Integrin Conformation Research Articles

The dual structural roles of the membrane distal region of the α-integrin cytoplasmic tail during integrin inside-out activation.

Studies on the mechanism of integrin inside-out activation have been focused on the role of β-integrin cytoplasmic tails, which are relatively conserved and bear binding sites for the intracellular activators including talin and kindlin. Cytoplasmic tails for α-integrins share a conserved GFFKR motif at the membrane-proximal region and this forms a specific interface with the β-integrin membrane-proximal region to keep the integrin inactive. The α-integrin membrane-distal regions, after the GFFKR motif, are diverse both in length and sequence and their roles in integrin activation have not been well-defined. In this study, we report that the α-integrin cytoplasmic membrane-distal region contributes to maintaining integrin in the resting state and to integrin inside-out activation. Complete deletion of the α-integrin membrane-distal region diminished talin- and kindlin-mediated integrin ligand binding and conformational change. A proper length and suitable amino acids in α-integrin membrane-distal region was found to be important for integrin inside-out activation. Our data establish an essential role for the α-integrin cytoplasmic membrane-distal region in integrin activation and provide new insights into how talin and kindlin induce the high-affinity integrin conformation that is required for fully functional integrins.

The Dual Structural Roles of the Membrane Distal Region of α Integrin Cytoplasmic Tail in Integrin inside-out Activation

Integrin inside-out activation is essential for platelet aggregation mediated by αIIbβ3 and leukocytes migration and arresting mediated by αLβ2. How integrin is activated by the inside-out stimulation is not completely understood. Integrin activation from inside the cell is regulated through the transmembrane and cytoplasmic domains. Mutagenesis and structural studies revealed that the inactive integrin conformation is maintained by the specific interactions at the transmembrane and cytoplasmic domains. Inside-out signals impinging on integrin cytoplasmic domain disturb the transmembrane and cytoplasmic associations, resulting in conformational change of extracellular domain that is required for binding ligands. Studies on the mechanism of integrin inside-out activation have been focused on β cytoplasmic tail that is relatively conserved and bears binding sites for the common intracellular activators including talin and kindlin. The integrin α cytoplasmic tails only share a conserved GFFKR motif at the membrane-proximal region that forms specific interface with the membrane-proximal region of β cytoplasmic tail. The membrane-distal regions after the GFFKR motif are diverse significantly both in length and sequence. Their roles in integrin activation have not been well characterized. In this study, by comprehensive mutagenesis, we defined the role of the membrane-distal region of α integrin cytoplasmic tail in maintaining integrin in the resting state and in integrin inside-out activation. We found that complete deletion of the αIIb cytoplasmic membrane-distal region greatly enhances αIIbβ3 activation induced by the active mutations such as β3-K716A and β3-G708L, indicating that the missing of membrane-distal region facilitates integrin activation, i.e. the αIIb membrane-distal region contributes to the inactive integrin conformation. On the other hand, complete deletion of the αIIb membrane-distal region abolished integrin activation induced by the active mutations of αIIb-R995 and β3-D723, indicating that the αIIb membrane-distal region also contributes to integrin inside-out activation. We demonstrated that deletion of the membrane-distal region of αIIb, αV, or αL integrin greatly diminished ligand binding induced by overexpression of talin-1 head and/or kindlin-2 or -3 in 293FT cells. We further confirmed the effect of α cytoplasmic membrane-distal region on integrin inside-out activation in K562 cells. In the absence of αIIb cytoplasmic membrane-distal region, PMA failed to induce ligand binding to αIIbβ3 integrin expressed in K562 cells. This effect was due to the lack of talin-1-head and kindlin-induced integrin conformational change (ectodomain extension and headpiece opening) in the absence of α cytoplasmic membrane-distal region as reported by the conformation-dependent monoclonal antibodies. Structural superposition of αIIbβ3 transmembrane-cytoplasmic heterodimer and talin-1-head/β-tail complex reveals steric clashes between talin-1 head and the αIIb membrane-distal residues (NR997) immediately follow the GFFKR motif, which has been suggested to play a role in talin-mediated integrin activation. To test this possibility, we retained two native residues, NR997 for the αIIb membrane-distal region and found that it partially restores talin-1-head-induced integrin activation. Replacing the NR997 with small amino acids, GG997 or AA997 has little effect, while with the bulky residues YY997 significantly reduced talin-1-head-induced αIIbβ3 activation. Interestingly, retaining two native residues for the membrane-distal region of αV or αL integrin failed to restore talin-1-head-induced αVβ3 or αLβ2 activation. Retaining as long as 8 native residues for the αL membrane-distal region is not sufficient to restore talin-1-head-induced αLβ2 activation to the level of intact αL. These data demonstrate that a steric clash might play a role but is not the sole mechanism by which the α cytoplasmic membrane-distal region participates in integrin inside-out activation. A proper length and amino acids of the membrane-distal region is required for talin-induced integrin activation. Our data established an essential role of the α integrin cytoplasmic membrane-distal region in integrin activation and provide new insight of how talin and kindlin induce the high affinity integrin conformation that is required for fully functional integrins. DisclosuresNo relevant conflicts of interest to declare.

Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation, but is independent of post-translational modifications of osteopontin

Osteopontin (OPN) is a ligand for the α4ß1 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of post-translational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines and compared OPN interaction with α4 integrin to that of VCAM and fibronectin. Jurkat cells, whose α4 integrins are inherently activated, adhered to different preparations of OPN in the presence of Mn2+: the EC50 of adhesion was not affected by phosphorylation or glycosylation status. Thrombin cleavage of OPN at the C-terminus of the α4 integrin-binding site also did not affect binding affinity. THP-1 cells express a low-affinity conformation of the integrin and adhered to OPN only in the presence of Mn2+ plus PMA or an activating antibody. This was in contrast to VCAM and fibronectin: THP-1 cells adhered to these ligands without integrin activation. Studies with ligand-induced binding site antibodies demonstrated that the SVVYGLR peptide of OPN bound to the α4 integrin with a similar affinity as the LDV peptide of fibronectin, suggesting that a high off-rate is responsible for the reduced binding of OPN to the low-affinity forms of this integrin. Together, the results suggest OPN has very low affinity for the α4 integrin on human leukocytes under physiological conditions.

Membrane tension drives ligand-independent integrin signaling.

Integrins anchor cells to the extracellular matrix (ECM) and control a multitude of essential cellular functions by activating a variety of signaling pathways. In this issue of The EMBO Journal, a study conducted by Ferraris and colleagues report that binding of urokinase-type plasminogen activator receptor (uPAR) to vitronectin is sufficient to trigger ligand-independent β1 and β3 integrin signaling. The coupling of uPAR and integrins occurs independent of lateral interaction between the two receptors, but relies on membrane tension (Ferraris et al, 2014).

α5β1 integrins in hepatocytes act as receptors for bile acids with a (nor)ursodeoxycholane scaffold

Ursodeoxycholic acid (UDCA) is a standard treatment in several cholestatic liver diseases (Figure 1A) [1]. In vivo, conjugation with taurine occurs rapidly and yields tauroursodeoxycholic acid (TUDC), which has been shown to promote choleresis by triggering the insertion of ATP-dependent transport proteins (e.g., the bile salt export pump (Bsep) and the multidrug resistance protein-2 (Mrp2)) into the canalicular membrane [2]. TUDC-induced recruitment of Bsep results from activation of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3 kinase), and c-Src, which leads to downstream activation of extracellular signal-regulated kinases (Erks) and p38 mitogen-activated protein kinase (p38). Upon hepatocyte swelling, either induced by exposure to a hypoosmotic environment or insulin, a5b1 integrins become activated and trigger similar signaling events towards choleresis. a5b1 Integrins may also become activated by a swelling-independent way as previously shown by exposing hepatocytes to pathophysiological concentrations of urea [3]. Both TUDC-induced and swelling-dependent signaling were abolished in the presence of an antagonistic, RGD-motif containing hexapeptide (GRGDSP). These findings led us to hypothesize that a5b1 integrin will act as a receptor for TUDC in hepatocytes. We tested this hypothesis in a combined experimental and computational study [4]. Immunofluorescence staining on cryosections of isolated perfused rat liver (IPRL) revealed the active conformation of b1 integrin within 1 min after addition of TUDC at a concentration of 20 μM. Furthermore, phosphorylation of Erk-1 and -2 as well as activation of the epidermal growth factor receptor were induced by TUDC within the same time span. These effects were sensitive to inhibition by GRGDSP but insensitive to the presence of an inactive control peptide (GRADSP). As TUDC does not affect hepatocyte volume, which excludes that TUDC triggers integrin activation osmotically, these findings demonstrated that TUDC directly activates a5b1 integrins and triggers signaling events towards choleresis. While swelling-induced b1 integrin activation occurs primarily in the plasma membrane, TUDC-induced b1 integrin activation occurs primarily in the cytosol of hepatocytes. We demonstrated that the presence of the Na/taurocholate cotransporting polypeptide (Ntcp) is required for the latter. The need to uptake and/or concentrate TUDC inside the hepatocyte for b1 integrin activation to occur may explain why TUDC primarily acts in the liver. In order to provide insights at a molecular level as to how TUDC activates a5b1-integrin, a complex structure of a homology model of the ectodomain of a5b1-integrin and TUDC was generated by molecular docking and subsequently subjected to molecular dynamics (MD) simulations of 200 ns length [4]. These simulations revealed pronounced conformational changes in three regions of the bA domain of the integrin (Figure 1B): I) Helix a1 straightens and becomes continuous; II) this leads to a tighter packing between the top of helix a7 and the center of a1, which has been characterized as “T-junction formation” in an X-ray structure of integrin aIIbb3 bound to a ligand as well as in computational studies of agonistbound integrins; III) as a result, helix a7 moves downwards and outwards, which imposes a torque on the hybrid domain. The induced rotational motion of the hybrid domain is a prerequisite for the unbending of the integrin ectodomain, which, in turn, is required for integrin activation according to current models. Neither did MD simulations of the ectodomain of a5b1 integrin bound to GRGDSP nor to taurocholic acid (TC) (Figure 1A) reveal such conformational changes, in line with results * Correspondence: gohlke@uni-duesseldorf.de Department of Mathematics and Natural Sciences, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, 40225 Dusseldorf, Germany Full list of author information is available at the end of the article Bonus et al. European Journal of Medical Research 2014, 19(Suppl 1):S13 http://www.eurjmedres.com/content/19/S1/S13 EUROPEAN JOURNAL OF MEDICAL RESEARCH

Abstract 54: PSI Domain of ß3 Integrin Has Endogenous Thiol Isomerase Function and is a Novel Antithrombotic Therapeutic Target

Integrin αIIbβ3 plays a critical role in platelet aggregation and adhesion, key events in hemostasis and thrombosis. Integrin activation involves complex signalling events that lead to conformational changes exposing ligand-binding sites; however, mechanisms underlying integrin activation remain poorly understood. The β subunit contains a PSI domain that is highly conserved across integrins and species, though its function is unknown. Integrin β subunits are cysteine-rich and endogenous thiol isomerase activity in integrin β3 has been reported. The PSI domain contains two CXXC sequences, the active site motif of protein disulfide isomerase (PDI). Based on this observation and the location of this domain at the knee region of the integrin, we hypothesized that integrin PSI domain has endogenous thiol isomerase function, which plays a key regulatory role in integrin conformation and function. Targeting the PSI domain may have therapeutic potential. Using reduced, denatured RNase, a recombinant murine integrin β3 PSI domain demonstrated endogenous PDI-like activity. This PDI-like activity was dose-dependently inhibited by the PDI inhibitor, bacitracin. Mutation of either CXXC motif within the integrin β3 PSI domain reduced PDI-like activity, while removal of both CXXC motifs completely abolished this activity. We developed unique mouse anti-mouse/anti-human β3 PSI domain monoclonal antibodies (anti-PSI mAbs) that inhibited the PDI-like activity of both the murine recombinant integrin β3 PSI domain and purified human platelet β3 integrin, in a dose-dependent manner. Interestingly, the anti-PSI mAbs blocked fibrinogen to human platelet β3 integrin in a cell free system. Furthermore, anti-PSI mAbs inhibited murine and human platelet aggregation in vitro and ex vivo and inhibited murine thrombus formation in vivo without significantly changing bleeding time or platelet count. In conclusion, we identified that the PSI domain has PDI function, is a fundamental regulator of platelet β3 integrin activation, and is a potential novel target for anti-thrombotic therapies. Since PSI domain is conserved in all integrin β subunits, our discovery may have broad implications for the role of integrins in cell biology of many human diseases.

RGD Labeled Ru(II) Polypyridyl Conjugates for Platelet Integrin αIIbβ3 Recognition and as Reporters of Integrin Conformation

The ability of two novel ruthenium(II) polypyridyl-Arg-Gly-Asp (RGD) peptide conjugates to act as molecular probes for reporting on the presence and conformation of integrin αIIbβ3 in solution and in live cells was described. The compounds are [Ru(bpy)2PIC-RGD](2+), bpy-RGD, and [Ru(dpp)2PIC-RGD](2+), dpp-RGD, where dpp is 4,7-diphenyl-1,10-phenanthroline, bpy is 2,2'-bipyridine, and PIC is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline. Bpy-RGD is hydrophilic, whereas dpp-RGD is comparatively hydrophobic. Both probes exhibited good affinity and high specificity for purified αIIbβ3 in solution. Binding of either complex to the resting integrin resulted in an approximately 8-fold increase of emission intensity from the metal center with dissociation constants (Kd) in the micromolar range for each complex. The Kd for each conjugate/αIIbβ3 assembly were compared following treatment of the integrin with the activating agents, Mn(2+) and dithiothreitol (DTT), which are commonly used to induce active-like conformational changes in the integrin. For bpy-RGD/αIIbβ3 Kd showed relatively little variation with integrin activation, presenting the following trend: denatured αIIbβ3 > resting αIIbβ3 = pretreated DTT = pretreated Mn(2+). Kd for dpp-RGD/ αIIbβ3 showed greater variation with integrin activation and the following trend was followed: denatured αIIbβ3 > resting αIIbβ3 > pretreated Mn(2+) = pretreated DTT. Time resolved luminescence anisotropy was carried out to obtain the rotational correlation time of bpy-RGD and dpp-RGD bound to resting or nominally activated integrin. The rotational correlation times of bpy-RGD and dpp-RGD, too fast to measure unbound, decreased to 1.50 ± 0.03 μs and 2.58 ± 0.04 μs, respectively, when the complexes were bound to resting integrin. Addition of Mn(2+) to bpy-RGD/αIIbβ3 or dpp-RGD/αIIbβ3 reduced the rotational correlation time of the ruthenium center to 1.29 ± 0.03 μs and to 1.72 ± 0.03 μs, respectively. Following treatment, the rotational correlation time decreased to 1.04 ± 0.01 μs and 1.29 ± 0.03 μs for bpy-RGD/αIIbβ3, and dpp-RGD/αIIbβ3, respectively. The large relative changes in rotational correlation times observed for Mn(2+) or DTT activated integrin indicates significant change in protein conformation compared with the resting integrin. The results also indicated that the metal complex itself affects the final conformational and/or aggregation status of the protein obtained. Furthermore, the extent of conformational change was influenced by whether the probe was bound to the integrin before or after activator treatment. Finally, in vitro studies indicated that both probes selectively bind to CHO cells expressing the resting form of αIIbβ3. In each case the probe colocalized with αIIb specific SZ22 antibody. Overall, this work indicates that bpy-RGD and dpp-RGD may be useful peptide-probes for rapid assessment of integrin structural status and localization in solution and cells.

Integrin clustering as a result of local membrane deformations and local signaling feedbacks

Integrins are essential receptors for the development and functioning of multicellular animals because they mediate cell adhesion and migration, and regulate cell proliferation and apoptosis. Ligand-dependent activation of integrins involves the formation of receptor clusters and this has been accounted both to extracellular forces as mediated by the glycocalyx as well as to intracellular forces mediated by the cytoskeleton. Here we describe a Monte Carlo simulation that considers both the binding processes on the membrane as well as the intracellular signaling processes that stabilize the open integrin conformation. We show that integrin clustering can result both from the effects of integrin avidity, as a result of membrane deformations, as well as from the locally enhanced availability of talins in the open conformation, as a result of local positive feedback signaling via PIPKIγ and PIP2. The model was carefully parameterized based on reported quantitative data and reproduces a wide range of experimental data, including results that previously appeared inconsistent.

Activation states of blood eosinophils in asthma

Asthma is characterized by airway inflammation rich in eosinophils. Airway eosinophilia is associated with exacerbations and has been suggested to play a role in airway remodelling. Recruitment of eosinophils from the circulation requires that blood eosinophils become activated, leading to their arrest on the endothelium and extravasation. Circulating eosinophils can be envisioned as potentially being in different activation states, including non-activated, pre-activated or 'primed', or fully activated. In addition, the circulation can potentially be deficient of pre-activated or activated eosinophils, because such cells have marginated on activated endothelium or extravasated into the tissue. A number of eosinophil surface proteins, including CD69, L-selectin, intercellular adhesion molecule-1 (ICAM-1, CD54), CD44, P-selectin glycoprotein ligand-1 (PSGL-1, CD162), cytokine receptors, Fc receptors, integrins including αM integrin (CD11b), and activated conformations of Fc receptors and integrins, have been proposed to report cell activation. Variation in eosinophil activation states may be associated with asthma activity. Eosinophil surface proteins proposed to be activation markers, with a particular focus on integrins, and evidence for associations between activation states of blood eosinophils and features of asthma are reviewed here. Partial activation of β1 and β2 integrins on blood eosinophils, reported by monoclonal antibodies (mAbs) N29 and KIM-127, is associated with impaired pulmonary function and airway eosinophilia, respectively, in non-severe asthma. The association with lung function does not occur in severe asthma, presumably due to greater eosinophil extravasation, specifically of activated or pre-activated cells, in severe disease.

The cytokine midkine supports neutrophil trafficking during acute inflammation by promoting adhesion via β2 integrins (CD11/CD18)

AbstractEmerging evidence suggests a role of the cytokine midkine (MK) in inflammation. In this study, its functional relevance for recruitment of polymorphonuclear neutrophils (PMNs) during acute inflammation was investigated. Intravital microscopy and histologic analysis of tumor necrosis factor-α-stimulated cremaster muscle venules revealed severely compromised leukocyte adhesion and extravasation in MK−/− mice compared with MK+/+ animals. Systemic administration of recombinant MK completely rescued the adhesion defect in MK−/− mice. In a hind limb ischemia model, leukocyte accumulation in MK−/− mice was significantly diminished compared with MK+/+ animals. However, MK did not lead to an inflammatory activation of PMNs or endothelial cells suggesting that it does not serve as classical proinflammatory cytokine. Unexpectedly, immobilized MK mediated PMN adhesion under static and flow conditions, whereas PMN-derived MK was dispensable for the induction of adhesion. Furthermore, adhesion strengthening remained unaffected by MK. Flow cytometry revealed that immobilized, but not soluble MK, significantly promoted the high affinity conformation of β2 integrins of PMNs. Blocking studies of low-density lipoprotein receptor-related protein 1 (LRP1) suggested that LRP1 may act as a receptor for MK on PMNs. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of β2 integrins, thereby facilitating PMN trafficking during acute inflammation.

An internal ligand-bound, metastable state of a leukocyte integrin, αXβ2

How is massive conformational change in integrins achieved on a rapid timescale? We report crystal structures of a metastable, putative transition state of integrin αXβ2. The αXβ2 ectodomain is bent; however, a lattice contact stabilizes its ligand-binding αI domain in a high affinity, open conformation. Much of the αI α7 helix unwinds, loses contact with the αI domain, and reshapes to form an internal ligand that binds to the interface between the β propeller and βI domains. Lift-off of the αI domain above this platform enables a range of extensional and rotational motions without precedent in allosteric machines. Movements of secondary structure elements in the β2 βI domain occur in an order different than in β3 integrins, showing that integrin β subunits can be specialized to assume different intermediate states between closed and open. Mutations demonstrate that the structure trapped here is metastable and can enable rapid equilibration between bent and extended-open integrin conformations and up-regulation of leukocyte adhesiveness.

Domain 1 of Mucosal Addressin Cell Adhesion Molecule Has an I1-set Fold and a Flexible Integrin-binding Loop

Mucosal addressin cell adhesion molecule (MAdCAM) binds integrin α4β7. Their interaction directs lymphocyte homing to mucosa-associated lymphoid tissues. The interaction between the two immunoglobulin superfamily (IgSF) domains of MAdCAM and integrin α4β7 is unusual in its ability to mediate either rolling adhesion or firm adhesion of lymphocytes on vascular surfaces. We determined four crystal structures of the IgSF domains of MAdCAM to test for unusual structural features that might correlate with this functional diversity. Higher resolution 1.7- and 1.4-Å structures of the IgSF domains of MAdCAM in a previously described crystal lattice revealed two alternative conformations of the integrin-binding loop, which were deformed by large lattice contacts. New crystal forms in the presence of two different Fabs to MAdCAM demonstrate a shift in IgSF domain topology from the I2- to I1-set, with a switch of integrin-binding loop from CC' to CD. The I1-set fold and CD loop appear biologically relevant. The different conformations seen in crystal structures suggest that the integrin-binding loop of MAdCAM is inherently flexible. This contrasts with rigidity of the corresponding loops in vascular cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, ICAM-2, ICAM-3, and ICAM-5 and may reflect a specialization of MAdCAM to mediate both rolling and firm adhesion by binding to different α4β7 integrin conformations.

Disruption of disulfide-restriction at integrin knees induces activation and ligand-independent signaling of α4β7

Control of integrin activation and signaling plays crucial roles in cell adhesion, spreading and migration. Here, we report that selective breakage of two conserved disulfide bonds located at the knees of integrin α4C589-C594 and β7C494-C526 activated α4β7. This activated integrin had a unique structure that was different from the typical extended conformation of active integrin. In addition, these activated α4β7 integrins spontaneously clustered on the cell membrane and triggered integrin downstream signaling independent of ligand binding. Although these disulfide bonds were not broken during α4β7 activation by inside-out signaling or Mn(2+), they could be specifically reduced by 0.1 mM dithiothreitol, a reducing strength that could be produced in vivo under certain conditions. Our findings reveal a novel mechanism of integrin activation under specific reducing conditions by which integrin can signal and promote cell spreading in the absence of ligand.

Novel α2β1 Integrin Inhibitors Reveal That Integrin Binding to Collagen under Shear Stress Conditions Does Not Require Receptor Preactivation

The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm(2)). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm(2)) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2β1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2β1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation.

Addition of Omega-3 α-Linolenic Acid to Platelet Apheresis Units Preserves Platelet Activatability Over Time and Reduces Baseline Activation Under Routine Storage Conditions: A Pilot Study

Abstract 3433 Background:Omega-3 fatty acids (W3-FA) have been shown to be cardioprotective by reducing plasmatic triglycerides, atherosclerosis, arrhythmias, inflammation, and thrombosis. We have previously demonstrated that the plant-derived W3-FA a-linolenic acid (ALA) can achieve similar effects as the marine-derived EPA and DHA; it reduced platelet activation and aggregation, atherosclerosis and thrombosis in mouse models. In particular, ALA increased platelet survival by reducing their clearance, thus resulting in an increased platelet count. Hypothesis:To translate this finding to transfusion medicine, we hypothesized that the addition of ALA to platelet apheresis units a) could reduce platelet baseline activation during storage and b) result in a better function/activatability over 5–10 days of storage. Methods:In this pilot study, 4 platelet apheresis units from 2 healthy volunteers were collected according to routine blood bank standards and stored under typical conditions at 22°C on a rocker for 10 days. The units were supplemented with 30 uM ALA (a concentration which can be achieved by dietary intake) or vehicle (ethanol). Samples were analyzed daily over 10 days for impedance aggregometry, thrombin generation, thrombus formation on collagen and adhesion to VWF under high shear conditions (2500 s−1). Receptor expression/activation/cleavage was determined by flow cytometry (GPIb, P-selectin, activated a2bb3 integrin, Annexin V binding) and loss of mitochondrial membrane potential by JC-1 staining. Results:After 10 days of storage, platelets from ALA-treated bags showed a better activatability compared to vehicle-treated bags. Thrombus formation on collagen in the flow chamber at d10 was higher for ALA-treated platelets (covered area: 102.779 um2 vs 68.729 um2) as well as the adhesion to VWF (platelet-covered area: 2072,2 um2 vs 427,3 um2). Thrombin generation (as measured by CAT assay) was also higher for ALA-treated samples on d10 (ETP: 15.5 nm thrombin/min vs <2 nm thrombin/min). Thrombin-induced aggregation was higher for the ALA-treated samples (AUC: 17.1±10.4 vs 3.65±0.21 with 0.25 U/ml thrombin; 59±51 vs 15.35±10.5 with 0.5 U/ml thrombin). ALA reduced GPIb cleavage from the platelet membrane (decrease of surface exposed GPIb: d1=100%, d10= 69±10% vs 63±7%). On the other hand, ALA reduced the spontaneous expression of the active conformation of a2bb3 integrin (1.18 vs 1.94 fold) and the P-selectin expression (0.9 vs 1.20 fold), while the thrombin-induced activatability of these receptors was preserved. ALA also reduced the platelet aggregation in response to collagen and thrombin on d1. Conclusions:The addition of ALA to single donor platelet apheresis units at a concentration which is nutritionally achievable results in preserved platelet activatability and function over time, while it reduces baseline activation under typical storage conditions. Both endpoints appear favorable in transfusion medicine and encourage further evaluation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Glycogen Synthase Kinase 3β Inhibition Prevents Monocyte Migration across Brain Endothelial Cells via Rac1-GTPase Suppression and Down-Regulation of Active Integrin Conformation

Glycogen synthase kinase (GSK) 3β has been identified as a regulator of immune responses. We demonstrated previously that GSK3β inhibition in human brain microvascular endothelial cells (BMVECs) reduced monocyte adhesion/migration across BMVEC monolayers. Herein, we tested the idea that GSK3β inhibition in monocytes can diminish their ability to engage the brain endothelium and migrate across the blood-brain barrier. Pretreatment of primary monocytes with GSK3β inhibitors resulted in a decrease in adhesion (60%) and migration (85%), with similar results in U937 monocytic cells. Monocyte-BMVEC interactions resulted in diminished barrier integrity that was reversed by GSK3β suppression in monocytic cells. Because integrins mediate monocyte rolling/adhesion, we detected the active conformational form of very late antigen 4 after stimulation with a peptide mimicking monocyte engagement by vascular cell adhesion molecule-1. Peptide stimulation resulted in a 14- to 20-fold up-regulation of the active form of integrin in monocytes that was suppressed by GSK3β inhibitors (40% to 60%). Because small GTPases, such as Rac1, control leukocyte movement, we measured active Rac1 after monocyte activation with relevant stimuli. Stimulation enhanced the level of active Rac1 that was diminished by GSK3β inhibitors. Monocytes treated with GSK3β inhibitors showed increased levels of inhibitory sites of the actin-binding protein, cofilin, and vasodilator-stimulated phosphoprotein-regulating conformational changes of integrins. These results indicate that GSK3β inhibition in monocytes affects active integrin expression, cytoskeleton rearrangement, and adhesion via suppression of Rac1-diminishing inflammatory leukocyte responses.

In Vivo Platelet Activation in Critically Ill Patients With Primary 2009 Influenza A(H1N1)

Changes in platelet reactivity during 2009 influenza A(H1N1) (A[H1N1]) have not been characterized. We prospectively examined platelet activation and cytokine responses in patients with A(H1N1) (n = 20), matched patients with bacterial pneumonia (n = 15), and nonhospitalized, healthy control subjects (n = 10). Platelet-monocyte aggregation was higher in patients with A(H1N1) (21.4% ± 4.7%) compared with patients with pneumonia (10.9% ± 3.7%) and control subjects (8.1% ± 4.5%, P < .05). Similarly, PAC-1 (antibody that binds to the active conformation of integrin α(IIb)β(3)) binding to platelets is increased in patients with A(H1N1) (9.5% ± 4.7%) compared with patients with pneumonia (1.0% ± 0.7%) and healthy subjects (0.61% ± 0.15%, P < .10). PAC-1 binding was twofold higher in patients with A(H1N1) with shock vs those without shock. IL-6 levels were elevated in patients with A(H1N1), indicating systemic inflammation consistent with activation of circulating platelets. These findings, derived from a small but documented cohort of patients, demonstrate that platelet activation responses during A(H1N1) are enhanced-exceeding responses in patients with bacterial pneumonia-and provide new evidence that platelets may contribute to inflammatory responses during A(H1N1).

Leukocyte CD47 plays a critical role in T‐cell recruitment in vivo through affinity regulation of LFA‐1 and VLA‐4 integrins

Host defense critically depends on effector T‐cell adhesive interaction with the vascular endothelium. CD47 is broadly expressed and participates in leukocyte recruitment and immune functions. CD47 (Integrin Associated Protein) functionally associates with multiple integrins. Here we examined the hypothesis that CD47 regulates LFA‐1 and VLA‐4 adhesive function in T‐cell recruitment. Intravital microscopy studies revealed that transferred CD47ko Th1 cells have reduced adhesion to WT TNFα‐inflamed cremaster microvessels versus WT Th1 cells. Studies in an in vitro flow model showed that CD47ko Th1 cells, as compared to WT cells, also have reduced adhesion and diapedesis of TNFα‐activated endothelium, and bind less to immobilized ICAM‐1 and VCAM‐1. Similarly, human Jurkat T‐cells lacking CD47 bind less to TNFα‐activated endothelium and to immobilized ICAM‐1 and VCAM‐1. Finally, Jurkat CD47 null cells have a reduction in Mn+2‐induced binding of mAb 24 (67%↓) and KIM127 (51%↓), which detect activated conformations of β2 integrins. However, the absence of CD47 does not affect their β2 integrin dependent “adhesion strengthening” to ICAM‐1 as determined by a cell detachment assay. Taken together, our results indicate CD47 primarily affects T‐cell VLA‐4 and LFA‐1 affinity regulation, and this function is necessary for T‐cell adhesion and diapedesis. Supported by NIH HL‐36028 and AHA fellowship 11POST7730055.

Distinct roles for talin‐1 and kindlin‐3 in LFA‐1‐dependent neutrophil rolling and arrest

β2 integrins, including LFA‐1 (αLβ2), play critical roles in leukocyte recruitment during inflammation by binding to ICAM‐1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA‐1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA‐1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin activating proteins, talin‐1 and kindlin‐3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post‐capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin‐1 is necessary for both LFA‐1‐dependent neutrophil rolling and arrest. In contrast, kindlin‐3 was required only for neutrophil arrest and was dispensable for rolling mediated by intermediate affinity LFA‐1. We further corroborate our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. These data indicate that talin‐1 is sufficient to induce LFA‐1 extension to an intermediate affinity state whereas kindlin‐3 is critical for the transition of LFA‐1 to a high affinity state.

Distinct Roles for Talin-1 and Kindlin-3 in Regulating LFA-1 Conformation during Neutrophil Rolling and Arrest

β2 integrins, including LFA-1 (αLβ2), play critical roles in leukocyte trafficking. LFA-1 mediates the recruitment of neutrophils during inflammation through interaction with ICAM-1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA-1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA-1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin tail-binding activating proteins, talin-1 and kindlin-3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post-capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin-1 is necessary for both LFA-1-dependent neutrophil rolling and arrest. In contrast, kindlin-3 is required only for neutrophil arrest and is dispensable for rolling mediated by intermediate affinity LFA-1. These data indicate that talin-1 is sufficient to induce LFA-1 extension to an intermediate affinity state whereas kindlin-3 is critical for the transition of LFA-1 to a high affinity state. We further corroborated our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. Our results are the first to demonstrate that members of the talin and kindlin protein families play distinct roles in regulating integrin conformation.