ABSTRACT: We found that mice infected with different isolates of lymphocytic choriomeningitis virus (LCMV) develop a mild hemorrhagic anemia, which becomes severe and eventually lethal in animals depleted of platelets or lacking integrin beta3. Lethal hemorrhagic anemia is mediated by virus-induced IFN-alpha/beta that causes platelet dysfunction, mucocutaneous blood loss and suppression of erythropoiesis. In addition to the life-threatening hemorrhagic anemia, platelet-depleted mice fail to mount an efficient cytotoxic T lymphocyte (CTL) response and cannot clear LCMV. Transfusion of functional platelets into these animals reduces hemorrhage, prevents death and restores CTL-induced viral clearance in a manner partially dependent on CD40 ligand (CD40L). These results indicate that, upon activation, platelets expressing integrin beta3 and CD40L are required for protecting the host against the induction of an IFN-alpha/beta-dependent lethal hemorrhagic diathesis and for clearing LCMV infection through CTLs.
Project description:Published data suggest that the tyrosine kinase syk participates in platelet signalling through the integrin alphaIIbbeta3. Our data show an association of syk and integrin beta3 in immunoprecipitates from unstimulated and stimulated platelets. We detected syk in anti-beta3 precipitates and, conversely, beta3 in anti-syk precipitates. In vitro kinase assays with anti-beta3 precipitates demonstrated that syk activity was enhanced in ADP-stimulated platelets.
Project description:The purpose of this work was to determine platelet and myeloid cell-specific requirements for beta3-containing integrins in hemostasis, bone resorption, and tumor growth. LoxP-flanked mice were generated to study the conditional deletion of beta3-integrin in platelets [knockout in platelets (KOP)] and myeloid cells [knockout in myeloid (KOM)]. Using the beta3KOP and beta3KOM strains of mice, we studied the role of beta3-integrin in hemostasis, bone resorption, and subcutaneous tumor growth. Tissue-specific deletion of platelet beta3-integrins in beta3KOP mice did not affect bone mass but resulted in a severe bleeding phenotype. No growth difference of tumor xenografts or in neoangiogenesis were found in beta3KOP mice, in contrast to the defects observed in germline beta3(-/-) mice. Conditional deletion of myeloid beta3-integrins in beta3KOM mice resulted in osteopetrosis but had no effect on hemostasis or mortality. Tumor growth in beta3KOM mice was increased and accompanied by decreased macrophage infiltration, without increase in blood vessel number. Platelet beta3-integrin deficiency was sufficient to disrupt hemostasis but had no effect on bone mass or tumor growth. Myeloid-specific beta3-integrin deletion was sufficient to perturb bone mass and enhance tumor growth due to reduced macrophage infiltration in the tumors. These results suggest that beta3-integrins have cell-specific roles in complex biological processes.-Morgan, E. A., Schneider, J. G., Baroni, T. E., Uluçkan, O., Heller, E., Hurchla, M. A., Deng, H., Floyd, D., Berdy, A., Prior, J. L., Piwnica-Worms, D., Teitelbaum, S. L., Ross, F. P., Weilbaecher, K. N. Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit.
Project description:The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.
Project description:Src tyrosine kinases transmit integrin-dependent signals pivotal for cell movement and proliferation. Here, we establish a mechanism for Src activation by integrins. c-Src is shown to bind constitutively and selectively to beta3 integrins through an interaction involving the c-Src SH3 domain and the carboxyl-terminal region of the beta3 cytoplasmic tail. Clustering of beta3 integrins in vivo activates c-Src and induces phosphorylation of Tyr-418 in the c-Src activation loop, a reaction essential for adhesion-dependent phosphorylation of Syk, a c-Src substrate. Unlike c-Src, Hck, Lyn, and c-Yes bind more generally to beta1A, beta2, and beta3 cytoplasmic tails. These results invoke a model whereby Src is primed for activation by direct interaction with an integrin beta tail, and integrin clustering stabilizes activated Src by inducing intermolecular autophosphorylation. The data provide a paradigm for integrin regulation of Src and a molecular basis for the similar functional defects of osteoclasts or platelets from mice lacking beta3 integrins or c-Src.
Project description:alphaIIbbeta3 interaction with fibrinogen promotes Src-dependent platelet spreading in vitro. To determine the consequences of this outside-in signaling pathway in vivo, a "beta3(Delta760-762)" knockin mouse was generated that lacked the 3 C-terminal beta3 residues (arginine-glycine-threonine [RGT]) necessary for alphaIIbbeta3 interaction with c-Src, but retained beta3 residues necessary for talin-dependent fibrinogen binding. beta3(Delta760-762) mice were compared with wild-type beta3(+/+) littermates, beta3(+/-) heterozygotes, and knockin mice where beta3 RGT was replaced by beta1 C-terminal cysteine-glycine-lysine (EGK) to potentially enable signaling by Src kinases other than c-Src. Whereas beta3(+/+), beta3(+/-) and beta3/beta1(EGK) platelets spread and underwent tyrosine phosphorylation normally on fibrinogen, beta3(Delta760-762) platelets spread poorly and exhibited reduced tyrosine phosphorylation of c-Src substrates, including beta3 (Tyr(747)). Unlike control mice, beta3(Delta760-762) mice were protected from carotid artery thrombosis after vessel injury with FeCl(3). Some beta3(Delta760-762) mice exhibited prolonged tail bleeding times; however, none demonstrated spontaneous bleeding, excess bleeding after surgery, fecal blood loss, or anemia. Fibrinogen binding to beta3(Delta760-762) platelets was normal in response to saturating concentrations of protease-activated receptor 4 or glycoprotein VI agonists, but responses to adenosine diphosphate were impaired. Thus, deletion of beta3 RGT disrupts c-Src-mediated alphaIIbbeta3 signaling and confers protection from arterial thrombosis. Consequently, targeting alphaIIbbeta3 signaling may represent a feasible antithrombotic strategy.
Project description:Thrombocytopenia and thrombosis following treatment with the integrin alphaIIbbeta3 antagonist eptifibatide are rare complications caused by patient antibodies specific for ligand-occupied alphaIIbbeta3. Whether such antibodies induce platelet clearance by simple opsonization, by inducing mild platelet activation, or both is poorly understood. To gain insight into the mechanism by which eptifibatide-dependent antibodies initiate platelet clearance, we incubated normal human platelets with patient serum containing an alphaIIbbeta3-specific, eptifibatide-dependent antibody. We observed that in the presence of eptifibatide, patient IgG induced platelet secretion and aggregation as well as tyrosine phosphorylation of the integrin beta3 cytoplasmic domain, the platelet FcgammaRIIa Fc receptor, the protein-tyrosine kinase Syk, and phospholipase Cgamma2. Each activation event was inhibited by preincubation of the platelets with Fab fragments of the FcgammaRIIa-specific mAb IV.3 or with the Src family kinase inhibitor PP2. Patient serum plus eptifibatide did not, however, activate platelets from a patient with a variant form of Glanzmann thrombasthenia that expressed normal levels of FcgammaRIIa and the alphaIIbbeta3 complex but lacked most of the beta3 cytoplasmic domain. Taken together, these data suggest a novel mechanism whereby eptifibatide-dependent antibodies engage the integrin beta3 subunit such that FcgammaRIIa and its downstream signaling components become activated, resulting in thrombocytopenia and a predisposition to thrombosis.
Project description:Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signalling, and the beta3 subunit of alphaIIbbeta3 integrin has been shown to interact with multiple SFK members. Here, we analyzed the interactions and functional consequences of Fyn and Src binding to alphaIIbbeta3. Fyn associated with the beta3 subunit in resting and thrombin-aggregated platelets, whereas interaction between Src and alphaIIbbeta3 was seen predominantly in resting but not in thrombin-aggregated platelets. We have also observed that Fyn but not Src localized to focal adhesions in CHO cells adherent to fibrinogen through alphaIIbbeta3. On the basis of these differences, we wanted to determine the sequence requirements for the interaction of Fyn and Src within the beta3-cytoplasmic domain. Whereas Src association required the C-terminal region of beta3, Fyn continued to interact with mutants that could no longer associate with Src and that contained as few as 13 membrane-proximal amino acids of the beta3-cytoplasmic tail. Using deletion mutants of beta3-cytoplasmic tails expressed as GST-fusion proteins, we narrowed down the Fyn-binding site even further to the amino acid residues 721-725 (IHDRK) of the beta3-cytoplasmic domain. On the basis of these observations, we explored whether Fyn-/- mice exhibited any abnormalities in hemostasis and platelet function. We found that Fyn-/- mice significantly differed in their second bleeding times compared with wild-type mice, and platelets from Fyn-/- mice exhibited delayed spreading on fibrinogen-coated surfaces. Using mutant forms of Fyn, it appears that its kinase activity is required for its localization to focal adhesions and to mediate alphaIIbbeta3-dependent cell spreading. Our results suggest that Fyn and Src have distinct requirements for interaction with alphaIIbbeta3; and, consequently, the two SFK can mediate different functional responses.
Project description:The alphavbeta3 integrins are linked to human bleeding disorders, and pathogenic hantaviruses regulate the function of alphavbeta3 integrins and cause acute vascular diseases. alphavbeta3 integrins are present in either extended (active) or dramatically bent (inactive) structures, and interconversion of alphavbeta3 conformers dynamically regulates integrin functions. Here, we show that hantaviruses bind human alphavbeta3 integrins and that binding maps to the plexin-semaphorin-integrin (PSI) domain present at the apex of inactive, bent, alphavbeta3-integrin structures. Pathogenic hantaviruses [New York-1 virus (NY-1V) and Hantaan virus (HTNV)] bind immobilized beta3 polypeptides containing the PSI domain, and human (but not murine) beta3 polypeptides inhibit hantavirus infectivity. Substitution of human beta3 residues 1-39 for murine beta3 residues directed pathogenic hantavirus infection of nonpermissive CHO cells expressing chimeric alphavbeta3 receptors. Mutation of murine beta3 Asn-39 to Asp-39 present in human beta3 homologues (N39D) permitted hantavirus infection of cells and specified PSI domain residue interactions with pathogenic hantaviruses. In addition, cell-surface expression of alphavbeta3 locked in an inactive bent conformation conferred hantavirus infectivity of CHO cells. Our findings indicate that hantaviruses bind to a unique domain exposed on inactive integrins and, together with prior findings, suggest that this interaction restricts alphavbeta3 functions that regulate vascular permeability. Our findings suggest mechanisms for viruses to direct hemorrhagic or vascular diseases and provide a distinct target for modulating alphavbeta3-integrin functions.
Project description:CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily, binds to CD40, leading to many effects depending on target cell type. Platelets express CD40L and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanisms. A family of proteins called TNF receptor associated factors (TRAFs) plays key roles in mediating CD40L-CD40 signaling. Platelets express several TRAFs. It has been shown that TRAF2 plays a role in CD40L-mediated platelet activation. Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression levels of collagen receptor GPVI and integrin ?IIb?3 in platelets were not affected by deletion of TRAF3, suggesting that increased platelet activation in the TRAF3 knockout mice was not due to increased expression platelet receptors. Time to formation of thrombi in a FeCl3-induced thrombosis model was significantly shortened in the TRAF3 knockout mice. However, mouse tail-bleeding times were not affected by deletion of TRAF3. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.
Project description:The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the alphaIIb cytoplasmic tail of platelet integrin alphaIIb beta3, which has a primary role in platelet aggregation. We also show that the WASP-CIB complex is important in alphaIIb beta3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces alphaIIb beta3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding.