Publications

2018
A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System
Qin, Y., et al. A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System. Cell (2018).Abstract
Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β.
qin-2018.pdf
Ligand and cation-induced structural alterations of the leukocyte integrin LFA-1
Sen, M., Koksal, A.C., Yuki, K., Wang, J. & Springer, T.A. Ligand and cation-induced structural alterations of the leukocyte integrin LFA-1. J Biol Chem (2018).Abstract

In aI integrins including leukocyte function-associated antigen-1 (LFA-1), ligand-binding function is delegated to the aI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the aI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the aI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the aI domain is linked to headpiece opening and engenders a preferred aI domain orientation. Using small-angle Xray scattering (SAXS) and negative-stain EM we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+. Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized aI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Iglike domains pointed in the opposite direction from the β-subunit leg of LFA-1. Mutations in the integrin βI domain α1/α1` helix stabilizing either the open or the closed βI-domain conformation indicated that α1/α1` helix movements are linked to ICAM-1 binding by the aI domain and to the extended-open conformation of the ectodomain. The LFA-1--ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 β-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton, and is consistent with the cytoskeletal force model of integrin activation.

Measuring integrin conformational change on the cell surface with super-resolution microscopy
Moore, T.I., Aaron, J., Chew, T.-L. & Springer, T.A. Measuring integrin conformational change on the cell surface with super-resolution microscopy . Cell Reports In Press, (2018). Publisher's VersionAbstract

We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.

moore-2018.pdf
High integrin αVβ6 affinity reached by hybrid domain deletion slows ligand-binding on-rate
Dong, X., et al. High integrin αVβ6 affinity reached by hybrid domain deletion slows ligand-binding on-rate. Proc Natl Acad Sci U S A (2018).Abstract
The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αVβ6 truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αVβ6 affinity, in contrast to β1 integrins. In integrin opening, rearrangement at the interface between the βI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the βI domain, which greatly increases in affinity in the open conformation. The larger size of the βI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn2+ with hybrid domain truncation, αVβ6 on-rate for both pro-TGF-β1 and fibronectin declined. The results suggest that the open conformation of αVβ6 has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αVβ6 structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.
dong-2018.pdf
Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation
Le, V.Q., et al. Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation. EMBO J (2018).Abstract
Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-β1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-β1 to the α1-helix, latency lasso, and β1-strand in the prodomain and to the β6'- and β7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.
le-2018.pdf
2017
Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration
Nordenfelt, P., et al. Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration. Nat Commun 8, 1, 2047 (2017).Abstract
Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.
nordenfelt-2017.pdf
Energy landscape differences among integrins establish the framework for understanding activation
Li, J. & Springer, T.A. Energy landscape differences among integrins establish the framework for understanding activation. J Cell Biol (2017).Abstract
Why do integrins differ in basal activity, and how does affinity for soluble ligand correlate with cellular adhesiveness? We show that basal conformational equilibrium set points for integrin α4β1 are cell type specific and differ from integrin α5β1 when the two integrins are coexpressed on the same cell. Although α4β1 is easier to activate, its high-affinity state binds vascular cell adhesion molecule and fibronectin 100- to 1,000-fold more weakly than α5β1 binds fibronectin. Furthermore, the difference in affinity between the high- and low-affinity states is more compressed in α4β1 (600- to 800-fold) than in α5β1 (4,000- to 6,000-fold). α4β1 basal conformational equilibria differ among three cell types, define affinity for soluble ligand and readiness for priming, and may reflect differences in interactions with intracellular adaptors but do not predict cellular adhesiveness for immobilized ligand. The measurements here provide a necessary framework for understanding integrin activation in intact cells, including activation of integrin adhesiveness by application of tensile force by the cytoskeleton, across ligand-integrin-adaptor complexes.
li-2017-25001.pdf
Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
Swaminathan, V., et al. Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions. Proc Natl Acad Sci U S A 114, 40, 10648-10653 (2017).Abstract
Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.
swaminathan-2017-24944.pdf
Prodomain-Growth Factor Swapping in the Structure of pro-TGF-β1
Zhao, B., Xu, S., Dong, X., Lu, C. & Springer, T.A. Prodomain-Growth Factor Swapping in the Structure of pro-TGF-β1. J Biol Chem (2017).Abstract
Transforming growth factor (TGF)-β is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-β is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked prior to proprotein convertase (PC) cleavage, and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-β1 with the PC cleavage site mutated, to mimic the structure of the TGF-β1 proprotein. Structure, mutation, and model building demonstrate that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure, i.e., the prodomain arm domain and GF domain in each monomer are swapped. Swapping has important implications for the mechanism of biosynthesis in the TGF-β family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-β family. Our structure, together with two previous ones, also provides insights into which regions of the prodomain-GF complex are highly structurally conserved, and which are perturbed by crystal lattice contacts.
zhao-2017-24928.pdf
Flow-induced elongation of von Willebrand factor precedes tension-dependent activation
Fu, H., et al. Flow-induced elongation of von Willebrand factor precedes tension-dependent activation. Nat Commun 8, 1, 324 (2017).Abstract
Von Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions. Re-compaction of Von Willebrand factor is accelerated by intramolecular interactions and increases GPIbα dissociation rate. This mechanism enables VWF to be locally activated by hydrodynamic force in hemorrhage and rapidly deactivated downstream, providing a paradigm for hierarchical mechano-regulation of receptor-ligand binding.Von Willebrand factor (VWF) is a blood protein involved in clotting and is proposed to be activated by flow, but the mechanism is unknown. Here the authors show that VWF is first converted from a compact to linear form by flow, and is subsequently activated to bind GPIbα in a tension-dependent manner.
fu-2017-24942.pdf
Rules of engagement between αvβ6 integrin and foot-and-mouth disease virus
Kotecha, A., et al. Rules of engagement between αvβ6 integrin and foot-and-mouth disease virus. Nat Commun 8, 15408 (2017).Abstract
Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvβ6, via a conserved arginine-glycine-aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvβ6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.
kotecha-2017-24923.pdf
Atypical interactions of integrin αVβ8 with pro-TGF-β1
Wang, J., et al. Atypical interactions of integrin αVβ8 with pro-TGF-β1. Proc Natl Acad Sci U S A (2017).Abstract

Integrins αVβ6 and αVβ8 are specialized for recognizing pro-TGF-β and activating its growth factor by releasing it from the latency imposed by its surrounding prodomain. The integrin αVβ8 is atypical among integrins in lacking sites in its cytoplasmic domain for binding to actin cytoskeleton adaptors. Here, we examine αVβ8 for atypical binding to pro-TGF-β1. In contrast to αVβ6, αVβ8 has a constitutive extended-closed conformation, and binding to pro-TGF-β1 does not stabilize the open conformation of its headpiece. Although Mn(2+) potently activates other integrins and increases affinity of αVβ6 for pro-TGF-β1 25- to 55-fold, it increases αVβ8 affinity only 2- to 3-fold. This minimal effect correlates with the inability of Mn(2+) and pro-TGF-β1 to stabilize the open conformation of the αVβ8 headpiece. Moreover, αVβ8 was inhibited by high concentrations of Mn(2+) and was stimulated and inhibited at markedly different Ca(2+) concentrations than αVβ6 These unusual characteristics are likely to be important in the still incompletely understood physiologic mechanisms that regulate αVβ8 binding to and activation of pro-TGF-β.

pnas-2017-wang-1705129114.pdf
Integrin extension enables ultrasensitive regulation by cytoskeletal force
Li, J. & Springer, T.A. Integrin extension enables ultrasensitive regulation by cytoskeletal force. Proc Natl Acad Sci U S A (2017).Abstract

Integrins undergo large-scale conformational changes upon activation. Signaling events driving integrin activation have previously been discussed conceptually, but not quantitatively. Here, recent measurements of the intrinsic ligand-binding affinity and free energy of each integrin conformational state on the cell surface, together with the length scales of conformational change, are used to quantitatively compare models of activation. We examine whether binding of cytoskeletal adaptors to integrin cytoplasmic domains is sufficient for activation or whether exertion of tensile force by the actin cytoskeleton across the integrin-ligand complex is also required. We find that only the combination of adaptor binding and cytoskeletal force provides ultrasensitive regulation. Moreover, switch-like activation by force depends on the large, >130 Å length-scale change in integrin extension, which is well tailored to match the free-energy difference between the inactive (bent-closed) and active (extended-open) conformations. The length scale and energy cost in integrin extension enable activation by force in the low pN range and appear to be the key specializations that enable cell adhesion through integrins to be coordinated with cytoskeletal dynamics.

pnas-2017-li-4685-90.pdf
Distinct recognition of complement iC3b by integrins αXβ2 and αMβ2
Xu, S., Wang, J., Wang, J.-H. & Springer, T.A. Distinct recognition of complement iC3b by integrins αXβ2 and αMβ2. Proc Natl Acad Sci U S A (2017).Abstract

Recognition by the leukocyte integrins αXβ2 and αMβ2 of complement iC3b-opsonized targets is essential for effector functions including phagocytosis. The integrin-binding sites on iC3b remain incompletely characterized. Here, we describe negative-stain electron microscopy and biochemical studies of αXβ2 and αMβ2 in complex with iC3b. Despite high homology, the two integrins bind iC3b at multiple distinct sites. αXβ2 uses the αX αI domain to bind iC3b on its C3c moiety at one of two sites: a major site at the interface between macroglobulin (MG) 3 and MG4 domains, and a less frequently used site near the C345C domain. In contrast, αMβ2 uses its αI domain to bind iC3b at the thioester domain and simultaneously interacts through a region near the αM β-propeller and β2 βI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXβ2 and the binding site of αMβ2 on iC3b. Distinctive binding sites on iC3b by integrins αXβ2 and αMβ2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors.

xu-2017-24910.pdf
Sorting zebrafish thrombocyte lineage cells with a Cd41 monoclonal antibody enriches hematopoietic stem cell activity
Gansner, J.M., et al. Sorting zebrafish thrombocyte lineage cells with a Cd41 monoclonal antibody enriches hematopoietic stem cell activity. Blood (2017). gansner-2017.pdf
Conformational equilibria and intrinsic affinities define integrin activation
Li, J., et al. Conformational equilibria and intrinsic affinities define integrin activation. EMBO J (2017).Abstract

We show that the three conformational states of integrin α5β1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1 On the surface of K562 cells, α5β1 is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.

li-2017-24745.pdf
Force interacts with macromolecular structure in activation of TGF-β
Dong, X., et al. Force interacts with macromolecular structure in activation of TGF-β. Nature 542, 7639, 55-59 (2017).Abstract

Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-β1 precursor (pro-TGF-β1), integrins bind to the prodomain, apply force, and release the TGF-β growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVβ6 binds pro-TGF-β1 in an orientation biologically relevant for force-dependent release of TGF-β from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-β orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin β-subunit. Simulations of force-dependent activation of TGF-β demonstrate evolutionary specializations for force application through the TGF-β prodomain and through the β- and not α-subunit of the integrin.

dong-2017-24772.pdf
2016
Samy, K.P., et al. Selective Targeting of High-Affinity LFA-1 Does Not Augment Costimulation Blockade in a Nonhuman Primate Renal Transplantation Model. Am J Transplant (2016).Abstract
Costimulation blockade (CoB) via belatacept is a lower-morbidity alternative to calcineurin inhibitor (CNI)-based immunosuppression. However, it has higher rates of early acute rejection. These early rejections are mediated in part by memory T cells, which have reduced dependence on the pathway targeted by belatacept and increased adhesion molecule expression. One such molecule is leukocyte function antigen (LFA)-1. LFA-1 exists in two forms: a commonly expressed, low-affinity form and a transient, high-affinity form, expressed only during activation. We have shown that antibodies reactive with LFA-1 regardless of its configuration are effective in eliminating memory T cells but at the cost of impaired protective immunity. Here we test two novel agents, leukotoxin A and AL-579, each of which targets the high-affinity form of LFA-1, to determine whether this more precise targeting prevents belatacept-resistant rejection. Despite evidence of ex vivo and in vivo ligand-specific activity, neither agent when combined with belatacept proved superior to belatacept monotherapy. Leukotoxin A approached a ceiling of toxicity before efficacy, while AL-579 failed to significantly alter the peripheral immune response. These data, and prior studies, suggest that LFA-1 blockade may not be a suitable adjuvant agent for CoB-resistant rejection.
Coordinated integrin activation by actin-dependent force during T-cell migration.
Nordenfelt, P., Elliott, H.L. & Springer, T.A. Coordinated integrin activation by actin-dependent force during T-cell migration. Nat Commun 7, 13119 (2016).Abstract

For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration.

nordenfelt-2016-24447.pdf
Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia.
Bury, L., et al. Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia. Haematologica 101, 1, 46-56 (2016).Abstract

Several patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin αIIbβ3. The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of αIIbβ3 lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β3 del647-686 mutation and Chinese hamster ovary cells expressing different αIIbβ3-activating mutations, we showed that reduced surface expression of αIIbβ3 is due to receptor internalization. Moreover, we demonstrated that permanent triggering of αIIbβ3-mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β3-transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated αIIbβ3 is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia.

bury-2016-24728.pdf

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