of platelet aggregates.
Most investigators report that the space between apposed platelet
membranes varies within the aggregate and that the platelets are
interconnected by proteinaceous bridges ranging in length from 20 to 60
nm. While the diameters of some of these bridges seem appropriate for
single fibrinogen molecules, many areas show larger masses of material
in which individual molecules are difficult to discern. Immediately
after aggregate formation narrow, 20-nm, protein-filled
interplatelet gaps are common. After 2 to 3 minutes, larger, 50-nm
interplatelet gaps predominate, with protein bridges connecting
platelets across gaps as large as 100 nm. Protein bound to the
platelet surface outside of the platelet-platelet adhesion
zones appears as discrete masses similar in width to the protein
bridges, but approximately half the height above the plasma membrane.
The chemical composition of these bridges is unknown. Although some
studies report that IIbß3 extends as much as 18 to 20 nm above the
plasma membrane, our observations of platelet surfaces with the low
voltage scanning electron microscope and the atomic force microscope,
as well as other published transmission electron microscopic images of
platelet surface membranes, show that receptor height above the
membrane surface is 7 to 8 nm. Thus, it is likely that in the 20-nm
interplatelet gaps, fibrinogen has bound in appressed orientation
to one or more receptors on one platelet, then attached to
receptors on the second platelet via either primary or secondary
receptor interaction sites within the fibrinogen molecule. On the other
hand, for a single fibrinogen molecule to bridge a gap of 50 to 100 nm,
the fibrinogen molecule would have to bind, most likely by the terminal
chain, to only one receptor on one platelet, leaving the rest
of the molecule free to interact with a receptor on a second
platelet. If, as in the study described here, cross-linking of two
or more fibrinogen receptors is necessary to initiate receptor movement
on adherent, spread platelets, then it is likely that a single
fibrinogen molecule binds to and cross-links two receptors on one
platelet. This would require the molecule to lie flat on the
platelet membrane surface, a view supported by previous
transmission electron microscopic studies, as well as recent scanning
electron microscopic images of platelet-bound
fibrinogen.47,57 It should be noted that the edge
to center translocation of ligand-receptor complexes seen in fully
spread, substrate adherent platelets differs from receptor-ligand
movement observed in nonsubstrate adherent
platelets.28 While the work described here
does not address the possible requirement for receptor cross-linking in
initiation of receptor movement in nonadherent platelets, the
implications for fibrinogen orientation on a platelet surface
remain the same. Fibrinogen molecules appear to bind initially in a
"side-on" orientation, lying flat on the platelet surface
membrane. The bound fibrinogen on surface-adherent, spread
platelets then coalesces to form small, nonfibrillar, branched
protein aggregates. Many of these develop globular protrusions that are
10 to 30 nm in diameter. These fibrinogen aggregates are similar in
size and form to structures seen on the membrane surface outside of
platelet-platelet adhesion zones in nonadherent platelet
aggregates. A possible explanation for the development of these
fibrinogen aggregates on the spread platelet surface and of the
thickened, fibrinogen or fibrinogen-containing bridges seen attaching
platelets in aggregates is that one or more of the receptor-induced
binding sites in fibrinogen are self-adhesive in nature, permitting
receptor-bound fibrinogen to interact with other receptor-bound or
surface-adsorbed fibrinogen. Thus, we propose a model for fibrinogen
mediation of platelet aggregation that combines direct linkage of
receptors on two platelets by a single fibrinogen molecule,
providing a close apposition of membranes, particularly at early time
points, and indirect linkage of platelet receptors via adhesive
interactions between fibrinogen molecules or between fibrinogen and
other adhesive ligands, producing proteinaceous bridges across 50- to
100-nm interplatelet spaces, which form over a time scale of
several minutes.
Received February 10, 1997; accepted June 10, 1997.
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