RESUMO
Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.
Assuntos
Desenvolvimento Ósseo/fisiologia , MAP Quinase Quinase Quinase 4/deficiência , Defeitos do Tubo Neural/enzimologia , Animais , Apoptose , Sequência de Bases , Padronização Corporal/genética , Padronização Corporal/fisiologia , Desenvolvimento Ósseo/genética , DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Humanos , MAP Quinase Quinase Quinase 4/genética , MAP Quinase Quinase Quinase 4/fisiologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/patologia , Fenótipo , Fosforilação , Gravidez , Rombencéfalo/anormalidades , Rombencéfalo/enzimologia , Rombencéfalo/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: The Mer receptor tyrosine kinase, cloned from a B-lymphoblastoid library, is the mammalian orthologue of the chicken retroviral oncogene v-eyk and sends antiapoptotic and transforming signals when activated. To determine if Mer expression is ectopic in T-cell acute lymphoblastic leukemia (ALL) and potentially important in leukemogenesis, we analyzed Mer expression in normal human thymocytes and lymphocytes and in pediatric ALL patient samples. EXPERIMENTAL DESIGN: Reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to determine expression of Mer in sorted human thymocyte populations, lymphocytes, and lymphocytes activated by phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. Mer expression in 34 T-cell ALL (T-ALL) patient samples was evaluated by reverse transcription-PCR, and Mer protein expression in a separate cohort of 16 patient samples was assayed by flow cytometry and Western blot. RESULTS: Mer expression was absent in normal thymocytes or lymphocytes, and in T cells activated with phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. In contrast, Jurkat cells and T-ALL patient samples expressed unique 180 to 185 kDa Mer protein glycoforms. Substantial Mer RNA levels were principally observed in a subset of T-ALL patient samples that expressed B220 (P = 0.004) but lacked surface expression of CD3 (P = 0.02) and CD4 (P = 0.006), a phenotypic profile consistent with immature lymphoblasts. In addition, 8 of 16 T-ALL patient samples had Mer protein detected by flow cytometry and Western blot. CONCLUSIONS: Transforming Mer signals may contribute to T-cell leukemogenesis, and abnormal Mer expression may be a novel therapeutic target in pediatric ALL therapy.
Assuntos
Leucemia-Linfoma de Células T do Adulto/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/patologia , Proto-Oncogene Mas , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/patologia , Transcrição Gênica , c-Mer Tirosina QuinaseRESUMO
The successes of targeted therapeutics against EGFR and ALK in non-small cell lung cancer (NSCLC) have demonstrated the substantial survival gains made possible by precision therapy. However, the majority of patients do not have tumors with genetic alterations responsive to these therapies, and therefore identification of new targets is needed. Our laboratory previously identified MER receptor tyrosine kinase as one such potential target. We now report our findings targeting MER with a clinically translatable agent--Mer590, a monoclonal antibody specific for MER. Mer590 rapidly and robustly reduced surface and total MER levels in multiple cell lines. Treatment reduced surface MER levels by 87%, and this effect was maximal within four hours. Total MER levels were also dramatically reduced, and this persisted for at least seven days. Mechanistically, MER down-regulation was mediated by receptor internalization and degradation, leading to inhibition of downstream signaling through STAT6, AKT, and ERK1/2. Functionally, this resulted in increased apoptosis, increased chemosensitivity to carboplatin, and decreased colony formation. In addition to carboplatin, Mer590 interacted cooperatively with shRNA-mediated MER inhibition to augment apoptosis. These data demonstrate that MER inhibition can be achieved with a monoclonal antibody in NSCLC. Optimization toward a clinically available anti-MER antibody is warranted.
Assuntos
Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Carboplatina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular Tumoral , Regulação para Baixo , Resistência a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Terapia de Alvo Molecular , Proteína Oncogênica v-akt/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , c-Mer Tirosina QuinaseRESUMO
We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined.
Assuntos
Adenina/análogos & derivados , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Adenina/administração & dosagem , Adenina/farmacocinética , Adenina/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral/efeitos dos fármacos , Técnicas de Química Sintética , Humanos , Concentração Inibidora 50 , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/metabolismo , Leucemia de Células B/patologia , Camundongos SCID , Terapia de Alvo Molecular , Piperazinas/administração & dosagem , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , c-Mer Tirosina Quinase , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Leucemia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Linhagem Celular Tumoral , Humanos , Leucemia/enzimologia , Leucemia/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos TransgênicosRESUMO
The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.
Assuntos
Cicloexanóis/síntese química , Fibrinolíticos/síntese química , Fibrinolíticos/uso terapêutico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirimidinas/síntese química , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Cicloexanóis/uso terapêutico , Desenho de Fármacos , Humanos , Modelos Moleculares , Pirimidinas/química , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , c-Mer Tirosina QuinaseRESUMO
Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although survival rates have improved, patients with certain biologic subtypes still have suboptimal outcomes. Current chemotherapeutic regimens are associated with short- and long-term toxicities and novel, less toxic therapeutic strategies are needed. Mer receptor tyrosine kinase is ectopically expressed in ALL patient samples and cell lines. Inhibition of Mer expression reduces prosurvival signaling, increases chemosensitivity, and delays development of leukemia in vivo, suggesting that Mer tyrosine kinase inhibitors are excellent candidates for targeted therapies. Brain and spinal tumors are the second most common malignancies in childhood. Multiple chemotherapy approaches and radiotherapies have been attempted, yet overall survival remains dismal. Mer is also abnormally expressed in atypical teratoid/rhabdoid tumors (AT/RT), providing a rationale for targeting Mer as a therapeutic strategy. We have previously described UNC569, the first small-molecule Mer inhibitor. This article describes the biochemical and biologic effects of UNC569 in ALL and AT/RT. UNC569 inhibited Mer activation and downstream signaling through ERK1/2 and AKT, determined by Western blot analysis. Treatment with UNC569 reduced proliferation/survival in liquid culture, decreased colony formation in methylcellulose/soft agar, and increased sensitivity to cytotoxic chemotherapies. MYC transgenic zebrafish with T-ALL were treated with UNC569 (4 µmol/L for two weeks). Fluorescence was quantified as indicator of the distribution of lymphoblasts, which express Mer and enhanced GFP. UNC569 induced more than 50% reduction in tumor burden compared with vehicle- and mock-treated fish. These data support further development of Mer inhibitors as effective therapies in ALL and AT/RT.
Assuntos
Antineoplásicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Tumor Rabdoide/metabolismo , Teratoma/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Terapia de Alvo Molecular , Neoplasias Experimentais , Fosforilação/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tumor Rabdoide/tratamento farmacológico , Tumor Rabdoide/patologia , Teratoma/tratamento farmacológico , Teratoma/patologia , Peixe-Zebra , c-Mer Tirosina QuinaseRESUMO
Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant hERG activity in the PatchXpress assay. Through structure-activity relationship studies, 35 (UNC1062) was identified as a potent (IC50 = 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells, UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as a therapeutic target, UNC1062 is a promising candidate for further drug development.
Assuntos
Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , c-Mer Tirosina QuinaseRESUMO
Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation based on high selectivity against other kinases and good pharmacokinetic properties. When applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein. Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation of this compound for therapeutic application in patients with cancer.
Assuntos
Antineoplásicos/síntese química , Cicloexanóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Piridinas/síntese química , Pirimidinas/síntese química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ciclização , Cicloexanóis/farmacologia , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-Atividade , c-Mer Tirosina QuinaseRESUMO
Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies.
Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Melanócitos/metabolismo , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Proto-Oncogene Mas , c-Mer Tirosina QuinaseRESUMO
Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.
RESUMO
The MerTK plays several important roles in normal macrophage physiology, including regulation of cytokine secretion and clearance of apoptotic cells. Mer signaling in other cell types, including malignant cells that ectopically overexpress the RTK, leads to downstream prosurvival pathway activation. We explored the hypothesis that Mer has a prosurvival role in macrophages exposed to oxidative stress. H(2)O(2) treatment of peritoneal exudate murine macrophages and J774 cells rapidly stimulated Mer phosphorylation in a concentration-dependent manner. Mer phosphorylation was dependent on the ligand Gas6, as treatment with warfarin or MerFc (a fusion protein of the extracellular domain of Mer and the Fc portion of human Ig), inhibitors of Gas6 activity, blocked H(2)O(2)-mediated activation of Mer. Antiapoptotic signals including pAkt and pErk 1/2 were increased dramatically (threefold and 4.5-fold, respectively) in WT Mer-positive macrophages compared with Mer KO macrophages stimulated with H(2)O(2). In a consistent manner, Mer expression led to decreased cleavage of proapoptotic indicators PARP and Caspase-3. Furthermore, Mer provided up to twofold enhanced cellular survival to primary macrophages exposed to H(2)O(2). These data represent the first report of Mer activation in response to oxidative stress and demonstrate the ability of Mer RTK to promote macrophage survival in disease states that involve an oxidative stress environment.
Assuntos
Macrófagos/citologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Sobrevivência Celular , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , c-Mer Tirosina QuinaseRESUMO
Membrane-bound receptors generate soluble ligand-binding domains either by proteolytic cleavage of the extracellular domain or alternative mRNA splicing yielding a secreted protein. Mertk (Mer) is in a receptor tyrosine kinase family with Axl and Tyro-3, and all 3 receptors share the Gas6 ligand. Mer regulates macrophage activation, promotes apoptotic cell engulfment, and supports platelet aggregation and clot stability in vivo. We have found that the membrane-bound Mer protein is cleaved in the extracellular domain via a metalloproteinase. The cleavage results in the production of a soluble Mer protein released in a constitutive manner from cultured cells. Significant amounts of the soluble Mer protein were also detected in human plasma, suggesting its physiologic relevance. Cleavage of Mer was enhanced by treatment with LPS and PMA and was specifically inhibited by a tumor necrosis factor alpha-converting enzyme metalloproteinase inhibitor. As a decoy receptor for Gas6, soluble Mer prevented Gas6-mediated stimulation of membrane-bound Mer. The inhibition of Gas6 activity by soluble Mer led to defective macrophage-mediated engulfment of apoptotic cells. Furthermore, soluble Mer decreased platelet aggregation in vitro and prevented fatal collagen/epinephrine-induced thromboembolism in mice, suggesting a potential therapeutic use for soluble Mer in the treatment of clotting disorders.