The macromolecular architecture of platelet-derived microparticles.

Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbß3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbß3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn(2+)), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.

ß-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbß3 Headpiece.

The platelet integrin αIIbß3 binds to a KQAGDV motif at the fibrinogen γ-chain C terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and ß-subunits; the basic Lys or Arg side chain hydrogen bonds to the αIIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the ß3-subunit. Ligand binding induces headpiece opening, with conformational change in the ß-subunit. During this opening, RGD slides in the ligand-binding pocket toward αIIb, with movement of the ßI-domain ß1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and αIIb. Here we test whether ligand interactions with ß3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbß3 headpiece with affinity comparable with the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbß3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α-subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the ß3-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required.

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand.

Increased ligand binding to integrin ("activation") underpins many biological processes, such as leukocyte trafficking, cell migration, host-pathogen interaction, and hemostasis. Integrins exist in several conformations, ranging from compact and bent to extended and open. However, the exact conformation of membrane-embedded, full-length integrin bound to its physiological macromolecular ligand is still unclear. Integrin αIIbß3, the most abundant integrin in platelets, has been a prototype for integrin activation studies. Using negative stain electron microscopy and nanodisc-embedding to provide a membrane-like environment, we visualized the conformation of full-length αIIbß3 in both a Mn(2+)-activated, ligand-free state and a Mn(2+)-activated, fibrin-bound state. Activated but ligand-free integrins exist mainly in the compact conformation, whereas fibrin-bound αIIbß3 predominantly exists in a fully extended, headpiece open conformation. Our results show that membrane-embedded, full-length integrin adopts an extended and open conformation when bound to its physiological macromolecular ligand.

Structural mechanism of integrin inactivation by filamin.

Activation of heterodimeric (αß) integrin is crucial for regulating cell adhesion. Binding of talin to the cytoplasmic face of integrin activates the receptor, but how integrin is maintained in a resting state to counterbalance its activation has remained obscure. Here, we report the structure of the cytoplasmic domain of human integrin αIIbß3 bound to its inhibitor, the immunoglobin repeat 21 of filamin A (FLNa-Ig21). The structure reveals an unexpected ternary complex in which FLNa-Ig21 not only binds to the C terminus of the integrin ß3 cytoplasmic tail (CT), as previously predicted, but also engages N-terminal helices of αIIb and ß3 CTs to stabilize an inter-CT clasp that helps restrain the integrin in a resting state. Combined with functional data, the structure reveals a new mechanism of filamin-mediated retention of inactive integrin, suggesting a new framework for understanding regulation of integrin activation and adhesion.

Three-dimensional reconstruction of intact human integrin αIIbß3: new implications for activation-dependent ligand binding.

Integrin αIIbß3 plays a central role in hemostasis and thrombosis. We provide the first 3-dimensional reconstruction of intact purified αIIbß3 in a nanodisc lipid bilayer. Unlike previous models, it shows that the ligand-binding head domain is on top, pointing away from the membrane. Moreover, unlike the crystal structure of the recombinant ectodomain, the lower legs are not parallel, straight, and adjacent. Rather, the αIIb lower leg is bent between the calf-1 and calf-2 domains and the ß3 Integrin-Epidermal Growth Factor (I-EGF) 2 to 4 domains are freely coiled rather than in a cleft between the ß3 headpiece and the αIIb lower leg. Our data indicate an important role for the region that links the distal calf-2 and ß-tail domains to their respective transmembrane (TM) domains in transmitting the conformational changes in the TM domains associated with inside-out activation.

Recreation of the terminal events in physiological integrin activation.

Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

Structure of a complete integrin ectodomain in a physiologic resting state and activation and deactivation by applied forces.

The complete ectodomain of integrin alpha(IIb)beta(3) reveals a bent, closed, low-affinity conformation, the beta knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The beta(3) knee passes between the beta(3)-PSI and alpha(IIb)-knob to bury the lower beta leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the beta subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state.

Integrin conformational regulation: uncoupling extension/tail separation from changes in the head region by a multiresolution approach.

Integrin-dependent adhesion and signaling are regulated by conformational changes whose details remain controversial. Crystallography revealed bent shapes for resting and primed integrin ectodomains, whereas large, ligand-induced rearrangements in other constructs suggested extension, "opening," and tail separation. We have used experimental/computed hydrodynamics to discriminate among different alpha(v)beta(3) and alpha(IIb)beta(3) atomic models built on X-ray, NMR, and EM data. In contrast with X-ray structures and EM maps, hydrodynamics indicate that resting integrins are already extended. Furthermore, the hydrodynamics of an alpha(v)beta(3) ectodomain-fibronectin fragment complex support opening via additional head region conformational changes (hybrid domain swing-out), but without tail separation. Likewise, frictional changes induced by priming agents in full-length alpha(IIb)beta(3) correlate well with the swing-out coupled to a simple transmembrane helix shift in an extended, electron tomography-based model. Extension and immediate tail separation are then uncoupled from head region rearrangements following activation, thus underscoring integrins' delicate, finely tuned plasticity.

Integrin alpha IIb beta 3 in a membrane environment remains the same height after Mn2+ activation when observed by cryoelectron tomography.

Integrins perform the critical function of signalling cell attachment to the extracellular matrix or to other cells. This signalling is done through a structural change propagated bidirectionally across the plasma membrane. Integrin activation has been extensively studied with ectodomain constructs, but the structural change within intact, membrane-bound molecules remains a subject of live debate. Using cryoelectron tomography, we examined the simplest predication of the different integrin activation models, i.e., the change in height of the molecules. Analysis using techniques that compensate for the missing wedge during alignment and averaging and that search for patterns in the structure of the aligned molecular subvolumes extracted from the tomogram reveals that the vast majority of molecules show no dramatic height change upon Mn(2+)-induced activation of membrane-bound integrins when compared with an inactive integrin control group. Thus, the result is inconsistent with the switchblade activation model.

Multi-step fibrinogen binding to the integrin (alpha)IIb(beta)3 detected using force spectroscopy.

The regulated ability of integrin alphaIIbbeta3 to bind fibrinogen plays a crucial role in platelet aggregation and hemostasis. We have developed a model system based on laser tweezers, enabling us to measure specific rupture forces needed to separate single receptor-ligand complexes. First of all, we performed a thorough and statistically representative analysis of nonspecific protein-protein binding versus specific alphaIIbbeta3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified alphaIIbbeta3 and fibrinogen, covalently attached to underlying surfaces, ranged from approximately 20 to 150 pN. This distribution could be fit with a sum of an exponential curve for weak to moderate (20-60 pN) forces, and a Gaussian curve for strong (>60 pN) rupture forces that peaked at 80-90 pN. The interactions corresponding to these rupture force regimes differed in their susceptibility to alphaIIbbeta3 antagonists or Mn2+, an alphaIIbbeta3 activator. Varying the surface density of fibrinogen changed the total binding probability linearly >3.5-fold but did not affect the shape of the rupture force distribution, indicating that the measurements represent single-molecule binding. The yield strength of alphaIIbbeta3-fibrinogen interactions was independent of the loading rate (160-16,000 pN/s), whereas their binding probability markedly correlated with the duration of contact. The aggregate of data provides evidence for complex multi-step binding/unbinding pathways of alphaIIbbeta3 and fibrinogen revealed at the single-molecule level.

Ligand binding promotes the entropy-driven oligomerization of integrin alpha IIb beta 3.

Integrin alpha(IIb)beta(3) clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of alpha(IIb)beta(3). The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at approximately 43 degrees C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen gamma-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for alpha(IIb)beta(3) complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 degrees C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for alpha(IIb)beta(3) protomers and dimers based on the ectodomain structure of alpha(v)beta(3). We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.

Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling.

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.

Tirofiban blocks platelet adhesion to fibrin with minimal perturbation of GpIIb/IIIa structure.

A biophysical approach tested the hypothesis that tirofiban, like eptifibatide, perturbs GpIIb/IIIa structure. Tirofiban bound tightly to platelet GpIIb/IIIa (EC50 approximately 24 nmol/L) and effectively inhibited platelet aggregation (IC50 approximately 37 nmol/L) but blocked platelet adhesion to clotted fibrin only at much higher doses (IC50 approximately 580 nmol/L). Electrophoretic analyses demonstrated that tirofiban protected GpIIb/IIIa from SDS-induced subunit dissociation. However, saturating tirofiban concentrations had little or no effect on GpIIb/IIIa secondary or tertiary structure, as determined by circular dichroic spectroscopy, dynamic light scattering, and sedimentation velocity measurements performed with purified receptors in octyl glucoside. Moderate dose-dependent effects on GpIIb/IIIa quaternary structure were detected by sedimentation equilibrium. Transmission electron microscopy showed minimal tirofiban-induced receptor activation or oligomerization. Thus, even at the increased concentrations needed to block platelet:fibrin adhesive interactions, tirofiban exhibited only limited effects on GpIIb/IIIa conformation and clustering. Our results provide new insights into the mechanisms and potential prothrombotic complications of integrin antagonists.

Binding strength and activation state of single fibrinogen-integrin pairs on living cells.

Integrin activation states determine the ability of these receptors to mediate cell-matrix and cell-cell interactions. The prototypic example of this phenomenon is the platelet integrin, alphaIIbbeta3. In unstimulated platelets, alphaIIbbeta3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables alphaIIbbeta3 to bind ligands such as fibrinogen and von Willebrand factor. To study the regulation of integrin activation states at the level of single molecules, we developed a model system based on laser tweezers, enabling us to determine the rupture forces required to separate single ligand-receptor pairs by using either purified proteins or intact living cells. Here, we show that rupture forces of individual fibrinogen molecules and either purified alphaIIbbeta3 or alphaIIbbeta3 on the surface of living platelets were 60 to 150 pN with a peak yield strength of 80-100 pN. Platelet stimulation using either ADP or the thrombin receptor-activating peptide enhanced the accessibility but not the adhesion strength of single alphaIIbbeta3 molecules, indicating that there are only two states of alphaIIbbeta3 activation. Thus, we found it possible to use laser tweezers to measure the regulation of forces between individual ligand-receptor pairs on living cells. This methodology can be applied to the study of other regulated cell membrane receptors using the ligand-receptor yield strength as a direct measure of receptor activation/inactivation state.

Binding of a fibrinogen mimetic stabilizes integrin alphaIIbbeta3's open conformation.

The platelet integrin alphaIIbbeta3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of alphaIIbbeta3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for alphaIIbbeta3. Eptifibatide binding promptly and reversibly perturbed the conformation of the alphaIIbbeta3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted alphaIIbbeta3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected alphaIIbbeta3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to alphaIIbbeta3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects alphaIIbbeta3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.

High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: detection of P-selectin (CD62P), GPI-IX complex (CD42A/CD42B alpha,B beta), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging.

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)

Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes.

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.

Effects of ligand-mimetic peptides Arg-Gly-Asp-X (X = Phe, Trp, Ser) on alphaIIbbeta3 integrin conformation and oligomerization.

The purpose of this investigation was to determine what structural changes convert "inert" alphaIIbbeta3 integrins into "activated" high-affinity receptors for adhesive proteins. Light scattering, analytical ultracentrifugation, electron microscopy, and molecular modeling were used to probe the conformational states of the alphaIIbbeta3 integrin. Isolated from human blood platelets in octyl glucoside, the alphaIIbbeta3 complex behaved as an asymmetric 230 kDa macromolecule with a z-average translational diffusion coefficient of 2.9 F and a weight-average sedimentation coefficient of 7.7 S. Dynamic light scattering showed that ligand-mimetic peptides (RGDX, X = F, W, S) caused prompt, concentration-dependent increases in the Stokes radius (R(s)) of the alphaIIbbeta3 complex, whereas control peptides of reversed sequence (XDGR, X = F, W, S) had no significant effect. Sedimentation velocity data coupled with time-derivative analyses showed that RGDX peptides shifted the distribution of alphaIIbbeta3 sedimenting species toward smaller s values. Sedimentation equilibrium measurements indicated that a slower increase in the alphaIIbbeta3 molecular weight distribution took place in the presence of RGDX ligand-mimetics. Electron microscopy showed a split of alphaIIbbeta3's globular domain into two distinct nodules in the presence of RGDX peptides; oligomers joined through their stalk regions were seen frequently. These observations suggest that receptor occupancy by ligand-mimetic RGDX peptides is tightly coupled to relatively large changes in the structure of the alphaIIbbeta3 complex. alphaIIbbeta3 bead models were developed to describe quantitatively the ligand-induced transition from a "closed" to an "open" integrin conformation and the limited oligomerization that follows. This provides a new mechanistic framework for understanding integrin activation and the formation of signaling clusters on the surface of stimulated platelets.