Glutaredoxin 1 regulates cholesterol metabolism and gallstone formation by influencing protein S-glutathionylation.

OBJECTIVE: Cholesterol gallstone disease (CGD) is closely related to cholesterol metabolic disorder. Glutaredoxin-1 (Glrx1) and Glrx1-related protein S-glutathionylation are increasingly being observed to drive various physiological and pathological processes, especially in metabolic diseases such as diabetes, obesity and fatty liver. However, Glrx1 has been minimally explored in cholesterol metabolism and gallstone disease. METHODS: We first investigated whether Glrx1 plays a role in gallstone formation in lithogenic diet-fed mice using immunoblotting and quantitative real-time PCR. Then a whole-body Glrx1-deficient (Glrx1-/-) mice and hepatic-specific Glrx1-overexpressing (AAV8-TBG-Glrx1) mice were generated, in which we analyzed the effects of Glrx1 on lipid metabolism upon LGD feeding. Quantitative proteomic analysis and immunoprecipitation (IP) of glutathionylated proteins were performed. RESULTS: We found that protein S-glutathionylation was markedly decreased and the deglutathionylating enzyme Glrx1 was greatly increased in the liver of lithogenic diet-fed mice. Glrx1-/- mice were protected from gallstone disease induced by a lithogenic diet because their biliary cholesterol and cholesterol saturation index (CSI) were reduced. Conversely, AAV8-TBG-Glrx1 mice showed greater gallstone progression with increased cholesterol secretion and CSI. Further studies showed that Glrx1-overexpressing greatly altered bile acid levels and/or composition to increase intestinal cholesterol absorption by upregulating Cyp8b1. In addition, liquid chromatography-mass spectrometry and IP analysis revealed that Glrx1 also affected the function of asialoglycoprotein receptor 1 (ASGR1) by mediating its deglutathionylation, thereby altering the expression of LXRα and controlling cholesterol secretion. CONCLUSION: Our findings present novel roles of Glrx1 and Glrx1-regulated protein S-glutathionylation in gallstone formation through the targeting of cholesterol metabolism. Our data advises Glrx1 significantly increased gallstone formation by simultaneously increase bile-acid-dependent cholesterol absorption and ASGR1- LXRα-dependent cholesterol efflux. Our work suggests the potential effects of inhibiting Glrx1 activity to treat cholelithiasis.

Phytochrome B enhances seed germination tolerance to high temperature by reducing S-nitrosylation of HFR1.

Light and ambient high temperature (HT) have opposite effects on seed germination. Light induces seed germination through activating the photoreceptor phytochrome B (phyB), resulting in the stabilization of the transcription factor HFR1, which in turn sequesters the suppressor PIF1. HT suppresses seed germination and triggers protein S-nitrosylation. Here, we find that HT suppresses seed germination by inducing the S-nitrosylation of HFR1 at C164, resulting in its degradation, the release of PIF1, and the activation of PIF1-targeted SOMNUS (SOM) expression to alter gibberellin (GA) and abscisic acid (ABA) metabolism. Active phyB (phyBY276H ) antagonizes HFR1 S-nitrosylation and degradation by increasing S-nitrosoglutathione reductase (GSNOR) activity. In line with this, substituting cysteine-164 of HFR1 with serine (HFR1C164S ) abolishes the S-nitrosylation of HFR1 and decreases the HT-induced degradation of HFR1. Taken together, our study suggests that HT and phyB antagonistically modulate the S-nitrosylation level of HFR1 to coordinate seed germination, and provides the possibility to enhance seed thermotolerance through gene-editing of HFR1.

COVID-19: a probable role of the anticoagulant Protein S in managing COVID-19-associated coagulopathy.

The COVID-19 pandemic has caused monumental mortality, and there are still no adequate therapies. Most severely ill COVID-19 patients manifest a hyperactivated immune response, instigated by interleukin 6 (IL6) that triggers a so called "cytokine storm" and coagulopathy. Hypoxia is also associated with COVID-19. So far overlooked is the fact that both IL6 and hypoxia depress the abundance of a key anticoagulant, Protein S. We speculate that the IL6-driven cytokine explosion plus hypoxemia causes a severe drop in Protein S level that exacerbates the thrombotic risk in COVID-19 patients. Here we highlight a mechanism by which the IL6-hypoxia curse causes a deadly hypercoagulable state in COVID-19 patients, and we suggest a path to therapy.

Amelioration of Diabetes by Protein S.

Protein S is an anticoagulant factor that also regulates inflammation and cell apoptosis. The effect of protein S on diabetes and its complications is unknown. This study compared the development of diabetes between wild-type and transgenic mice overexpressing human protein S and the development of diabetic glomerulosclerosis between mice treated with and without human protein S and between wild-type and protein S transgenic mice. Mice overexpressing protein S showed significant improvements in blood glucose level, glucose tolerance, insulin sensitivity, and insulin secretion compared with wild-type counterparts. Exogenous protein S improved insulin sensitivity in adipocytes, skeletal muscle, and liver cell lines in db/db mice compared with controls. Significant inhibition of apoptosis with increased expression of BIRC3 and Bcl-2 and enhanced activation of Akt/PKB was induced by protein S in islet ß-cells compared with controls. Diabetic wild-type mice treated with protein S and diabetic protein S transgenic mice developed significantly less severe diabetic glomerulosclerosis than controls. Patients with type 2 diabetes had significantly lower circulating free protein S than healthy control subjects. This study shows that protein S attenuates diabetes by inhibiting apoptosis of ß-cells and the development of diabetic nephropathy.

Evidence for a subventricular zone neural stem cell phagocytic activity stimulated by the vitamin K-dependent factor protein S.

Neural stem cells, whose major reservoir in the adult mammalian brain is the subventricular zone (SVZ), ensure neuropoiesis, a process during which many generated cells die. Removal of dead cells and debris by phagocytes is necessary for tissue homeostasis. Using confocal and electron microscopy, we demonstrate that cultured SVZ cells phagocytose both 1 and 2 µm latex beads and apoptotic cell-derived fragments. We determine by flow cytometry that phagocytic cells represent more than 10% of SVZ cultured cells. Phenotyping of SVZ cells using nestin, GFAP, Sox2, or LeX/SSEA and quantification of aldehyde dehydrogenase (ALDH) activity, reveals that cells with neural stem-cell features phagocytose and represent more than 30% of SVZ phagocytic cells. In vivo, nestin-, Sox2-, and ALDH-expressing neural stem-like cells engulfed latex beads or apoptotic cell-derived fragments that were injected into mice lateral brain ventricles. We show also that SVZ cell phagocytic activity is an active process, which depends both on cytoskeleton dynamic and on recognition of phosphatidylserine eat-me signal, and is stimulated by the vitamin K-dependent factor protein S (ProS). ProS neutralizing antibodies inhibit SVZ cell phagocytic activity and exposure of SVZ cells to apoptotic cell-derived fragments induces a transient Mer tyrosine kinase receptor (MerTK) phosphorylation. Conversely, MerTK blocking antibodies impair both basal and ProS-stimulated SVZ cell phagocytic activity. By revealing that neural stem-like cells act within the SVZ neurogenic niche as phagocytes and that the ProS/MerTK path represents an endogenous regulatory mechanism for SVZ cell phagocytic activity, the present report opens-up new perspectives for both stem cell biology and brain physiopathology.

Erythrocyte-derived microparticles supporting activated protein C-mediated regulation of blood coagulation.

Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte-derived microparticle surface is suitable for the anticoagulant reactions of the protein C system, which may be important to balance the initiation and propagation of coagulation in vivo.

Vitamin K and the nervous system: an overview of its actions.

The role of vitamin K in the nervous system has been somewhat neglected compared with other physiological systems despite the fact that this nutrient was identified some 40 y ago as essential for the synthesis of sphingolipids. Present in high concentrations in brain cell membranes, sphingolipids are now known to possess important cell signaling functions in addition to their structural role. In the past 20 y, additional support for vitamin K functions in the nervous system has come from the discovery and characterization of vitamin K-dependent proteins that are now known to play key roles in the central and peripheral nervous systems. Notably, protein Gas6 has been shown to be actively involved in cell survival, chemotaxis, mitogenesis, and cell growth of neurons and glial cells. Although limited in number, studies focusing on the relationship between vitamin K nutritional status and behavior and cognition have also become available, pointing to diet and certain drug treatments (i.e., warfarin derivatives) as potential modulators of the action of vitamin K in the nervous system. This review presents an overview of the research that first identified vitamin K as an important nutrient for the nervous system and summarizes recent findings that support this notion.

Function of the activated protein C (APC) autolysis loop in activated FVIII inactivation.

Activated protein C (APC) binds to its substrates activated factor V (FVa) and activated factor VIII (FVIIIa) with a basic exosite that consists of loops 37, 60, 70 and the autolysis loop. These loops have a high density of basic residues, resulting in a positive charge on the surface of APC. Many of these residues are important in the interaction of APC with FVa and FVIIIa. The current study focused on the function of the autolysis loop in the interaction with FVIIIa. This loop was previously shown to interact with FVa, and it inhibits APC inactivation by plasma serpins. Charged residues of the autolysis loop were individually mutated to alanine and the activity of these mutants was assessed in functional FVIIIa inactivation assays. The autolysis loop was functionally important for FVIIIa inactivation. Mutation of R306, K311 and R314 each resulted in significantly reduced FVIIIa inactivation. The inactivating cleavages of FVIIIa at R336 and R562 were affected equally by the mutations. Protein S and FV stimulated cleavage at R562 more than cleavage at R336, independent of mutations in the autolysis loop. Together, these results confirmed that the autolysis loop plays a significant role as part of the basic exosite on APC in the interaction with FVIIIa.

Species-specific anticoagulant and mitogenic activities of murine protein S.

BACKGROUND: The protein C pathway down-regulates thrombin generation and promotes cytoprotection during inflammation and stress. In preclinical studies using models of murine injury (e.g., sepsis and ischemic stroke), murine protein S may be required because of restrictive species specificity. DESIGN AND METHODS: We prepared and characterized recombinant murine protein S using novel coagulation assays, immunoassays, and cell proliferation assays. RESULTS: Purified murine protein S had good anticoagulant co-factor activity for murine activated protein C, but not for human activated protein C, in mouse or rat plasma. In human plasma, murine protein S was a poor co-factor for murine activated protein C and had no anticoagulant effect with human activated protein C, suggesting protein S species specificity for factor V in addition to activated protein C. We estimated that mouse plasma contains 22+/-1 microg/mL protein S and developed assays to measure activated protein C co-factor activity of the protein S in murine plasma. Activated protein C-independent anticoagulant activity of murine protein S was demonstrable and quantifiable in mouse plasma, and this activity was enhanced by exogenous murine protein S. Murine protein S promoted the proliferation of mouse and human smooth muscle cells. The potency of murine protein S was higher for mouse cells than for human cells and similarly, human protein S was more potent for human cells than for mouse cells. CONCLUSIONS: The spectrum of bioactivities of recombinant murine protein S with mouse plasma and smooth muscle cells is similar to that of human protein S. However, in vitro and in vivo studies of the protein C pathway in murine disease models are more appropriately performed using murine protein S. This study extends previous observations regarding the remarkable species specificity of protein S to the mouse.

Direct effects of protein S in ameliorating acute lung injury.

OBJECTIVE: Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse. METHODS: Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation. RESULTS: Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS-treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor-alpha, interleukin-6 and monocyte chemoattractant protein-1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor-alpha and interleukin-6 in the lung compared with untreated animals. Thrombin-antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells. CONCLUSION: Protein S protects against LPS-induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.

Human protein S inhibits the uptake of AcLDL and expression of SR-A through Mer receptor tyrosine kinase in human macrophages.

Human protein S is an anticoagulation protein. However, it is unknown whether protein S could regulate the expression and function of macrophage scavenger receptor A (SR-A) in macrophages. Human THP-1 monocytes and peripheral blood monocytes were differentiated into macrophages and then treated with physiological concentrations of human protein S. We found that protein S significantly reduced acetylated low-density lipoprotein (AcLDL) uptake and binding by macrophages and decreased the intracellular cholesteryl ester content. Protein S suppressed the expression of the SR-A at both mRNA and protein levels. Protein S reduced the SR-A promoter activity primarily through inhibition in the binding of transcription factors to the AP-1 promoter element in macrophages. Furthermore, human protein S could bind and induce phosphorylation of Mer receptor tyrosine kinase (Mer RTK). Soluble Mer protein or tyrosine kinase inhibitor herbimycin A effectively blocked the effects of protein S on AcLDL uptake. Immunohistochemical analysis revealed that the level of protein S was substantially increased in human atherosclerotic arteries. Thus, human protein S can inhibit the expression and activity of SR-A through Mer RTK in macrophages, suggesting that human protein S is a modulator for macrophage functions in uptaking of modified lipoproteins.

Protein S down-regulates factor Xase activity independent of activated protein C: specific binding of factor VIII(a) to protein S inhibits interactions with factor IXa.

Protein S functions as an activated protein C (APC)-independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa-dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17-fold increase in K(m) for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in K(m) for factor X. Surface plasmon resonance-based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (K(d); c. 10 nmol/l). Competition binding assays using Glu-Gly-Arg-active-site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg(336) by factor IXa was c. 1.8-fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down-regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.

Protein S is a cofactor for tissue factor pathway inhibitor.

Protein S is a vitamin K-dependent protein that acts as a cofactor of the anticoagulant protein APC. However, protein S also exhibits anticoagulant activity in the absence of APC. Thrombin generation experiments in normal plasma and in plasma deficient in tissue factor pathway inhibitor (TFPI) and/or protein S demonstrated that protein S stimulates the inhibition of TF by TFPI. Kinetic analysis in model systems containing purified proteins showed that protein S enhances the formation of the binary FXa:TFPI complex by reducing the Ki of TFPI from approximately 4 nM to approximately 0.5 nM. Enhancement of inhibitory activity of TFPI by protein S is only observed with full-length TFPI and in the presence of a negatively charged phospholipid surface. The Ki decrease brings the TFPI concentration necessary for FXa:TFPI complex formation within range of the plasma TFPI concentration which increases FXa:TFPI complex formation and accelerates feedback inhibition of the TF pathway by enhancing the formation of the quaternary TFPI:FXa:TF:FVIIa complex. Thus, protein S is not only a cofactor of APC, but also of TFPI. A reduced TFPI cofactor activity may contribute to the increased risk of venous thrombosis in protein-S deficient individuals. Using calibrated automated thrombography we have developed two assays that enable quantification of the functional activity of the TFPI/protein S system in plasma. These assays show that the activity of the TFPI/protein S system is greatly impaired in oral contraceptive users.

Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor.

Tissue factor (TF) plays an important role in hemostasis, inflammation, angiogenesis, and the pathophysiology of atherosclerosis and cancer. In this article we uncover a mechanism in which protein S, which is well known as the cofactor of activated protein C, specifically inhibits TF activity by promoting the interaction between full-length TF pathway inhibitor (TFPI) and factor Xa (FXa). The stimulatory effect of protein S on FXa inhibition by TFPI is caused by a 10-fold reduction of the K(i) of the FXa/TFPI complex, which decreased from 4.4 nM in the absence of protein S to 0.5 nM in the presence of protein S. This decrease in K(i) not only results in an acceleration of the feedback inhibition of the TF-mediated coagulation pathway, but it also brings the TFPI concentration necessary for effective FXa inhibition well within range of the concentration of TFPI in plasma. This mechanism changes the concept of regulation of TF-induced thrombin formation in plasma and demonstrates that protein S and TFPI act in concert in the inhibition of TF activity. Our data suggest that protein S deficiency not only increases the risk of thrombosis by impairing the protein C system but also by reducing the ability of TFPI to down-regulate the extrinsic coagulation pathway.

Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels.

Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.

Protein S multimers and monomers each have direct anticoagulant activity.

BACKGROUND AND OBJECTIVES: Plasma protein S (PS) is an essential anticoagulant that has activated protein C-independent, direct anticoagulant activity (PS-direct). It was reported that monomeric purified PS has poor PS-direct and that a subpopulation of multimeric purified PS has high PS-direct and high affinity for phospholipids. We independently examined the relative PS-direct and affinity for phospholipids of monomeric and multimeric PS and we obtained contrasting results. METHODS AND RESULTS: Unpurified recombinant protein S (rPS) was monomeric and had PS-direct potency similar to that of both PS in plasma and multimeric affinity-purified PS, as measured in plasma assays for PS-direct and in thrombin-generation assays. Multimers of unpurified rPS were not induced by ethylenediaminetetraacetic acid (EDTA), pH 2.5, NaSCN, or barium adsorption/elution. Multimers were induced by chromatography in the presence of EDTA and thus may be concentration-dependent. In contrast to a different report, monomers, dimers, trimers, and higher-order PS forms were clearly separated in sedimentation velocity experiments and multimers were not dissociated by adding Ca(2+). Active plasma-derived and recombinant immunoaffinity-purified PS were fractionated into monomers and multimers. On a mass basis, monomers and multimers had similar specific PS-direct and ability to compete with prothrombinase components (factors Xa/Va) for limiting phospholipids. FXa ligand blotted to both monomers and multimers. CONCLUSIONS: Plasma PS-direct is similar to that of affinity-purified PS and unpurified rPS. Under our conditions, monomeric and multimeric PS have similar PS-direct and ability to compete for phospholipids. Discordant earlier findings are likely due to loss of PS-direct during conventional purification procedures.

Protein S modulates the anticoagulant action of recombinant human activated protein C: a comparison between neonates and adults.

Recombinant human-activated protein C (rhAPC, Drotrecogin alpha (activated), Xigris) has been shown to reduce organ damage and decrease mortality in severe sepsis. Since protein S (PS) serves as a potentiating cofactor of activated protein C and since PS levels are low in neonatal plasma, we hypothesized that the anticoagulant effect of rhAPC would be decreased in cord plasma compared to adult plasma. We demonstrate that the anticoagulant action of 0.3 microg ml(-1) rhAPC (5 nmol l(-1)) was decreased in cord plasma compared to adult plasma, and dose dependently increased in cord plasma in the presence of increasing activities of PS. Correspondingly, the anticoagulant action of rhAPC decreased in adult plasma in the presence of decreasing activities of PS. The low anticoagulant action of rhAPC in cord compared to adult plasma is attributable to low neonatal levels of PS, and as previously shown, to low neonatal levels of TFPI and AT. Our laboratory experiments do not allow definite conclusions for clinical situations. However, we speculate that the anticoagulant efficacy of rhAPC is impaired in neonates and in clinical situations associated with consumption and/or inhibition of PS, AT, and TFPI, such as severe sepsis.

Protein S attenuates the invasive potential of THP-1 cells by interfering with plasminogen binding on cell surface via a protein C-independent mechanism.

Protein S, a cofactor for activated protein C (aPC) to inactivate coagulation factors, also plays a pivotal role in inflammation. Based on our recent findings that aPC and protein S modifies tissue plasminogen activator (tPA)-catalyzed activation of Glu-plasminogen (Glu-plg), we analyzed possible role of protein S in cell-associated plasminogen activation and invasive potential of inflammatory cells. Monocyte-like THP-1 cells, to which both plasminogen and tPA bind, enhanced tPA-catalyzed plasminogen activation, which was partially abolished by protein S but not by aPC. Protein S attenuated both the plasminogen binding to THP-1 cells and associated their invasive potential through Matrigel.

Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells.

Survival of the retina requires the daily phagocytosis of photoreceptor outer segments (OS) by the overlying retinal pigment epithelium (RPE). OS phagocytosis by cultured RPE requires serum and we have recently shown that the vitamin K-dependent serum protein, Gas6, can completely replace serum in this process. Surprisingly, however, we show here that 4-month-old Gas6 knockout mice have normal appearing retinas, except for a reduced ratio of outer segment to inner segment length. We also show that removal of Gas6 from serum does not abrogate the ability of serum to support OS phagocytosis by rat RPE. Both of these findings suggest the presence of an additional serum ligand that is able to support OS phagocytosis by RPE cells. Protein S (PS) is a vitamin K-dependent serum protein with a high degree of structural similarity to Gas6, and a well characterized role in blood coagulation. We report here that recombinant rat PS is able to stimulate OS phagocytosis, and similar to Gas6, it does so through a Mer-dependent mechanism. This is the first demonstration of a common role for Gas6 and PS in any biological process. The existence of redundant ligands for Mer-dependent OS phagocytosis underscores the critical role of this process in the maintenance of retinal function.