Brian G. Petrich

Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation

In inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III. Both kindlin-3 and talin-1, another FERM domain-containing cytoskeletal protein, are required for integrin activation, but their individual roles in the induction of specific integrin conformers are unclear. Here, we induce differential LFA-1 activation in neutrophils through engagement of the selectin ligand P-selectin glycoprotein ligand-1 or the chemokine receptor CXCR2. We find that talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling, whereas both talin-1 and kindlin-3 are required for induction of the high-affinity conformation of LFA-1 with an open headpiece, which results in neutrophil arrest. In vivo, both slow rolling and arrest are defective in talin-1-deficient neutrophils, whereas only arrest is defective in kindlin-3-deficient neutrophils. We conclude that talin-1 and kindlin-3 serve distinct functions in LFA-1 activation.

Microfluidic devices for studies of shear-dependent platelet adhesion

Adhesion of platelets to blood vessel walls is a shear stress dependent process that promotes arrest of bleeding and is mediated by the interaction of receptors expressed on platelets with various extracellular matrix (ECM) proteins that may become exposed upon vascular injury. Studies of dynamic platelet adhesion to ECM-coated substrates in conventional flow chambers require substantial fluid volumes and are difficult to perform with blood samples from a single laboratory mouse. Here we report dynamic platelet adhesion assays in two new microfluidic devices made of PDMS. Small cross-sections of the flow chambers in the devices reduce the blood volume requirements to <100 microl per assay, making the assays compatible with samples of whole blood obtained from a single mouse. One device has an array of 8 flow chambers with shear stress varying by a factor of 1.93 between adjacent chambers, covering a 100-fold range from low venous to arterial. The other device allows simultaneous high-resolution fluorescence imaging of dynamic adhesion of platelets from two different blood samples. Adhesion of platelets in the devices to three common ECM substrate coatings was verified to conform with published results. The devices were subsequently used to study the roles of extracellular and intracellular domains of integrin alphaIIbbeta3, a platelet receptor that is a central mediator of platelet aggregation and thrombus formation. The study involved wild-type mice and two genetically modified mouse strains and showed that the absence of the integrin impaired adhesion at all shear stresses, whereas a mutation in its intracellular domain reduced the adhesion only at moderate and high stresses. Because of small sample volumes required, the devices could be employed in research with genetically-modified model organisms and for adhesion tests in clinical settings with blood from neonates.

The antithrombotic potential of selective blockade of talin-dependent integrin αIIbβ3 (platelet GPIIb–IIIa) activation

Abstract In vitro studies indicate that binding of talin to the beta(3) integrin cytoplasmic domain (tail) results in integrin alpha(IIb)beta(3) (GPIIb-IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the beta(3) tail. We introduced a beta(3)(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a beta(3)(L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate alpha(IIb)beta(3) require binding of talin to the beta(3) tail. beta(3)(L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride-induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of beta(3)(Y747A) and virtually all beta(3)-null animals examined. Remarkably, less than 5% of beta(3)(L746A) animals exhibited this form of bleeding. These results establish that alpha(IIb)beta(3) activation in vivo is dependent on the interaction of talin with the beta(3) integrin cytoplasmic domain. Furthermore, they suggest that modulation of beta(3) integrin-talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.

Loss of PIP5KIγ, unlike other PIP5KI isoforms, impairs the integrity of the membrane cytoskeleton in murine megakaryocytes

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the alpha, beta, and gamma isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of PIP(2) in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KIgamma(-/-) megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wild-type PIP5KIgamma, but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KIbeta- and PIP5KIgamma(-/-) cells had impaired PIP(2) synthesis. However, PIP5KIbeta-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KIbeta in PIP5KIgamma(-/-) cells failed to revert this defect. Megakaryocytes lacking the PIP5KIgamma-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KIgamma(-/-) cells. These findings demonstrate a unique role for PIP5KIgamma in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells.

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Talin-dependent integrin activation is required for fibrin clot retraction by platelets

Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton.

Talin1 and Rap1 Are Critical for Osteoclast Function

ABSTRACT To determine talin1s role in osteoclasts, we mated TLN1fl/fl mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss.

Caspase-1–mediated pathway promotes generation of thromboinflammatory microparticles

Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway-dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell-induced thrombosis.

Blocking neutrophil integrin activation prevents ischemia–reperfusion injury

Yago et al. find that neutrophils expressing a talin1 mutant that perturbs integrin activation fail to undergo chemokine-induced arrest, spreading, and migration, thus preventing ischemia–reperfusion injury.

Endothelial cell talin1 is essential for embryonic angiogenesis

Using Tln1fl/fl;CreER mice, we show that tamoxifen-induced inactivation of the talin1 gene throughout the embryo produces an angiogenesis phenotype that is restricted to newly forming blood vessels. The phenotype has a rapid onset in early embryos, resulting in vessel defects by 48 h and death of the embryo within 72 h. Very similar vascular defects were obtained using a Tie2-Cre endothelial cell-specific Tln1 knockout, a phenotype that was rescued by expression of a Tln1 mini-gene in endothelial cells. We show that endothelial cells, unlike most other cell types, do not express talin2, which can compensate for loss of talin1, and demonstrate for the first time that endothelial cells in vivo lacking talin1 are unable to undergo the cell spreading and flattening required to form vessels.