Mitochondrially Mediated Integrin αIIbβ3 Protein Inactivation Limits Thrombus Growth

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin α_IIbβ₃ or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin α_IIbβ₃ and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin α_IIbβ₃ epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin α_IIbβ₃ function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β₃ cytoplasmic domain, coincided closely with integrin α_IIbβ₃ inactivation. Calpain inhibition blocked integrin β₃ cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β₃ and associated regulatory proteins, resulting in integrin α_IIbβ₃ inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.     

Tyrosine Phosphorylation of Integrin β3 Regulates Kindlin-2 Binding and Integrin Activation

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY⁷⁵⁹ motif in the β₃ CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β₃ CT interacted well with kindlin-2, whereas the Tyr⁷⁵⁹-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β₃ CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.     

Dok-2 Adaptor Protein Regulates the Shear-dependent Adhesive Function of Platelet Integrin αIIbβ3 in Mice

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin α_IIbβ₃, raising the possibility that it participates in integrin α_IIbβ₃ outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin α_IIbβ₃-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P₂ accumulation. Although agonist-induced integrin α_IIbβ₃ affinity regulation was unaltered in Dok-2^−/− platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin α_IIbβ₃ adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.     

Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

Structural data of integrin αIIbβ3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a “bent,” low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that “clasping” the αIIb head domain to the β3 tail decreased fibrinogen binding. To study the role of αIIb extension about the genu, we introduced a disulfide “clamp” between the αIIb thigh and calf-1 domains. Clamped αIIbβ3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn²⁺ or by coexpressing the clamped αIIb with a β3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M_r 7,500) or αIIbβ3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for αIIb extension about the genu in the binding of ligands of 340,000 and 900,000 M_r with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with αIIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in β3 that affect the intrinsic affinity of the binding pocket for ligand.     

The Structure of the N-Terminus of Kindlin-1: A Domain Important for αIIbβ3 Integrin Activation

The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the β-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced αIIbβ3 integrin activation and for the localization of kindlin-1 to focal adhesions.     

Phosphorylation of Zona Occludens-2 by Protein Kinase Cε Regulates Its Nuclear Exportation

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCε. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.     

Cross-talk between Serine/Threonine Protein Phosphatase 2A and Protein Tyrosine Phosphatase 1B Regulates Src Activation and Adhesion of Integrin αIIbβ3 to Fibrinogen

Integrin α_IIbβ₃ signaling mediated by kinases and phosphatases participate in hemostasis and thrombosis, in part, by supporting stable platelet adhesion. Our previous studies indicate that the genetic manipulation of PP2Acα (α isoform of the catalytic subunit of protein phosphatase 2A) negatively regulate the adhesion of human embryonal kidney 293 cells expressing α_IIbβ₃ to fibrinogen. Here, we demonstrated that small interference RNA (siRNA) mediated knockdown of PP2Acα in 293 α_IIbβ₃ cells led to the dephosphorylation of Src Tyr-529, phosphorylation of Src Tyr-418 and an increased Src kinase activity. Conversely, overexpression of PP2Acα decreased the basal Src activity. Pharmacological inhibition of PP2Ac in human platelets or PP2Acα knockdown in primary murine megakaryocytes resulted in Src activation. PP2Acα-depleted 293 α_IIbβ₃ cells did not alter the serine (Ser) phosphorylation of Src but enhanced the Ser-50 phosphorylation of protein tyrosine phosphatase 1B (PTP-1B) with a concomitant increase in the PTP-1B activity. Src activation in the PP2Acα-depleted 293 α_IIbβ₃ cells was abolished by siRNA mediated knockdown of PTP-1B. Pharmacological inhibition of Src or knockdown of Src, PTP-1B blocked the enhanced activation of extracellular signal-regulated kinase (ERK1/2) and the increased adhesiveness of PP2Acα-depleted 293 α_IIbβ₃ cells to fibrinogen, respectively. Thus, inactivation of PP2Acα promotes hyperphosphorylation of PTP-1B Ser-50, elevates PTP-1B activity, which dephosphorylates Src Tyr-529 to activate Src and its downstream ERK1/2 signaling pathways that regulate α_IIbβ₃ adhesion. Moreover, these studies extend the notion that a cross-talk between Ser/Thr and Tyr phosphatases can fine-tune α_IIbβ₃ outside-in signaling.     

The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

Background

Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin α_IIbβ₃. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with α_IIbβ₃, the role of PP1c in platelet reactivity is unclear.

Methodology/Principal Findings

Using γ isoform of PP1c deficient mice (PP1cγ^−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ^−/− platelets showed decreased α_IIbβ₃ activation despite comparable levels of α_IIbβ₃, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin α_IIbβ₃ signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ^−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ^−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ^−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ^−/− platelets.

Conclusions/Significance

These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.     

Integrin αIIbβ3 intermediates: From molecular dynamics to adhesion assembly

The platelet integrin α_IIbβ₃ undergoes long-range conformational transitions associated with its functional conversion from inactive (low-affinity) to active (high-affinity) during hemostasis. Although new conformations that are intermediate between the well-characterized bent and extended states have been identified, their molecular dynamic properties and functions in the assembly of adhesions remain largely unexplored. In this study, we evaluated the properties of intermediate conformations of integrin α_IIbβ₃ and characterized their effects on the assembly of adhesions by combining all-atom simulations, principal component analysis, and mesoscale modeling. Our results show that in the low-affinity, bent conformation, the integrin ectodomain tends to pivot around the legs; in intermediate conformations, the headpiece becomes partially extended, away from the lower legs. In the fully open, active state, α_IIbβ₃ is flexible, and the motions between headpiece and lower legs are accompanied by fluctuations of the transmembrane helices. At the mesoscale, bent integrins form only unstable adhesions, but intermediate or open conformations stabilize the adhesions. These studies reveal a mechanism by which small variations in ligand binding affinity and enhancement of the ligand-bound lifetime in the presence of actin retrograde flow stabilize α_IIbβ₃ integrin adhesions.     

Intermolecular Transmembrane Domain Interactions Activate Integrin αIIbβ3

Integrins are the major cell adhesion molecules responsible for cell attachment to the extracellular matrix. The strength of integrin-mediated adhesion is controlled by the affinity of individual integrins (integrin activation) as well as by the number of integrins involved in such adhesion. The positive correlation between integrin activation and integrin clustering had been suggested previously, but several trials to induce integrin clustering by dimerization of the transmembrane domain or tail region of integrin α subunits failed to demonstrate any change in integrin activation. Here, using platelet integrin αIIbβ3 as a model system, we showed that there is intermolecular lateral interaction between integrins through the transmembrane domains, and this interaction can enhance the affinity state of integrins. In addition, when integrin clustering was induced through heteromeric lateral interactions using bimolecular fluorescence complementation, we could observe a significant increase in the number of active integrin molecules. Because the possibility of intermolecular interaction would be increased by a higher local concentration of integrins, we propose that integrin clustering can shift the equilibrium in favor of integrin activation.     

Three-Dimensional Structures of Full-Length,Membrane-Embedded Human αIIbβ3 Integrin Complexes

Integrins are bidirectional, allosteric transmembrane receptors that play a central role in hemostasis and arterial thrombosis. Using cryo-electron microscopy, multireference single-particle reconstruction methods, and statistics-based computational fitting approaches, we determined three-dimensional structures of human integrin α_IIbβ₃ embedded in a lipid bilayer (nanodiscs) while bound to domains of the cytosolic regulator talin and to extracellular ligands. We also determined the conformations of integrin in solution by itself to localize the membrane and the talin-binding site. To our knowledge, our data provide unprecedented three-dimensional information about the conformational states of intact, full-length integrin within membrane bilayers under near-physiological conditions and in the presence of cytosolic activators and extracellular ligands. We show that α_IIbβ₃ integrins exist in a conformational equilibrium clustered around four main states. These conformations range from a compact bent nodule to two partially extended intermediate conformers and finally to a fully upright state. In the presence of nanodiscs and the two ligands, the equilibrium is significantly shifted toward the upright conformation. In this conformation, the receptor extends ∼20 nm upward from the membrane. There are no observable contacts between the two subunits other than those in the headpiece near the ligand-binding pocket, and the α- and β-subunits are well separated with their cytoplasmic tails ∼8 nm apart. Our results indicate that extension of the ectodomain is possible without separating the legs or extending the hybrid domain, and that the ligand-binding pocket is not occluded by the membrane in any conformations of the equilibrium. Further, they suggest that integrin activation may be influenced by equilibrium shifts.     

Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-β Pathway

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Integrin αIIbβ3: A novel effector of Gα13

Under physiological conditions, circulating platelets are discoid in shape.¹ On these platelets, the fibrinogen receptor (integrin α_IIbβ₃) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin α_IIbβ₃ through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling.² Outside-in signaling control processes, such as platelet spreading and clot retraction, by regulating small G-proteins such as RhoA, Rac and cdc42.Key words: platelets, integrin α_IIbβ₃, Galpha13, RhoA, clot retraction, thrombin, fibrinogenThe majority of the physiological platelet agonists (except collagen) induce inside-out signaling by binding to specific G-protein-coupled receptors (GPCRs). A G-protein plays a crucial role in translating the signal from GPCR to downstream effector molecules, ultimately leading to affinity modulation of integrin α_IIbβ₃. Platelets express nine Gα subunits; namely G_q, G_i1, G_i2, G_i3, G_z, G₁₂, G₁₃, G_s and G₁₆. Previous studies have shown that a small G-protein, RhoA, is activated by the G_12/13 family and plays a crucial role in calcium-independent platelet shape change.³ However, RhoA is also activated by α_IIbβ₃ and inhibits platelet spreading to trigger clot retraction.⁴ Recently, in a series of elegant experiments, Gong et al. have described the dynamic regulation of RhoA through a signaling crosstalk between Gα₁₃ and α_IIbβ₃.⁵By generating mice in which the platelets were depleted of Gα₁₃ using siRNA technology, Gong et al. investigated the role of Gα₁₃-mediated signaling on platelet spreading on immobilized Fg.⁵ The confocal images very clearly showed that, in the absence of Gα₁₃, platelets spread poorly on Fg, which was rescued by pretreatment with the Rho-kinase inhibitor Y27632, confirming previous findings that RhoA activated downstream of integrin α_IIbβ₃ inhibits platelet spreading. Interestingly, Gα₁₃-depleted platelets failed to activate c-Src but accelerated RhoA activation. From these observations, the authors infer that Gα₁₃ is important for integrin-mediated c-Src activation and RhoA inhibition, leading to increased cell spreading.⁵Since Gα₁₃ regulates integrin-mediated cell spreading and c-Src activation, Gong et al. examined the interaction of Gα₁₃ with α_IIbβ₃ using co-immunoprecipitation and GST pull-down assays.⁵ They found that the GTP-bound form of Gα₁₃ shows enhanced interaction with the integrin β₃ subunit. This interaction is required for the activation of c-Src and the inhibition of RhoA. However, they found that the inhibition of RhoA is transient. RhoA activation is suppressed for the first 15 min of platelet spreading, after which RhoA is activated. This initial suppression is rescued by blocking Gα₁₃ and β₃ cytoplasmic domain (β3-CD) interaction. Furthermore, they observed that RhoA activation parallels clot retraction.⁵ These findings indicate that Gα₁₃ is a key regulator of platelet spreading and clot retraction phenomena.According to Gong et al., thrombin-induced inside-out signaling through GPCR leads to GTP loading of Gα₁₃ (Fig. 1A). This GTP-bound Gα₁₃ interacts with integrin β3-CD of ligand-bound integrin, thus facilitating c-Src activation, which leads to platelet spreading. Blockade of the interaction between Gα₁₃ and β3-CD or cleavage of β3-CD by calpain results in clot retraction (Fig. 1B).Open in a separate windowFigure 1Schematic representation of the dynamic regulation of RhoA by Gα₁₃ during platelet activation. (A) Activation of platelets by thrombin receptors coupled to Gα₁₃ leads to the activation of RhoA, leading to platelet shape change. (B) The change in the conformation of integrin to a high-affinity form results in fibrinogen binding to α_IIbβ₃. Active Gα13 binds to the cytoplasmic domain of β₃ leading to the activation of c-Src, resulting in platelet spreading. The rise in intracellular calcium activates calpain, which cleaves the β₃ cytoplasmic domain, releasing c-Src, which, resulting in the activation of RhoA, leads to cell retraction. *Denotes GTP-bound active form of G-proteins.Perhaps the most significant and novel finding of the study is the identification of integrin α_IIbβ₃ as an effector of Gα₁₃. The study also convincingly shows that Gα₁₃ bound to integrin regulates RhoA via c-Src. Furthermore, achieving 80% knockdown of Gα₁₃ in an in vivo setting using siRNA represents a technological advancement. Since Gα₁₃ binds to integrin β3-CD in a 1:1 stoichiometry, it appears that only a small population of integrin is regulated by Gα₁₃, as there are far less Gα₁₃ molecules in a single platelet than the number of α_IIbβ₃ molecules. This will require further investigation. Gong et al. also finds that an appreciable amount of Gα₁₃ is associated with β₃ in resting platelets, which requires some explanation.⁵ It is also not clear if Gα₁₃ remains bound to β3-CD or dissociates from the integrin during clot retraction.Overall, this is a paradigm-shifting study that establishes the importance of the dynamic regulation of RhoA by Gα₁₃ in order to achieve efficient platelet spreading and clot retraction.     

β-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbβ3 Headpiece

The platelet integrin α_IIbβ₃ 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 β-subunits; the basic Lys or Arg side chain hydrogen bonds to the α_IIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the β₃-subunit. Ligand binding induces headpiece opening, with conformational change in the β-subunit. During this opening, RGD slides in the ligand-binding pocket toward α_IIb, with movement of the βI-domain β1-α1 loop toward α_IIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and α_IIb. Here we test whether ligand interactions with β₃ suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the α_IIbβ₃ 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 α_IIbβ₃ 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 β₃-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the α_IIb subunit on Arg is required.     

The Integrin-Linked Kinase-PINCH-Parvin Complex Supports Integrin αIIbβ3 Activation

Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (αIIbα6Bβ3) and mutant CHO cells expressing inactive αIIbα6Bβ3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and α-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and α-parvin, resulting in the restoration of αIIbα6Bβ3 activation. In the parental cells expressing active αIIbα6Bβ3, ILK, PINCH, and α-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both α- and β-parvin mRNA in the parent cells impaired the αIIbα6Bβ3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore αIIbα6Bβ3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired αIIbα6Bβ3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type αIIbβ3 that is an inactive form, overexpression of a talin head domain (THD) induced αIIbβ3 activation and the THD-induced αIIbβ3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the αIIbβ3-expressing CHO cells further augmented THD-induced αIIbβ3 activation, whereas they did not induce αIIbβ3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.     

Phosphoinositide 3-Kinase p110�� Regulates Integrin ��IIb��3 Avidity and the Cellular Transmission of Contractile Forces

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin α_IIbβ₃, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin α_IIbβ₃ activation (affinity modulation) primarily occurs downstream of G_i-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin α_IIbβ₃ bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin α_IIbβ₃ activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin α_IIbβ₃ association with the contractile cytoskeleton. Analysis of integrin α_IIbβ₃ adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin α_IIbβ₃ bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin α_IIbβ₃ receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors.     

On the Activation of Integrin αIIbβ3: Outside-in and Inside-out Pathways

Ataxin-1 is a human protein responsible for spinocerebellar ataxia type 1, a hereditary disease associated with protein aggregation and misfolding. Essential for ataxin-1 aggregation is the anomalous expansion of a polyglutamine tract near the protein N-terminus, but the sequence-wise distant AXH domain modulates and contributes to the process. The AXH domain is also involved in the nonpathologic functions of the protein, including a variety of intermolecular interactions with other cellular partners. The domain forms a globular dimer in solution and displays a dimer of dimers arrangement in the crystal asymmetric unit. Here, we have characterized the domain further by studying its behavior in the crystal and in solution. We solved two new structures of the domain crystallized under different conditions that confirm an inherent plasticity of the AXH fold. In solution, the domain is present as a complex equilibrium mixture of monomeric, dimeric, and higher molecular weight species. This behavior, together with the tendency of the AXH fold to be trapped in local conformations, and the multiplicity of protomer interfaces, makes the AXH domain an unusual example of a chameleon protein whose properties bear potential relevance for the aggregation properties of ataxin-1 and thus for disease.     

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, ^p1YMESRADR⁸, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (^pE315-^pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.