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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3311-3320

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3311-3320.)
© 1997 American Heart Association, Inc.

Articles

{alpha}IIbß3 Redistribution Triggered by Receptor Cross-Linking

Scott R. Simmons; Paul A. Sims; ; Ralph M. Albrecht

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.


*    Abstract
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 *Abstract
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Abstract Fibrinogen binding to {alpha}IIbß3 on adherent, spread platelets triggers active, cytoskeletally-directed redistribution of fibrinogen/{alpha}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, {alpha}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/{alpha}IIbß3 complex redistribution on platelet surfaces.


Key Words: platelet aggregation • fibrinogen • integrin • {alpha}IIbß3 • signal transduction


*    Introduction
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 up arrowAbstract
 *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 {alpha}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 {gamma}400–411 (HHLGGAKQAGDV) receptor-binding portions of the molecule, induces conformational changes in {alpha}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 {gamma} 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 {alpha}IIbß3, thus may provide the signal that initiates receptor clustering. Some further signal, also provided by binding of whole fibrinogen to {alpha}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 {alpha}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 {alpha}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{alpha}, Bß, and {gamma} chains.33 Several receptor-recognition sequences have been identified within the molecule. The fibrinogen {alpha} chains each contain two RGD sequences. The position of the {alpha} 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 {gamma} chains, {gamma} 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 {alpha}IIbß3 with fibrinogen revealed that 85% of the isolated receptors bound specifically to the distal {gamma} domains of the fibrinogen molecule.40 The remaining 15% bound, presumably to the {alpha} 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 {alpha} chain and the relationship between the RGD and HHLGGAKQAGDV binding sites on {alpha}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 {gamma} 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 {alpha}IIbß3 have been shown to expose neoepitopes within both the {alpha} and {gamma} 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.


*    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 {gamma} 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-{alpha}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 {alpha}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 {alpha}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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 1Down), 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 {alpha}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 2Down). 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 3Down).



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  		Figure 1. Platelet treated with 15-nm gold-conjugated fibrinogen, showing the typical centralized pattern of fibrinogen/{alpha}IIbß3 complexes on surface-adherent, spread platelet. Receptors are initially dispersed over much of platelet surface. Fibrinogen binding triggers receptor redistribution. Bound receptors move centripetally in the plane of the membrane, forming a band of receptor-bound fibrinogen gold (arrows) on the platelet surface. Bar indicates 1.0 µm.



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  		Figure 2. Monoclonal antibody AP3 binds to {alpha}IIbß3 in the presence of fibrinogen. Platelet treated with fibrinogen, rinsed, treated with AP3, fixed, and labeled with 15-nm gold-conjugated goat anti-mouse IgG. Fibrinogen has triggered centripetal movement of bound receptors. Gold-labeled AP3 (arrows) is bound to receptors in a centralized pattern typical of fibrinogen binding, as seen in Fig 1Up. Bar indicates 1.0 µm.



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  		Figure 3. Treatment of platelets with GRGDS and/or H12 blocks fibrinogen binding. Platelet treated with a mixture of GRGDS and H12, then with fibrinogen conjugated to colloidal gold. No fibrinogen-gold has bound to the platelet surface. Bar indicates 1.0 µm.

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 4Down). 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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  		Figure 4. Fibrinogen receptor-binding fragments do not trigger receptor movement. Platelet treated as in the legend to Fig 3Up with GRGDS and H12. Final receptor locations are identified by treatment with AP3, fixation to prevent further receptor movement, and detection of bound AP3 with goat anti-mouse IgG gold. Gold-labeled, peptide-bound receptors remain dispersed on platelet surface. Bar indicates 1.0 µm.

Treatment of platelets with antibodies to {alpha}IIbß3, whether as whole molecule or Fab fragments, did not trigger receptor redistribution (Fig 5Down). 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 6Down). 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 7Down). Addition of the secondary antibody in non-cross-linking, monovalent, Fab form did not trigger receptor redistribution.



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  		Figure 5. Platelet treated with 10E5 for 5 minutes, rinsed, and incubated in buffer for an additional 5 minutes, fixed with glutaraldehyde to prevent further receptor movement, and then labeled with gold-conjugated goat anti-mouse IgG. 10E5-bound receptors remain dispersed on platelet surface. Bar indicates 1.0 µm.



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  		Figure 6. Receptor movement induced by receptor cross-linking. Platelet treated as in the legend to Fig 5Up, omitting the fixation before treatment with goat anti-mouse IgG gold. Gold-conjugated goat anti-mouse IgG has cross-linked AP3-bound receptors, triggering receptor redistribution identical in pattern to that seen with fibrinogen-gold in Fig 1Up. Bar indicates 1.0 µm.



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  		Figure 7. Platelet treated with Fab fragments of 10E5, then with F(ab')2 goat anti-mouse IgG conjugated to 5-nm gold. F(ab')2 anti-mouse IgG-gold has cross-linked Fab 10E5-bound {alpha}IIbß3, triggering centripetal receptor redistribution. Platelet margin at right. Bar indicates 0.1 µm.

A second approach to cross-linking of the receptor by anti-{alpha}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 {alpha}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 8Down).



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  		Figure 8. Binding of single gold-conjugated IgG molecules (ie, one molecule conjugated to one 5-nm gold particle) to {alpha}IIbß3 does not trigger receptor movement, but binding of larger gold conjugates, as in 15-nm colloidal gold, where each gold particle is large enough to adsorb several IgG molecules, does cross-link receptors and trigger receptor redistribution. Only cross-linked receptors move, while antibody-bound but non-cross-linked receptors remain in place. Platelet treated simultaneously with 10E5 conjugated to 5- and 15-nm colloidal gold. Small, 5-nm, 10E5 gold labels do not trigger receptor redistribution and remain dispersed on the platelet surface, while larger, 15-nm 10E5 gold labels at lower right have centralized. Platelet margin at upper left. Bar indicates 0.1 µm.

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-{alpha}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-{alpha}IIbß3 to the receptor is insufficient to initiate receptor redistribution (Fig 9bDown). 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-{alpha}IIbß3 triggered receptor redistribution identical to that previously seen with fibrinogen-gold or 15-nm anti-{alpha}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 9cDown through 9f). Location and identity of gold labels followed in the light microscope were subsequently confirmed by electron microscopy (Fig 10Down). 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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  		Figure 9. Video-enhanced differential interference contrast light micrographic series showing binding and movement of 18-nm gold-conjugated goat anti-mouse IgG on AP3-treated platelet. Although 18-nm-diameter gold particles are too small to be resolved and clearly imaged in light microscopy, individual particles as well as groups of particles can be detected and followed via their inflated diffraction images in interference-based light microscopy. An individual gold particle appears as a diffuse shadow approximately 0.1 µm in diameter. a, Platelet treated with unlabeled, soluble AP3, showing the appearance of the platelet before gold labeling. b, Anti-mouse IgG gold binds to AP3/{alpha}IIbß3 complexes dispersed over platelet surface. Individual 18-nm gold labels (arrows) can be detected via their inflated diffraction images. c through e, Receptor/antibody complexes cross-linked by anti-mouse IgG gold move across the platelet surface toward the central granulomere, forming a band of recep-tor-bound gold-antibody complexes on the platelet surface. As this band of centralized, gold-labeled receptors forms, more gold (arrows) binds to receptor/antibody complexes remaining near the platelet perimeter, demonstrating that only cross-linked receptors move, while antibody-bound receptors, which have not yet been cross-linked by the binding of gold-conjugated second antibody, remain in place. f, Dark band surrounding the granulomere represents a concentration of cross-linked, gold-labeled antibody/receptor complexes on the platelet surface. Identity and location of the gold labels are subsequently confirmed by electron microscopy (Fig 10Up). Bar indicates 1.0 µm.



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  		Figure 10. Same platelet as was followed in the light microscope in Fig 9Up. Scanning electron microscopic examination confirms position and identity of gold labels in band on platelet surface. Arrow indicates same gold label as arrow in Fig 9fUp. Bar indicates 1.0 µm.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 {alpha}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 {alpha}IIbß3 binding to fibrinogen, which is immobilized on a surface or converted to fibrin.41,54 The {gamma} chain terminal dodecapeptides are the primary receptor-binding sequences in soluble fibrinogen. However, once the molecule binds to {alpha}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 {alpha}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 {alpha}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 {alpha}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 {alpha}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 {alpha} and {gamma} chains.33,40,43 In electron microscopic examination of isolated {alpha}IIbß3 bound to fibrinogen, single fibrinogen molecules were seen to bind to two receptor complexes, primarily via the {gamma} 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-{alpha}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 {alpha}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 {gamma} 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.


*    References
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