Yi Wu

Efficient phagocytosis of apoptotic cells is crucial for many cellular processes. One of earliest signals to the phagocyte is the expression of phosphatidylserine (PS) on the outer surface of the apoptotic cell that provides a potent 'eat-me' signal. Recognition of PS occurs either directly, via PS receptor (PS-R), or indirectly via alphavbeta5(3) integrin or Mer-family tyrosine kinases through the opsonizing proteins milk fat globule-EGF factor 8 protein (MFG-E8), or growth arrest specific factor-6 (Gas6), respectively. Because Mer and alphavbeta5 integrin share PS-dependent recognition signals, we investigated their post-receptor signaling cascades following receptor activation. Using a constitutively active form for Mer (CDMer) or Gas6 as a ligand to stimulate Mer, we found that Mer activation induced a post-receptor signaling cascade involving Src-mediated tyrosine phosphorylation of FAK on Tyr(861), the recruitment of FAK(Tyr861) to the alphavbeta5 integrin, and increased formation of p130(CAS)/CrkII/Dock180 complex to activate Rac1. Coexpression of Mer with alphavbeta5 integrin had a synergistic effect on Rac1 activation, lamellipodial formation and the phagocytosis of apoptotic cells. Interestingly, Gas6 or CDMer failed to stimulate p130(CAS) tyrosine phosphorylation or phagocytosis in beta5-deficient CS-1 cells or in mutant beta5DeltaC-expressing cells, suggesting that Mer is directionally and functionally linked to the integrin pathway. The present data indicate that receptors that recognize apoptotic cells in the context of PS functionally crosstalk to amplify intracellular signals to internalize apoptotic cells. Moreover, our data link another PS-dependent signal to the CrkII/Dock180/Rac1 module.

The contact system, also named as plasma kallikrein-kinin system, consists of three serine proteinases: coagulation factors XII (FXII) and XI (FXI), and plasma prekallikrein (PK), and the nonenzymatic cofactor high molecular weight kininogen (HK). This system has been investigated actively for more than 50 years. The components of this system and their interactions have been elucidated from in vitro experiments, which indicates that this system is prothrombotic by activating intrinsic pathway, and proinflammatory by producing bioactive peptide bradykinin. Although the activation of the contact system have been implicated in various types of human disease, in only a few instances is its role clearly defined. In the last 10 years, our understanding of the contact system, particularly its biology and (patho)physiology has greatly increased through investigations using gene-modified animal models. In this review we will describe a revitalized view of the contact system as a critical (patho)physiologic mediator of coagulation and inflammation.

Efficient clearance of apoptotic cells is essential for tissue homeostasis, allowing for cellular turnover without inflammatory consequences. The Mer (Nyk and c-Eyk) receptor tyrosine kinase (Mertk) is involved in two aspects of apoptotic cell clearance by acting as a receptor for Gas6, a γ-carboxylated phosphatidylserine-binding protein that bridges apoptotic and viable cells. First, Mertk acts in a bona fide engulfment pathway in concert with αvβ5 integrin by regulating cytoskeletal assemblages, and second, it acts as a negative regulator for inflammation by down-modulating pro-inflammatory signals mediated from bacterial lipopolysaccharide-Toll-like receptor 4 (TLR4) signaling, and hence recapitulating anti-inflammatory immune modulation by apoptotic cells. Here we describe Mertk post-receptor events that govern phagocytosis and cytoskeletal signaling are principally mediated by autophosphorylation site Tyr-867. Using the Mertk Y867F mutant and pharmacological inhibitors, we show that Tyr-867 is required for phosphatidylinositol 3-kinase and phospholipase Cγ2 activation; their activation in turn elicits protein kinase C-dependent signals that act on the actin cytoskeleton. Although MertkY867F blocked the tyrosine phosphorylation of FAK on Tyr-861 and p130cas and also abrogated the phagocytosis of apoptotic cells, this mutant did not suppress lipopolysaccharide-inducible NF-κB transcription, nor was NF-κB activation dependent on the protein kinase C inhibitor, calphostin C. Finally, unlike the cytoskeletal events associated with Tyr-867 autophosphorylation, the trans-inhibition of NF-κB occurred in a postnuclear-dependent fashion independent of cytosolic IκB phosphorylation and p65/RelA sequestration. Taken together, these data suggest that Mertk has distinct and separable effects for phagocytosis and for resolving inflammation, providing a molecular rationale for how immune licensing and inflammation can be dissociated from phagocytosis in a single phagocytic receptor. Efficient clearance of apoptotic cells is essential for tissue homeostasis, allowing for cellular turnover without inflammatory consequences. The Mer (Nyk and c-Eyk) receptor tyrosine kinase (Mertk) is involved in two aspects of apoptotic cell clearance by acting as a receptor for Gas6, a γ-carboxylated phosphatidylserine-binding protein that bridges apoptotic and viable cells. First, Mertk acts in a bona fide engulfment pathway in concert with αvβ5 integrin by regulating cytoskeletal assemblages, and second, it acts as a negative regulator for inflammation by down-modulating pro-inflammatory signals mediated from bacterial lipopolysaccharide-Toll-like receptor 4 (TLR4) signaling, and hence recapitulating anti-inflammatory immune modulation by apoptotic cells. Here we describe Mertk post-receptor events that govern phagocytosis and cytoskeletal signaling are principally mediated by autophosphorylation site Tyr-867. Using the Mertk Y867F mutant and pharmacological inhibitors, we show that Tyr-867 is required for phosphatidylinositol 3-kinase and phospholipase Cγ2 activation; their activation in turn elicits protein kinase C-dependent signals that act on the actin cytoskeleton. Although MertkY867F blocked the tyrosine phosphorylation of FAK on Tyr-861 and p130cas and also abrogated the phagocytosis of apoptotic cells, this mutant did not suppress lipopolysaccharide-inducible NF-κB transcription, nor was NF-κB activation dependent on the protein kinase C inhibitor, calphostin C. Finally, unlike the cytoskeletal events associated with Tyr-867 autophosphorylation, the trans-inhibition of NF-κB occurred in a postnuclear-dependent fashion independent of cytosolic IκB phosphorylation and p65/RelA sequestration. Taken together, these data suggest that Mertk has distinct and separable effects for phagocytosis and for resolving inflammation, providing a molecular rationale for how immune licensing and inflammation can be dissociated from phagocytosis in a single phagocytic receptor. The Mer/Nyk receptor tyrosine kinase (Mertk) 4The abbreviations used are: MertkMer (Nyk and c-Eyk) receptor tyrosine kinaseRTKreceptor tyrosine kinaseGas6growth arrest-specific factor-6PSphosphatidylserineEGFepidermal growth factorTNFtumor necrosis factorLPSlipopolysaccharideTLR4Toll-like receptor 4PI3Kphosphatidylinositol 3-kinasePLCphospholipase CPKCprotein kinase CmAbmonoclonal antibodyGFPgreen fluorescent proteinEGFPenhanced GFPFBSfetal bovine serumKDkinase-deadSHSrc homologyITIMimmunoreceptor tyrosine-based inhibitory motifCBPCREB-binding proteinCREBcAMP-response element-binding proteinRCSRoyal College of Surgeons. was first described as a tyrosine kinase retroviral oncogene (RPL30), called v-Ryk and later v-Eyk, that contained the retroviral envelope gp37-coding region fused to the intracellular domain of Eyk (1Jia R. Hanafusa H. J. Biol. Chem. 1994; 269: 1839-1844Abstract Full Text PDF PubMed Google Scholar, 2Jia R. Mayer B.J. Hanafusa T. Hanafusa H. J. Virol. 1992; 66: 5975-5987Crossref PubMed Google Scholar). Overexpression of the v-eyk gene product by infectious virus caused transformation of chicken embryo fibroblasts and produced sarcomas and lymphomas in vivo. Cloning of the cellular form of v-Eyk, c-Eyk, revealed an unusual extracellular domain structure, consisting of novel repeating units containing two C2-type Ig domains and two type III FN-like domains that were truncated in the viral genome (3Graham D.K. Dawson T.L. Mullaney D.L. Snodgrass H.R. Earp H.S. Cell Growth Differ. 1994; 5: 647-657PubMed Google Scholar). Based on homology of the ectodomains, Mertk is part of a subfamily of RTKs together with Tyro-3/Sky and Axl that have atypical ectodomains resembling the adhesion molecules neural cell adhesion molecule and L1 (4Graham D.K. Bowman G.W. Dawson T.L. Stanford W.L. Earp H.S. Snodgrass H.R. Oncogene. 1995; 10: 2349-2359PubMed Google Scholar, 5Rescigno J. Mansukhani A. Basilico C. Oncogene. 1991; 6: 1909-1913PubMed Google Scholar). Mertk, which is predominantly expressed in cells of myeloid, epithelial, and reproductive (Mer) origin, induces transformation when ectopically expressed in fibroblasts (6Ling L. Kung H.J. Mol. Cell. Biol. 1995; 15: 6582-6592Crossref PubMed Scopus (68) Google Scholar) but has also been shown to be overexpressed in neoplastic T and B cells (7Graham D.K. Salzberg D.B. Kurtzberg J. Sather S. Matsushima G.K. Keating A.K. Liang X. Lovell M.A. Williams S.A. Dawson T.L. Schell M.J. Anwar A.A. Snodgrass H.R. Earp H.S. Clin. Cancer Res. 2006; 12: 2662-2669Crossref PubMed Scopus (87) Google Scholar, 8Keating A.K. Salzberg D.B. Sather S. Liang X. Nickoloff S. Anwar A. Deryckere D. Hill K. Joung D. Sawczyn K.K. Park J. Curran-Everett D. McGavran L. Meltesen L. Gore L. Johnson G.L. Graham D.K. Oncogene. 2006; 25: 6092-6100Crossref PubMed Scopus (77) Google Scholar), as well as in mantle cell lymphomas (9Ek S. Hogerkorp C.M. Dictor M. Ehinger M. Borrebaeck C.A. Cancer Res. 2002; 62: 4398-4405PubMed Google Scholar). Although Axl and Mertk were first identified and associated with various human malignancies, it is now clear that they have complex and pleiotrophic functions depending on the cell type where they are expressed as well as in the context in which they are activated (10Hafizi S. Dahlback B. Cytokine Growth Factor Rev. 2006; 17: 295-304Crossref PubMed Scopus (210) Google Scholar). Mer (Nyk and c-Eyk) receptor tyrosine kinase receptor tyrosine kinase growth arrest-specific factor-6 phosphatidylserine epidermal growth factor tumor necrosis factor lipopolysaccharide Toll-like receptor 4 phosphatidylinositol 3-kinase phospholipase C protein kinase C monoclonal antibody green fluorescent protein enhanced GFP fetal bovine serum kinase-dead Src homology immunoreceptor tyrosine-based inhibitory motif CREB-binding protein cAMP-response element-binding protein Royal College of Surgeons. The biological ligands for Axl, Tyro-3/Sky, and Mertk are two homologous vitamin K-modified proteins, called growth arrest-specific factor-6 (Gas6) and Protein S (11Varnum B.C. Young C. Elliott G. Garcia A. Bartley T.D. Fridell Y.W. Hunt R.W. Trail G. Clogston C. Toso R.J. Nature. 1995; 373: 623-626Crossref PubMed Scopus (414) Google Scholar, 12Hafizi S. Dahlback B. FEBS J. 2006; 273: 5231-5244Crossref PubMed Scopus (289) Google Scholar), the latter also being a negative regulator of blood coagulation (12Hafizi S. Dahlback B. FEBS J. 2006; 273: 5231-5244Crossref PubMed Scopus (289) Google Scholar). Both proteins contain signal sequences that promote their secretion, followed by an N-terminal segment that is post-translationally modified by vitamin K to produce γ-carboxyglutamic acid residues (Gla domain). γ-Carboxylation of Gas6 is responsible for calcium-dependent interaction with PS and bridging of the apoptotic cell to the phagocyte (13Nagata K. Ohashi K. Nakano T. Arita H. Zong C. Hanafusa H. Mizuno K. J. Biol. Chem. 1996; 271: 30022-30027Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). To mediate Mertk binding and autophosphorylation, Gas6 has central EGF-like repeats, followed by a C-terminal sex hormone binding globulin-like domain consisting of tandem laminin G-like domains (14Manfioletti G. Brancolini C. Avanzi G. Schneider C. Mol. Cell. Biol. 1993; 13: 4976-4985Crossref PubMed Scopus (532) Google Scholar). Recent have the of a which an for and autophosphorylation and signaling L. D. Kung H.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, T. D. A. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). novel of Mertk has been identified in on and from and G. PubMed Scopus Google Scholar) and independent from a Mertk mutant that a in the intracellular kinase domain T.D. Earp H.S. Matsushima G.K. J. PubMed Google Scholar, R. Earp H.S. Matsushima G.K. Nature. PubMed Scopus Google Scholar, R. T.D. Earp H.S. Matsushima G. J. 2002; PubMed Scopus Google Scholar). that Tyro-3/Sky, Axl, and Mertk a by G. PubMed Scopus Google Scholar). that mutant Mertk when the show in and an by the of the to apoptotic cells R. T.D. Earp H.S. Matsushima G. J. 2002; PubMed Scopus Google Scholar). that apoptotic in the of pro-inflammatory in and a of inflammation and of intracellular that can to show to and with to R. T.D. Earp H.S. Matsushima G. J. 2002; PubMed Scopus Google Scholar). a in has been in the Royal College of H. D. D. Earp H.S. Matsushima G.K. Biol. PubMed Scopus Google Scholar, D. H. Earp H.S. Matsushima G.K. D. PubMed Scopus Google Scholar), associated with a of of Mertk in cells. the from an to clear in an pathway as described for apoptotic cells M.J. Res. PubMed Scopus Google Scholar, M.J. B.J. Res. PubMed Scopus Google Scholar). Although these effects on apoptotic cell clearance were first to a to Mertk, now suggest that Axl and also but are expressed in cell and have phagocytic functions in cells T.D. G. Earp H.S. Matsushima G.K. J. PubMed Scopus Google Scholar). from the and kinase-dead that Mertk two aspects of apoptotic cell First, it is involved in a bona fide cytoskeletal signaling pathway that tyrosine with αvβ5 and in the activation of and phagocytosis S. J. Cell PubMed Scopus Google Scholar, Cell Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. 2006; PubMed Scopus Google Scholar). Mertk is in the of inflammation and modulation of immune of this pathway is by the of Mertk to NF-κB activation and the of inflammatory that and T.D. Earp H.S. Matsushima G.K. J. PubMed Google Scholar, M.A. M. Earp H.S. Matsushima G. PubMed Scopus Google Scholar). Although cytoskeletal signals and signals that pro-inflammatory signals on the activation of Mertk and tyrosine it is not clear is engulfment is required for down-modulating the we that the tyrosine phosphorylation of and signals that on cytoskeletal and are mediated by a single Tyr-867 autophosphorylation site in and pharmacological inhibitors, we identified a Tyr-867 and activation of cytoskeletal to of Tyr-867 in the activation of and which to the activation of and effects on the actin that tyrosine phosphorylation of FAK and we for the Tyr-867 pathway and of NF-κB of signaling to be a was by but not IκB phosphorylation of of the Taken together, data suggest that Mertk has distinct and separable effects for phagocytosis and resolving inflammation, providing a molecular rationale for how engulfment and immune licensing can be dissociated a single phagocytic receptor. and were from the and p130cas were from from antibody was from antibody for was from Cell antibody Mertk was from and were from The to FAK and were from IκB and were from Cell The was from from was from was from was from calphostin C was from inhibitory and were from The retroviral a receptor from the intracellular part of Mertk and the extracellular and domains of the human to and mutant the binding as well as to and were as described T. Zong C. Hanafusa H. Mol. Cell. Biol. PubMed Google Scholar). in the motif and in were by The are as and The Mertk was from a embryo and the of and The mutant of Mertk, was by with the and was used as and were for with the The was to that the was and that were by Cell and cells were in modified with fetal bovine serum and in a containing in cells was the as by the cells were in with and and were with that in these cells with and cells were in containing and and on for the were 4 The were for protein was by the with of proteins were with antibody for 4 followed by with protein for 4 The were in to the of The were by and The were blocked with for which the were with for 4 and in containing binding was by and was with enhanced with the antibody to was with for the were blocked with and with the phagocytosis was as described S. J. Cell PubMed Scopus Google Scholar). for human T cells were with a fluorescent cell and to by for cells were by with cells were with that contained and to for with apoptotic cells. The cells were with apoptotic cells for a of which the cells were with containing to apoptotic cells. was used to the and fluorescent of green fluorescent that fluorescent cells. inhibitory was the of apoptotic cells. The cell in of intracellular was the and as by of was a M. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with of a containing the the of which is by a to of the motif together with of a the of which is dependent on the region The cells were to for 4 and with without Gas6 calphostin C for the 4 of this was used and in cells were the Cell were and were by the in an was in and the in cells from well were The in was with to the and the of with the in an was as a of of NF-κB in cells were used The cells were in with and containing The cells were with Gas6 with Gas6 followed by The cells were and in in in and in in for the cells were in in containing NF-κB antibody for The cells were with and in containing for were in and in containing for for were and for NF-κB an single cells were identified and with cells with and NF-κB were and for NF-κB the which the the NF-κB and for cell S. K. D. J. 2006; PubMed Scopus (210) Google Scholar). the two be and have NF-κB is the two be and have negative of the shown are from a single of independent Mertk for of and of NF-κB Both on functions in two aspects of apoptotic cell of which the tyrosine kinase domain and tyrosine phosphorylation shown in when Mertk was expressed in cells and with apoptotic T cells, Mertk phagocytosis by but this was to by the of a Mertk kinase-dead mutant containing a to the of Mertk to suppress NF-κB we used a cell shown in and activation of Mertk, by of Gas6 of Mertk activated suppress activation of the NF-κB in this a was as in gene in on a of with Mertk in the of Gas6 of activation required a when we cells with Gas6 and the the Gas6 on that was required to a NF-κB later in the this of Mertk trans-inhibition on signaling was when we a with the NF-κB but not data that the of Mertk as a phagocytic receptor and to suppress NF-κB activation are dependent on Mertk kinase and tyrosine of and Mertk to by the Tyr-867 that the of NF-κB signaling and by apoptotic cells dependent licensing but not phagocytosis M. J. A. J. PubMed Scopus Google Scholar, M. A. K. M. R. J. A. J. 2006; PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar). were we a molecular rationale for this by post-receptor phagocytosis and the of required the kinase of Mertk, we that proteins with tyrosine in the intracellular domain of Mertk to signal was also by by the of autophosphorylation site signals in cells, that the motif and that the of T. Zong C. Hanafusa H. Mol. Cell. Biol. PubMed Google Scholar). have been shown to be involved in biological cell K. M. B. G. B. J. 2006; PubMed Scopus Google Scholar), as well as phagocytosis of B.J. Biol. PubMed Scopus Google Scholar) and apoptotic cells Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). To the of Mertk autophosphorylation Tyr-867 site for phagocytosis and cytoskeletal signaling, we Mertk and an Y867F Mertk mutant with a for binding shown in activation of Mertk in of MertkY867F abrogated this to were when we expressed activated not when these cells were with apoptotic T cells, cells MertkY867F phagocytosis of apoptotic cells to cells was to with to signaling Mertk also in the activation of and B. A. J. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. Biol. PubMed Scopus (68) Google Scholar). Although are in that they contain two tandem Src homology which interaction with proteins that contain tyrosine it is also well that activation of phosphatidylinositol and to the M. G. M.A. J. J. 17: PubMed Scopus Google Scholar). phosphatidylinositol to produce and in the activation of have shown that the of cells with apoptotic cells tyrosine phosphorylation of but not and with Mertk to a in phagocytosis in and in cells B. J. Biol. PubMed Scopus (68) Google Scholar). To the of Mertk Y867F on the phosphorylation and activation of we expressed and as well as autophosphorylation site Mertk Mertk and Mertk in cells in the cells tyrosine phosphorylation of which was abrogated by the Y867F mutant but not by were expressed with the of the kinase-dead Mertk, a mutant that is to the of Mertk data that is a site for the of and which to Tyr-867 on of for Mertk to FAK and the is a of a product of phospholipase and is required for phagocytosis of apoptotic cells in B. A. J. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. S. T. J. PubMed Scopus Google Scholar), we activation is required for Mertk signaling to the and phagocytosis of apoptotic cells. have also shown that can on αvβ5 integrin and cytoskeletal B. A. J. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar), to integrin binding to To the of in Mertk signaling, we first used a shown in of apoptotic cells was by with this phagocytosis in cells the by the was in cells without Mertk of of cells with the pharmacological inhibitor, calphostin also tyrosine phosphorylation of cytoskeletal proteins p130cas and which have been in phagocytosis S. J. Cell PubMed Scopus Google Scholar) and Finally, to these signals were from the Tyr-867 autophosphorylation we the phosphorylation of FAK on this was shown to be required for binding in Mertk and MertkY867F to the effects of Y867F were not The that calphostin C abrogated phosphorylation and and to and to cytoskeletal and phagocytosis and of NF-κB by of Tyr-867 and that the autophosphorylation site mutant Tyr-867 a the was to autophosphorylation site the is required for the anti-inflammatory of by Matsushima and that Mertk NF-κB activation in and in Mertk mutant are to and have of when with T.D. Earp H.S. Matsushima G.K. J. PubMed Google Scholar). have shown that apoptotic cell without is to NF-κB activation and M. A. K. M. R. J. A. J. 2006; PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar). to the signaling and Mertk autophosphorylation site we the of and to suppress NF-κB activation as described in shown in of in cells a and in NF-κB activation when these cells were with MertkY867F did not the of Mertk to suppress NF-κB activation of the site to on the of Mertk to signaling the were for the Mertk in NF-κB we the of immunoreceptor tyrosine-based inhibitory in have the be by the J. Google Scholar). and first the of an in Axl, but we identified and in Mertk the are in signaling proteins that immune by binding cellular and J. PubMed Scopus Google Scholar), and can IκB phosphorylation and L. A. Cell Biol. PubMed Scopus Google Scholar). a as Mertk J. Cell PubMed Scopus Google Scholar), it is to the of proteins in Mertk To their in we to to and shown in of of these single did not the Mertk trans-inhibition on NF-κB when Mertk of the was with we to a interaction Mertk and not Mertk for NF-κB to the that cytoskeletal pathway on we the of Mertk on IκB phosphorylation and NF-κB NF-κB is by the inhibitory protein IκB in the IκB and allowing and to the we the of Gas6 on IκB phosphorylation and we in the IκB phosphorylation with without Gas6 we also the of the of NF-κB Gas6 and Using on of cells we that to the as a of with without Gas6 B and of cells with and Gas6, a that did not on the of Mertk activation to suppress NF-κB activation suggest in to the tyrosine events associated with Mertk and the the tyrosine events by Mertk that on NF-κB are in a Efficient phagocytosis is the of apoptotic cells in complex and clearance of apoptotic cells has been to and inflammatory Mertk is the receptor in clearance and in anti-inflammatory and we this to the post-receptor events of Mertk that are responsible for cytoskeletal signaling, and in to the immune functions by signaling these events can be and identified a phagocytic signaling by the autophosphorylation Tyr-867 site that involved phosphorylation of and activation of and tyrosine phosphorylation of have shown that phosphorylation is to integrin and involved in a pathway to apoptotic cells S. J. Cell PubMed Scopus Google Scholar). the of Mertk to suppress signaling the of NF-κB required a also independent of and to the are with a in which phagocytosis is not to mediate and a molecular of how phagocytosis can be dissociated from immune licensing and of suggest that Mertk can on the and of apoptotic cells S. J. Cell PubMed Scopus Google Scholar). this we that tyrosine phosphorylation of and FAK a single autophosphorylation site Tyr-867. in cells that Mertk Tyr-867 was required for cell growth and a binding site for which the of T. Zong C. Hanafusa H. Mol. Cell. Biol. PubMed Google Scholar). with this we that the Y867F blocked a for it is not how Tyr-867 induces the motif to Tyr-867 not to a motif for the domain of is that activation not tyrosine but also phosphatidylinositol and phosphatidylinositol binding of to a site on the on activation and phosphatidylinositol the of the M. G. M.A. J. J. 17: PubMed Scopus Google Scholar). and that can with tyrosine Mertk B. J. Biol. PubMed Scopus (68) Google Scholar), it was not this is a interaction binding was by a a of a clear of how and are by the autophosphorylation site the of these in phagocytosis and activation is by the that Y867F which a to the actin cytoskeleton. we have shown that when on with the αvβ5 integrin pathway for have shown that activation is required for phagocytosis of apoptotic B. A. J. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of in phagocytosis in cytoskeletal proteins involved in cell and actin by the that PS of to cytoskeletal and has also shown that segment binding to αvβ5 integrin and a J. PubMed Scopus Google Scholar). The show that a for integrin is dependent on and an signal from that activation be a in the Mertk activation and αvβ5 integrin with apoptotic cells anti-inflammatory but cell that phagocytosis can be from the anti-inflammatory signals from apoptotic cells. M. J. PubMed Scopus Google Scholar) pharmacological of engulfment that for of to apoptotic cells. is to by and as licensing M. A. K. M. R. J. A. J. 2006; PubMed Scopus Google Scholar). Using a well in which apoptotic cells NF-κB we a molecular for how engulfment and can be dissociated in Mertk, mediated by distinct in the events that the Mertk Tyr-867 autophosphorylation be to a in MertkY867F in of their and inflammation, to the licensing and phagocytosis on the inflammatory also a in the these signaling The pathway was in and on and cytoskeletal assemblages, the anti-inflammatory pathway to be by of NF-κB of Gas6 NF-κB transcription, we that IκB the Gas6 a on which is with in cell in T. Zong C. Hanafusa H. Mol. Cell. Biol. PubMed Google Scholar). the by the NF-κB activation was to binding to but the to the pathway described is from the that Mertk on a pathway are also in to by Matsushima and M.A. M. Earp H.S. Matsushima G. PubMed Scopus Google Scholar) with apoptotic cells, show the of IκB in the cytosolic The for these are not show that can negative by that and G. D. S. A. PubMed Scopus Google Scholar, D. S. A. 2006; PubMed Scopus Google Scholar). be two of and that distinct inhibitory data suggest that the events that signaling in a are for how Mertk on NF-κB by M. K. 6: PubMed Scopus Google Scholar) that the of p65/RelA can this these that kinase a central in by the binding of from to to this of kinase by in of with and the of the with Although the be it is complex and that is which blocked activation and did not NF-κB a by also of NF-κB anti-inflammatory signaling and of signaling M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that phosphorylation and with but also that a NF-κB complex that to but did not promote be required to Gas6 apoptotic cells mediate events that promote inhibitory to phosphorylation and in by of the tumor can NF-κB can also be by and L. K. B. G. Biol. PubMed Scopus Google Scholar). to how Gas6 on the NF-κB are and to novel for inflammation and immune we have a molecular of how engulfment can be dissociated from the anti-inflammatory events that govern the of apoptotic cells with The is by events the autophosphorylation site and is a cytosolic The latter is independent of Tyr-867 and and to be the be in the of novel for and for and the

A close homologue to protein disulfide isomerase (PDI) called ERp57 forms disulfide bonds in glycoproteins in the endoplasmic reticulum and is expressed on the platelet surface. We generated 2 rabbit Abs to ERp57. One Ab strongly inhibited ERp57 in a functional assay and strongly inhibited platelet aggregation. There was minimal cross-reactivity of this Ab with PDI by Western blot or in the functional assay. This Ab substantially inhibited activation of the αIIbβ3 fibrinogen receptor and P-selectin expression. Furthermore, adding ERp57 to platelets potentiated aggregation. In contrast, adding a catalytically inactive ERp57 inhibited platelet aggregation. When infused into mice the inactive ERp57 prolonged the tail bleeding times. We generated 2 IgG2a mAbs that reacted with ERp57 by immunoblot. One of these Abs inhibited both ERp57 activity and platelet aggregation. The other Ab did not inhibit ERp57 activity or platelet aggregation. The inhibitory Ab inhibited activation of αIIbβ3 and P-selectin expression, prolonged tail bleeding times, and inhibited FeCl(3)-induced thrombosis in mice. Finally, we found that a commonly used mAb to PDI also inhibited ERp57 activity. We conclude that a glycoprotein-specific member of the PDI family, ERp57, is required for platelet aggregation, hemostasis, and thrombosis.