Post-translational modifications of the ligands: Requirement for TAM receptor activation.

The Tyro3, Axl, and MerTK (TAM) receptors are three homologous Type I Receptor Tyrosine Kinases that have important homeostatic functions in multicellular organisms by regulating the clearance of apoptotic cells (efferocytosis). Pathologically, TAM receptors are overexpressed in a wide array of human cancers, and often associated with aggressive tumor grade and poor overall survival. In addition to their expression on tumor cells, TAMs are also expressed on infiltrating myeloid-derived cells in the tumor microenvironment, where they appear to act akin to negative immune checkpoints that impair host anti-tumor immunity. The ligands for TAMs are two endogenous proteins, Growth Arrest-Specific 6 (Gas6) and Protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PtdSer) on apoptotic cells and the TAM ectodomains. One interesting feature of TAMs biology is that their ligand proteins require specific post-translational modifications to acquire activities. This chapter summarized these important modifications and explained the molecular mechanisms behind such phenomenon. Current evidences suggest that these modifications help Gas6/Pros1 to achieve optimal PtdSer-binding capacities. In addition, this chapter included recent discovery of regulating machineries of PtdSer dynamic across the plasma membrane, as well as their potential impacts in the tumor microenvironment. Taken together, this review highlights the importance of the upstream PtdSer and Gas6 in regulating TAMs' function and hope to provide researchers with new perspectives to inspire future studies of TAM receptors in human disease models.

Vacunas para SARS-CoV-2: diferentes estrategias de los desarrollos en curso / Vaccines for SARS-CoV-2: different strategies of ongoing developments

El objetivo de este artículo es proporcionar una guía que sirva para la interpretación y seguimiento de los esfuerzos que se están desarrollando en todo el mundo con el objetivo de obtener una vacuna que pueda generar inmunidad contra el nuevo coronavirus SARS-CoV-2 de 2019, el agente causante de la enfermedad por coronavirus denominada COVID-19. Cinco meses después de haber sido detectada la enfermedad, ya hay 102 vacunas en distintos estadios de desarrollo, registradas por la Organización Mundial de la Salud (OMS), correspondientes a 8 plataformas vacunales con diferentes estrategias, y todos los días aparecen nuevas. Esto representará un enorme desafío de organismos internacionales, para la evaluación, comparación y selección de aquellas que cumplan con los criterios regulatorios indispensables de seguridad y eficacia y que, por otro lado, puedan ser producidas en cantidades suficientes para abastecer la demanda mundial. (AU)

The objective of this article is to provide a guide to help the interpretation and monitoring the efforts that are being carried out worldwide to obtain a vaccine that will be able to generate immunity against the new 2019 SARS-CoV-2 coronavirus, the viral agent causes the disease named COVID-19. Five months after the disease was detected, there are already 102 vaccines at different stages of development, registered by World Health Organization (WHO), corresponding to 8 vaccination platforms base on different strategies, and every day new ones appear. This will represent a huge challenge for international organizations, to evaluate, compare and selects those that will meet the essential regulatory criteria of safety and efficacy and that, would be able to be produced in enough quantities to supply the worldwide demand. Key words: SARS-Cov-2 vaccine, vaccine platform, COVID-19 strategy, attenuated virus, viral vector, viral proteins, viral DNA, viral RNA, nucleic acids, viral like particles, WHO. (AU)

Plasma phenotypes of protein S Lys196Glu and protein C Lys193del variants prevalent among young Japanese women.

: Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations, respectively; however, their diagnosis by plasma analyses is difficult because of the type II deficiency phenotype. Three gene variant genotypes were examined in young Japanese women (n = 231). Plasma total protein S activity and total protein S antigen levels were measured using a total protein S assay system, protein C and protein S activities by clot-based methods, and protein C and free protein S antigen levels by latex agglutination methods. protein S Tokushima (p.Lys196Glu) and protein C p.Lys193del variants were prevalent among participants with allele frequencies of 1.08 and 0.86%, respectively, whereas any carrier of protein C p.Arg189Trp variant was not identified. The plasma phenotype of the type II deficiency of protein S Tokushima heterozygotes was demonstrated by decreased total protein S activity with a normal total protein S antigen level; however, the protein C activities of protein C p.Lys193del heterozygotes were within reference intervals, whereas their protein C antigen levels were elevated. We compared the diagnostic accuracy of the total protein S activity/total protein S antigen ratio for identifying protein S Tokushima heterozygotes with that of the clot-based protein S activity/free protein S antigen ratio and found that sensitivity and specificity of 100% each was only achieved by the former. Protein S Tokushima and protein C p.Lys193del are prevalent among young Japanese women, and a plasma analysis using the total protein S assay system is more accurate than the clot-based protein S activity/free protein S antigen ratio for diagnosing protein S Tokushima carriers.

Effects of low-dose combined oral contraceptives and protein S K196E mutation on anticoagulation factors: a prospective observational study.

The association between low-dose combined oral contraceptives (COCs) and anticoagulation factors in Japanese women has been rarely studied. A total of 394 Japanese women with a new beginning cycle of COC use were enrolled, of whom 335 women visited the clinic within 4 weeks after starting the first cycle of COC. Visits occurred in the active phase (272 women) and the placebo phase (63 women). Free protein S (PS) antigen and activity levels and antithrombin activity levels decreased significantly in both the active and placebo phase groups. Protein C (PC) activity levels increased significantly in both groups. Larger reductions in free PS antigen and activity levels occurred with COC comprising either 30 µg ethinylestradiol/desogestrel or 20 µg ethinylestradiol/drospirenone than that comprising 35 µg ethinylestradiol/norethisterone. In four women with the Japanese-specific PS K196E mutation, mean PS activity was 65% before COC use and 57% during COC use, indicating further decrease with COC use. In conclusion, decreased antigen and activity levels of PS and antithrombin and increased activity levels of PC were observed even during the first cycle of low-dose COC use. The effects on PS and PC activities were also observed in the hormone-free interval.

Augmentation of Human Monocyte Responses to Lipopolysaccharide by the Protein S and Mer/Tyro3 Receptor Tyrosine Kinase Axis.

Resolution of the inflammatory response requires coordinated regulation of pro- and anti-inflammatory mediator production, together with clearance of recruited inflammatory cells. Many different receptors have been implicated in phagocytosis of apoptotic cells (efferocytosis), including Mer, a receptor tyrosine kinase that can mediate recognition and subsequent internalization of apoptotic cells. In this manuscript, we examine the expression and function of the Tyro3/Axl/Mer (TAM) family of receptors by human monocytes. We demonstrate that the Mer ligand, protein S, binds to the surface of viable monocytes via phosphatidylserine-dependent and -independent mechanisms. Importantly, we have identified a novel role for receptor tyrosine kinase signaling in the augmentation of monocyte cytokine release in response to LPS. We propose that low-level phosphatidylserine exposure on the plasma membrane of viable monocytes allows protein S binding that leads to TAM-dependent augmentation of proinflammatory cytokine production. Our findings identify a potentially important role for TAM-mediated signaling during the initiation phase of inflammation.

The cross-sectional association between vasomotor symptoms and hemostatic parameter levels in postmenopausal women.

OBJECTIVE: Vasomotor symptoms (VMS) may be a marker of cardiovascular risk. We aimed to evaluate the cross-sectional association of VMS presence and severity with hemostatic parameter levels measured at baseline among Women's Health Initiative (WHI) Hormone Therapy trial postmenopausal participants. METHODS: This cross-sectional analysis included 2,148 postmenopausal women with measures of VMS presence and severity reported in the 4 weeks before WHI baseline, who were not using warfarin or hormone therapy and for whom the following baseline hemostatic parameters were measured within the WHI Cardiovascular Disease Biomarker Case-Control Study: antithrombin, plasminogen activator inhibitor-1, protein C antigen, total and free protein S antigen, total and free tissue factor pathway inhibitor, D-dimer, normalized activated protein C sensitivity ratio, and thrombin generation. Using multiple linear regression, we estimated the adjusted average difference in each hemostatic parameter associated with VMS presence and severity. A multiple comparisons-corrected P value was computed using the P-min procedure to determine statistical significance of our smallest observed P value. RESULTS: Women were 67 years of age on average and 33% reported VMS presence at baseline. There was some suggestion that VMS presence may be associated with a -0.34 adjusted difference in normalized activated protein C sensitivity ratio compared with no VMS (95% CI, -0.60 to -0.087; P = 0.009), but this association was not significant after correction for multiple comparisons (P = 0.073). VMS presence or severity was not significantly associated with the other hemostatic parameters. CONCLUSIONS: We found no convincing evidence that VMS presence or severity was associated with levels of hemostatic parameters among postmenopausal women.

ELISA-Based Detection System for Protein S K196E Mutation, a Genetic Risk Factor for Venous Thromboembolism.

Protein S (PS) acts as a cofactor for activated protein C in the plasma anticoagulant system. PS Lys196-to-Glu (K196E) mutation is a genetic risk factor for venous thromboembolism in Japanese individuals. Because of the substantial overlap in PS anticoagulant activity between KK (wild-type) and KE (heterozygous) genotypes, it is difficult to identify PS K196E carriers by measuring PS activity. Here, we generated monoclonal antibodies specific to the PS K196E mutant and developed a simple and reliable method for the identification of PS K196E carriers. We immunized mice with a keyhole limpet hemocyanin-conjugated synthetic peptide with Glu196. The hybridoma cells were screened for the binding ability of the produced antibodies to recombinant mutant EGF-like domains of PS (Ile117-Glu283). We obtained three hybridoma cell lines producing PS K196E mutation-specific antibodies. We established a sandwich enzyme-linked immunosorbent assay (ELISA) system in which the PS K196E mutation-specific monoclonal antibody was used as a detection antibody. We measured human plasma samples by using this system and successfully discriminated 11 individuals with the KE genotype from 122 individuals with the KK genotype. The ELISA system using the PS K196E mutation-specific antibody is a useful tool for the rapid identification of PS K196E carriers, who are at a higher risk for venous thromboembolism.

The Axl receptor tyrosine kinase is a discriminator of macrophage function in the inflamed lung.

Much of the biology surrounding macrophage functional specificity has arisen through examining inflammation-induced polarizing signals, but this also occurs in homeostasis, requiring tissue-specific environmental triggers that influence macrophage phenotype and function. The TAM receptor family of receptor tyrosine kinases (Tyro3, Axl and MerTK) mediates the non-inflammatory removal of apoptotic cells by phagocytes through the bridging phosphatidylserine-binding molecules growth arrest-specific 6 (Gas6) or Protein S. We show that one such TAM receptor (Axl) is exclusively expressed on mouse airway macrophages, but not interstitial macrophages and other lung leukocytes, under homeostatic conditions and is constitutively ligated to Gas6. Axl expression is potently induced by granulocyte-macrophage colony-stimulating factor expressed in the healthy and inflamed airway, and by type I interferon or Toll-like receptor-3 stimulation on human and mouse macrophages, indicating potential involvement of Axl in apoptotic cell removal under inflammatory conditions. Indeed, an absence of Axl does not cause sterile inflammation in health, but leads to exaggerated lung inflammatory disease upon influenza infection. These data imply that Axl allows specific identification of airway macrophages, and that its expression is critical for macrophage functional compartmentalization in the airspaces or lung interstitium. We propose that this may be a critical feature to prevent excessive inflammation because of secondary necrosis of apoptotic cells that have not been cleared by efferocytosis.

An unexpected etiology of priapism: infection-related anti-protein s antibodies.

INTRODUCTION: In adolescents, the occurrence of priapism is commonly related to sickle cell disease and rarely to other causes. We hereby report a case of priapism due to an acquired protein S (PS) deficiency. AIM: The aim of this study was to describe a young man who developed a priapism with a thrombosis of the corpora cavernosa associated with an anti-PS antibody (anti-PS Ab). METHODS: One week after the onset of an influenza-like illness, a young male developed multiple extensive venous thromboses including a thrombosis of the corpora cavernosa causing painful partial priapism. These thromboses along with purpuric lesions with necrotic vesicles of the feet skin were linked to an acquired PS deficiency due to an anti-PS Ab. The optimal treatment of anti-PS Ab-associated thrombosis is debated but we chose to initiate (i) heparin; (ii) corticosteroids; and (iii) plasmapheresis. RESULTS: Even if priapism lasted more than 4 days, a full recovery of erectile function was observed within 3 months. As compared with priapism due to sickle cell disease, which is commonly associated with definitive erectile dysfunction, this favorable outcome is noteworthy. The skin healing was complete only 6 months later. CONCLUSION: Acquired PS deficiency complicating an infectious disease is a rare, life-threatening condition, associated with substantial morbidity related to amputations of limbs or digits. This is the first report of priapism due to acquired PS deficiency.

Anti S enigma in a pregnant patient.

Among the antibodies of the MNS blood group system, anti S antibody is generally IgG antibody reacting at 37 °C. It is rarely implicated in hemolytic transfusion reaction; however, it can lead to potentially severe transfusion reactions. Anti S is also capable of causing mild to severe fatal hemolytic disease of newborn. We report a case of anti S antibody in a pregnant patient with complicated falciparum malaria.

Anti-human protein S antibody induces tissue factor expression through a direct interaction with platelet phosphofructokinase.

INTRODUCTION: Autoantibodies including anti-human protein S antibody (anti-hPS Ab) and anti-human protein C antibody (anti-hPC Ab) can be detected in patients with autoimmune diseases with hypercoagulability. The objective of the present study was to determine the effects and molecular pathways of these autoantibodies on tissue factor (TF) expression in human coronary artery endothelial cells (HCAECs). MATERIALS AND METHODS: HCAECs were treated with anti-hPS Ab or anti-hPC Ab for 3 hours. TF expression was measured by real-time PCR and Western blot. TF-mediated procoagulant activity was determined by a commercial kit. MAPK phosphorylation was analyzed by Bio-Plex luminex immunoassay and Western blot. The potential proteins interacting with anti-hPS Ab were studied by immunoprecipitation, mass spectrometry and in vitro pull-down assay. RESULTS: Anti-hPS Ab, but not anti-hPC Ab, specifically induced TF expression and TF-mediated procoagulant activity in HCAECs in a concentration-dependent manner. This effect was confirmed in human umbilical endothelial cells (HUVECs). ERK1/2 phosphorylation was induced by anti-hPS Ab treatment, while inhibition of ERK1/2 by U0216 partially blocked anti-hPS Ab-induced TF upregulation (P<0.05). In addition, anti-hPS Ab specifically cross-interacted with platelet phosphofructokinase (PFKP) in HCAECs. Anti-hPS Ab was able to directly inhibit PFKP activities in HCAECs. Furthermore, silencing of PFKP by PFKP shRNA resulted in TF upregulation in HCAECs, while activation of PFKP by fructose-6-phosphate partially blocked the effect of anti-hPS Ab on TF upregulation (P<0.05). CONCLUSIONS: Anti-hPS Ab induces TF expression through a direct interaction with PFKP and ERK1/2 activation in HCAECs. Anti-hPS Ab may directly contribute to vascular thrombosis in the patient with autoimmune disorders.

Patients with antineutrophil cytoplasmic antibodies associated vasculitis in remission are hypercoagulable.

OBJECTIVES: The risk of venous thromboembolism (VTE) is increased in patients with antineutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) as compared to healthy subjects. The mechanisms underlying this increased occurrence of VTE are not completely understood. We hypothesize that AAV patients in remission are more procoagulant than healthy controls. METHODS: Patients with AAV in remission and no VTE for the last 6 months were included. Patients with severe renal impairment (serum creatinine > 250 µmol/l) were excluded. Age and sex matched healthy controls were included. The endogenous thrombin potential (ETP) was determined together with hemostatic variables: fibrinogen, D-dimers, factor VIII (FVIII), tissue factor pathway inhibitor (TFPI), protein C, and free protein S. RESULTS: Thirty-one patients were included. In 27 patients not taking anticoagulants, ETP was measured and found to be elevated: 137.1% as compared to a median of 90.0% for healthy controls (p < 0.01). Fibrinogen and D-dimer levels were not elevated in patients (median 3.5 g/l and 279 µg/l, respectively). FVIII and TFPI levels were also significantly increased in patients as compared to healthy controls (159% vs 137%; 122.5% vs 101%, respectively), whereas protein C and free protein S levels were not elevated (126.5% vs 118.6% and 124.6% vs 118.3%, respectively). CONCLUSION: Patients with AAV in remission are more procoagulant than healthy controls, as indicated by an increased ETP. The increased FVIII level measured in these patients suggests persistence of endothelial activation and/or dysfunction. This endothelial dysfunction may cause a continuous low-grade procoagulant state.

Plasma protein S residues 37-50 mediate its binding to factor Va and inhibition of blood coagulation.

Protein S (PS) is an anticoagulant plasma protein whose deficiency is associated with increased risk of venous thrombosis. PS directly inhibits thrombin generation by the blood coagulation pathways by several mechanisms, including by binding coagulation factors (F) Va and Xa. To identify PS sequences that mediate inhibition of FVa activity, antibodies and synthetic peptides based on PS sequence were prepared and employed in plasma coagulation assays, purified component prothrombinase assays, binding assays, and immunoblots. In the absence of activated protein C, monoclonal antibody (Mab) S4 shortened FXa-induced clotting in normal plasma but not in PS-depleted plasma. Mab S4 also blocked PS inhibition of FVa-dependent prothrombinase activity in purified component assays in the absence or presence of phospholipids and inhibited binding of PS to immobilised FVa. Epitope mapping identified N-terminal region residues 37-67 of PS as this antibody's epitope. A peptide representing PS residues 37-50 inhibited FVa-dependent prothrombinase activity in a non-competitive manner, with 50% inhibition observed at 11 µM peptide, whereas a peptide with a D-amino acid sequence of 37-50 was ineffective. FVa, but not FXa, bound specifically to the immobilised peptide representing residues 37-50, and the peptide inhibited binding of FVa to immobilised PS. These data implicate PS residues 37-50 as a binding site for FVa that mediates, at least in part, the direct inhibition of FVa-dependent procoagulant activity by PS.

T cell-derived protein S engages TAM receptor signaling in dendritic cells to control the magnitude of the immune response.

Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse T cells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to T cell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human T cells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response.

Thrombophilia and varicella zoster in children.

From 2005 to 2011, 25 children of both sexes (13 boys and 12 girls, mean age 7.8 ± 2.5 years, 5-12.4 years) with acute varicella zoster virus (VZV) infection were selected. Five patients showed venous thromboembolism characterized by deep venous thrombosis (DVT). Comparison of activated partial thromboplastin time, antithrombin III, D-dimer, lupus anticoagulant, free S protein (PS), C protein, and antiphospholipid and PS antibodies was performed on children with acute VZV and DVT (group I), acute uncomplicated VZV (group II), and 30 healthy controls of both sexes (15 boys and 15 girls, mean age 7.5 ± 2.6 years, group III). Genetic thrombophilic mutations (Factor V Leiden, MTHFR C677T, and Prothrombin G20210A) were evaluated. Coagulation disorders and PS antibody were found in children with acute VZV (groups I and II). Significant differences were shown among the three groups (P < 0.05). Acute VZV infection could be associated with coagulation disorders and production of inhibitory PS antibodies in many uncomplicated cases.

Anti-S antibodies: an unusual cause of haemolytic disease of the fetus and newborn (HDFN).

We report a case of haemolytic disease of the fetus and newborn due to anti-S antibodies. Baby G was born by emergency caesarean section at 35 weeks due to reduced fetal movement. Prior to delivery, antenatal screening revealed the mother's blood group was AB rhesus positive with anti-S antibody titres. The baby was pale but non-hydropic at birth with hepatosplenomegaly. Haemoglobin at birth was 5.23 g/dl and serum bilirubin 138 µmol/l. The baby required phototherapy, γ-globulin infusion and exchange transfusion with post-transfusion complications.

Platelet protein S directly inhibits procoagulant activity on platelets and microparticles.

Anticoagulant plasma protein S (PS) is essential for maintaining haemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the "thrombin-sensitive region", abolishing activated protein C (APC) cofactor activity. However, we showed that cleaved PS retains APC-independent anticoagulant activities ("PS-direct"). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p<0.0004, n=20) more thrombin generation upon addition of FXa and prothrombin. PltPS exerted PS-direct that was similar to or greater than that of Zn(2+)-containing pdPS and much greater than that of Zn(2+)-deficient pdPS. Findings were confirmed using purified pltPS. Platelet-bound pltPS and microparticle-bound pltPS had similar PS-direct. Finally, platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 1.5 ± 0.2-fold (p<0.0001, n=11) more FXa generation upon addition of TF/FVIIa and FX. Thus, pltPS inhibits both prothrombinase and extrinsic FXase activities. Neutralising antibodies against APC and TFPI had no effect on the PS-direct of pltPS or pdPS on platelets. This study indicates that pltPS may be an essential pool of PS that counterbalances procoagulant activities on platelets.

Incorporation of disulfide containing protein modules into multivalent antigenic conjugates: generation of antibodies against the thrombin-sensitive region of murine protein S.

Antigenic peptide conjugates can be used as vaccines and for the production of antibodies for clinical and research use. A method is presented here for the construction of conjugates incorporating oxidatively folded protein domains in their native conformation. This method was used to prepare multiple antigenic peptide constructs of the thrombin-sensitive loop region of murine anticoagulant protein S.

Studies on coagulation incompatibilities for xenotransplantation.

Microvascular thrombosis, following the activation of clotting cascade, is a hallmark of porcine solid organ xenograft rejection. The analysis of differences between human, monkey, and pig coagulation systems is crucial when monkey is used as animal model and pig as organ donor in xenotransplantation. Thrombosis, according to many authors, may be due to the molecular incompatibilities between natural anticoagulants present on pig endothelium and primate activated coagulation factors. The generation of activated protein C (PC) is critical for the physiological anticoagulation. One of the major incompatibilities may be related to the inability of pig thrombomodulin (TM) and endothelial protein C receptor to activate the recipient (primate) circulating PC in the presence of thrombin. Tissue factor pathway inhibitor (TFPI), is the primary inhibitor of tissue factor (TF)-induced coagulation. TFPI directly inhibits the activated factor X (FXa) and blocks the procoagulant activity of the TF/factor VIIa (FVIIa) complex by forming a quaternary TF/FVIIa/FXa/TFPI complex. Microvascular thrombosis, observed in the organ transplant, may also be due to the failure of pig TFPI to bind human FXa efficiently and inhibit human FVIIa/TF activity. The methods described in this chapter can be useful for the identification and characterization of primate and pig coagulation factors (isolated from a small volume of blood) by using SDS-PAGE and immunoblotting. Differences in molecular weight can help in the identification of the origin (pig or primate) of coagulation proteins in plasma from the recipient of xenografts. On the other hand, in vitro models of PC pathway and TFPI on human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) are described which can be used for studying incompatibilities between primate and pig.