Th1 Cells Rolling on Selectins Trigger DAP12-Dependent Signals That Activate Integrin αLß2.

During inflammation, both neutrophils and effector T cells use selectins to roll and integrins to arrest in postcapillary venules. In both cell types, chemokines can transduce signals that convert integrin αLß2 to a high-affinity conformation, which interacts with ICAM-1 to mediate arrest. In neutrophils, selectins also trigger an immunoreceptor-like signaling cascade that converts integrin αLß2 to an intermediate-affinity conformation, which interacts with ICAM-1 to slow rolling. It is not known whether selectins induce similar signaling events in T cells. Ag engagement causes phosphorylation of ITAMs on the TCR; these motifs recruit kinases and adaptors that lead to the activation of αLß2. We found that mouse Th1 cells rolling on P- or E-selectin triggered signals that promoted αLß2-dependent slow rolling on ICAM-1 in vitro and in vivo. The selectin signaling cascade resembled that used by the TCR, except that unexpectedly, Th1 cells employed the ITAM-bearing protein DAP12, which was not known to be expressed in these cells. Importantly, outside-in signaling through ligand-occupied αLß2 also required DAP12. Cooperative selectin and chemokine signaling in Th1 cells promoted αLß2-dependent slow rolling and arrest in vitro and in vivo and migration into Ag-challenged tissues in vivo. Our findings reveal an important function for DAP12 in Th1 cells and a new mechanism to recruit effector T cells to sites of inflammation.

Cooperative PSGL-1 and CXCR2 signaling in neutrophils promotes deep vein thrombosis in mice.

Inflammation is a major contributor to deep vein thrombosis (DVT). Flow restriction of the inferior vena cava (IVC) in mice induces DVT like that in humans. In this model, P-selectin-dependent adhesion of neutrophils and monocytes leads to release of neutrophil extracellular traps (NETs) and expression of tissue factor. However, it is not known what signals cause myeloid cells to generate these procoagulant effectors. Using ultrasonography and spinning-disk intravital microscopy in genetically engineered mice, we found that engagement of P-selectin glycoprotein ligand-1 (PSGL-1) and the chemokine receptor CXCR2 on rolling neutrophils propagated signals that cooperated to induce ß2 integrin-dependent arrest in flow-restricted IVCs. Unlike previous reports, PSGL-1 signaling in neutrophils did not require L-selectin, and it used tyrosine 145 rather than tyrosines 112 and 128 on the adaptor Src homology domain-containing leukocyte phosphoprotein of 76 kDa. PSGL-1 and CXCR2 signaling cooperated to increase the frequency and size of thrombi, in part by stimulating release of NETs. Unlike in neutrophils, blocking PSGL-1 or CXCR2 signaling in monocytes did not affect their recruitment into thrombi or their expression of tissue factor. Our results demonstrate that neutrophils cooperatively signal through PSGL-1 and CXCR2 to promote DVT.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells.

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1-/- ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1-/- mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1-/- platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1-/- platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1-/- platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.

Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2.

Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α) in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC1B). Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking sphingosine-1-phosphate had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local sphingosine-1-phosphate release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

O-glycans direct selectin ligands to lipid rafts on leukocytes.

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1(-/-) neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1(-/-) neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti-PSGL-1 or anti-CD44 F(ab')2. Furthermore, C1galt1(-/-) neutrophils incubated with anti-PSGL-1 F(ab')2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1(-/-) neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Multi-Inhibitory Effects of A2A Adenosine Receptor Signaling on Neutrophil Adhesion Under Flow.

A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, ß2 integrin-dependent slow rolling and chemokine-induced, ß2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced ß2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain "swing-out," which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-ß2 integrin Ab. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1, and ß2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A and its substrate C-terminal Src kinase, an inhibitor of SFKs. Treating neutrophils with a protein kinase A inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases.

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets.

O-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. In this study, we report that core 1 O-glycan-deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases, including metalloproteinases (MMP)-2/9. We found that the lymph contained activated MMP-2/9 and incubation of the lymph reduced surface levels of PDPN on core 1 O-glycan-deficient endothelial cells, but not on wild-type ECs. The lymph from mice with sepsis induced by cecal ligation and puncture, which contained bacteria-derived sialidase, reduced PDPN levels on wild-type ECs. The MMP inhibitor, GM6001, rescued these reductions. Additionally, GM6001 treatment rescued the reduction of PDPN level on lymphatic endothelial cells in mice lacking endothelial core 1 O-glycan or cecal ligation and puncture-treated mice. Furthermore, core 1 O-glycan-deficient or desialylated PDPN impaired platelet interaction under physiological flow. These data indicate that sialylated O-glycans of PDPN are essential for platelet adhesion and prevent PDPN from proteolytic degradation primarily mediated by MMPs in the lymph.

Cytoskeletal regulation of CD44 membrane organization and interactions with E-selectin.

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling.

Elevated CXCL1 expression in gp130-deficient endothelial cells impairs neutrophil migration in mice.

Neutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6 family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro, and even more upon stimulation with tumor necrosis factor-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered ß2 integrin-dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti-P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion but hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation.

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion.

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIß, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion.

Transcriptional regulation of podoplanin expression by Prox1 in lymphatic endothelial cells.

Transcription factor prospero homeobox 1 (Prox-1) and podoplanin (PDPN), mucin-type transmembane protein, are both constantly expressed in lymphatic endothelial cells (LECs) and appear to function in an LEC-autonomous manner. Mice globally lacking PDPN (Pdpn(-/-)) develop abnormal and blood-filled lymphatic vessels that highly resemble those in inducible mice lacking Prox-1 (Prox1(-/-)). Prox1 has also been reported to induce PDPN expression in cultured ECs. Thus, we hypothesize that PDPN functions downstream of Prox1 and that its expression is regulated by Prox1 in LECs at the transcriptional level. We first identified four putative binding elements for Prox1 in the 5' upstream regulatory region of Pdpn gene and found that Prox1 directly binds to the 5' regulatory sequence of Pdpn gene in LECs by chromatin immunoprecipitation assay. DNA pull down assay confirmed that Prox1 binds to the putative binding element. In addition, luciferase reporter assay indicated that Prox1 binding to the 5' regulatory sequence of Pdpn regulates Pdpn gene expression. We are therefore the first to experimentally demonstrate that Prox1 regulates PDPN expression at the transcriptional level in the lymphatic vascular system.

Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLß2.

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)ß(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)ß(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)ß(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)ß(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal ß(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the ß(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)ß(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)ß(2)-mediated arrest.

Core 1-derived O-glycans are essential E-selectin ligands on neutrophils.

Neutrophils roll on E-selectin in inflamed venules through interactions with cell-surface glycoconjugates. The identification of physiologic E-selectin ligands on neutrophils has been elusive. Current evidence suggests that P-selectin glycoprotein ligand-1 (PSGL-1), E-selectin ligand-1 (ESL-1), and CD44 encompass all glycoprotein ligands for E-selectin; that ESL-1 and CD44 use N-glycans to bind to E-selectin; and that neutrophils lacking core 2 O-glycans have partially defective interactions with E-selectin. These data imply that N-glycans on ESL-1 and CD44 and O-glycans on PSGL-1 constitute all E-selectin ligands, with neither glycan subset having a dominant role. The enzyme T-synthase transfers Gal to GalNAcalpha1-Ser/Thr to form the core 1 structure Galbeta1-3GalNAcalpha1-Ser/Thr, a precursor for core 2 and extended core 1 O-glycans that might serve as selectin ligands. Here, using mice lacking T-synthase in endothelial and hematopoietic cells, we found that E-selectin bound to CD44 and ESL-1 in lysates of T-synthase-deficient neutrophils. However, the cells exhibited markedly impaired rolling on E-selectin in vitro and in vivo, failed to activate beta2 integrins while rolling, and did not emigrate into inflamed tissues. These defects were more severe than those of neutrophils lacking PSGL-1, CD44, and the mucin CD43. Our results demonstrate that core 1-derived O-glycans are essential E-selectin ligands; that some of these O-glycans are on protein(s) other than PSGL-1, CD44, and CD43; and that PSGL-1, CD44, and ESL-1 do not constitute all glycoprotein ligands for E-selectin.

Regulation of catch bonds by rate of force application.

The current paradigm for receptor-ligand dissociation kinetics assumes off-rates as functions of instantaneous force without impact from its prior history. This a priori assumption is the foundation for predicting dissociation from a given initial state using kinetic equations. Here we have invalidated this assumption by demonstrating the impact of force history with single-bond kinetic experiments involving selectins and their ligands that mediate leukocyte tethering and rolling on vascular surfaces during inflammation. Dissociation of bonds between L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) loaded at a constant ramp rate to a constant hold force behaved as catch-slip bonds at low ramp rates that transformed to slip-only bonds at high ramp rates. Strikingly, bonds between L-selectin and 6-sulfo-sialyl Lewis X were impervious to ramp rate changes. This ligand-specific force history effect resembled the effect of a point mutation at the L-selectin surface (L-selectinA108H) predicted to contact the former but not the latter ligand, suggesting that the high ramp rate induced similar structural changes as the mutation. Although the A108H substitution in L-selectin eliminated the ramp rate responsiveness of its dissociation from PSGL-1, the inverse mutation H108A in P-selectin acquired the ramp rate responsiveness. Our data are well explained by the sliding-rebinding model for catch-slip bonds extended to incorporate the additional force history dependence, with Ala-108 playing a pivotal role in this structural mechanism. These results call for a paradigm shift in modeling the mechanical regulation of receptor-ligand bond dissociation, which includes conformational coupling between binding pocket and remote regions of the interacting molecules.

Cytoplasmic domain of P-selectin glycoprotein ligand-1 facilitates dimerization and export from the endoplasmic reticulum.

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation, reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization, and the cytoplasmic domain propagates signals that activate ß(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in "ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly, they have 10-fold less PSGL-1 on their surfaces than WT leukocytes. Using glycosidases, proteases, Western blotting, confocal microscopy, cell-surface cross-linking, FRET, and pulse-chase metabolic labeling, we demonstrate that deleting the cytoplasmic domain impaired dimerization and delayed export of PSGL-1 from the endoplasmic reticulum (ER), markedly increasing a monomeric precursor in the ER and decreasing mature PSGL-1 on the cell surface. A monomeric full-length PSGL-1 made by substituting the transmembrane domain with that of CD43 exited the ER normally, revealing that dimerization was not required for ER export. Thus, the transmembrane and cytoplasmic domains cooperate to promote dimerization of PSGL-1. Furthermore, the cytoplasmic domain provides a key signal to export precursors of PSGL-1 from the ER to the Golgi apparatus en route to the cell surface.

E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin alphaLbeta2-mediated slow leukocyte rolling.

In inflamed venules, neutrophils rolling on E-selectin induce integrin alpha(L)beta(2)-dependent slow rolling on intercellular adhesion molecule-1 by activating Src family kinases (SFKs), DAP12 and Fc receptor-gamma (FcRgamma), spleen tyrosine kinase (Syk), and p38. E-selectin signaling cooperates with chemokine signaling to recruit neutrophils into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) as the essential E-selectin ligand and Fgr as the only SFK that initiate signaling to slow rolling. In contrast, we found that E-selectin engagement of PSGL-1 or CD44 triggered slow rolling through a common, lipid raft-dependent pathway that used the SFKs Hck and Lyn as well as Fgr. We identified the Tec kinase Bruton tyrosine kinase as a key signaling intermediate between Syk and p38. E-selectin engagement of PSGL-1 was dependent on its cytoplasmic domain to activate SFKs and slow rolling. Although recruiting phosphoinositide-3-kinase to the PSGL-1 cytoplasmic domain was reported to activate integrins, E-selectin-mediated slow rolling did not require phosphoinositide-3-kinase. Studies in mice confirmed the physiologic significance of these events for neutrophil slow rolling and recruitment during inflammation. Thus, E-selectin triggers common signals through distinct neutrophil glycoproteins to induce alpha(L)beta(2)-dependent slow rolling.

Platelet glycoprotein Ibalpha forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF.

Arterial blood flow enhances glycoprotein Ibalpha (GPIbalpha) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbalpha/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbalpha dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbalpha-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif-13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbalpha on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.

Flow-enhanced adhesion regulated by a selectin interdomain hinge.

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin-ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.

Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte rolling and signaling under flow.

In inflamed venules, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to roll on P-selectin and E-selectin and to activate integrin alphaLbeta2 (lymphocyte function-associated antigen-1, LFA-1) to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Studies in cell lines have suggested that PSGL-1 requires its cytoplasmic domain to localize in membrane domains, to support rolling on P-selectin, and to signal through spleen tyrosine kinase (Syk). We generated "DeltaCD" mice that express PSGL-1 without the cytoplasmic domain. Unexpectedly, neutrophils from these mice localized PSGL-1 normally in microvilli, uropods, and lipid rafts. DeltaCD neutrophils expressed less PSGL-1 on their surfaces because of inefficient export from the endoplasmic reticulum. Limited digestion of wild-type neutrophils with O-sialoglycoprotein endopeptidase was used to reduce the PSGL-1 density to that on DeltaCD neutrophils. At matched PSGL-1 densities, both DeltaCD and wild-type neutrophils rolled similarly on P-selectin. However, DeltaCD neutrophils rolling on P-selectin did not trigger Syk-dependent activation of LFA-1 to slow rolling on ICAM-1. These data demonstrate that the PSGL-1 cytoplasmic domain is dispensable for leukocyte rolling on P-selectin but is essential to activate beta2 integrins to slow rolling on ICAM-1.

Neutrophils lacking ERM proteins polarize and crawl directionally but have decreased adhesion strength.

Ezrin/radixin/moesin (ERM) proteins are adaptors that link the actin cytoskeleton to the cytoplasmic domains of membrane proteins. Leukocytes express mostly moesin with lower levels of ezrin but no radixin. When leukocytes are activated, ERMs are postulated to redistribute membrane proteins from microvilli into uropods during polarization and to transduce signals that influence adhesion and other responses. However, these functions have not been tested in leukocytes lacking all ERMs. We used knockout (KO) mice with neutrophils lacking ezrin, moesin, or both proteins (double knockout [DKO]) to probe how ERMs modulate cell shape, adhesion, and signaling in vitro and in vivo. Surprisingly, chemokine-stimulated DKO neutrophils still polarized and redistributed ERM-binding proteins such as PSGL-1 and CD44 to the uropods. Selectin binding to PSGL-1 on moesin KO or DKO neutrophils activated kinases that enable integrin-dependent slow rolling but not those that generate neutrophil extracellular traps. Flowing neutrophils of all genotypes rolled normally on selectins and, upon chemokine stimulation, arrested on integrin ligands. However, moesin KO and DKO neutrophils exhibited defective integrin outside-in signaling and reduced adhesion strength. In vivo, DKO neutrophils displayed normal directional crawling toward a chemotactic gradient, but premature detachment markedly reduced migration from venules into inflamed tissues. Our results demonstrate that stimulated neutrophils do not require ERMs to polarize or to move membrane proteins into uropods. They also reveal an unexpected contribution of moesin to integrin outside-in signaling and adhesion strengthening.