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BACKGROUND: Protein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factor V (FVa) and VIII (FVIIIa), but also for tissue factor pathway inhibitor α (TFPIα) in the inhibition of activated factor X (FXa).OBJECTIVE: For therapeutic purposes, we aimed at generating single-domain antibodies (sdAbs) that could specifically modulate the APC-cofactor activity of PS in vivo.METHODS: A llama-derived immune library of sdAbs was generated and screened on recombinant human PS by phage-display. PS binders were tested in a global activated partial thromboplastin time (APTT)-based APC-cofactor activity assay.RESULTS: A PS-specific sdAb (PS003) was found to enhance the APC-cofactor activity of PS in our APTT-based assay, and this enhancing effect was greater for a bivalent form of PS003 (PS003biv). Further characterization of PS003biv demonstrated that PS003biv also enhanced the APC-cofactor activity of PS in a tissue factor (TF)-induced thrombin generation assay and stimulated APC in the inactivation of FVa, but not FVIIIa, in plasma-based assays. Furthermore, PS003biv was directed against the sex hormone-binding globulin (SHBG)-like domain but did not inhibit the binding of PS to C4b-binding protein (C4BP) and did not interfere with the TFPIα-cofactor activity of PS. In mice, PS003biv exerted an antithrombotic effect in a FeCl3 -induced thrombosis model, while not affecting physiological hemostasis in a tail-clip bleeding model.DISCUSSION: Altogether, these results showed that pharmacological enhancement of the APC-cofactor activity of PS through an original anti-PS sdAb might constitute a promising and safe antithrombotic strategy.

Phosphomannomutase 2 (PMM2) deficiency is the most prevalent congenital disorder of glycosylation. It is associated with coagulopathy, including protein C deficiency. Since all components of the anticoagulant and cytoprotective protein C system are glycosylated, we sought to investigate the impact of an N-glycosylation deficiency on this system as a whole. To this end, we developed a PMM2 knockdown model in the brain endothelial cell line hCMEC/D3. The resulting PMM2low cells were less able to generate activated protein C (APC), due to lower surface expression of thrombomodulin and endothelial protein C receptor. The low protein levels were due to downregulated transcription of the corresponding genes (THBD and PROCR, respectively), which itself was related to downregulation of transcription regulators Krüppel-like factors 2 and 4 and forkhead box C2. PMM2 knockdown was also associated with impaired integrity of the endothelial cell monolayer—partly due to an alteration in the structure of VE-cadherin in adherens junctions. The expression of protease-activated receptor 1 (involved in the cytoprotective effects of APC on the endothelium) was not affected by PMM2 knockdown. Thrombin stimulation induced hyperpermeability in PMM2low cells. However, pretreatment of cells with APC before thrombin simulation was still associated with a barrier-protecting effect. Taken as a whole, our results show that the partial loss of PMM2 in hCMEC/D3 cells is associated with impaired activation of protein C and a relative increase in barrier permeability.

Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre− mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p &lt, 0.05) and an increase in pseudo-tube width (41 ± 4.5%, p &lt, 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p &lt, 0.01), which is associated to a higher Ca2+ mobilization (p &lt, 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p &lt, 0.001, p &lt, 0.01, and p &lt, 0.01, respectively) as well as actin/tubulin distribution (p &lt, 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p &lt, 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.

The Transient Receptor Potential Vanilloid 4 (TRPV4) of endothelial cells contributes to many important functions including the regulation of Ca2+ homeostasis, cell volume, endothelial barrier permeability, and smooth muscle tone. However, its role in the transition of endothelial cells toward a pro‐inflammatory phenotype has not been studied so far. Using both arterial and venous endothelial cells, we first show that the pharmacological activation of TRPV4 channels with GSK1016790A, a potent TRPV4 agonist, triggers robust and sustained Ca2+ increases, which are blocked by both TRPV4 antagonists HC067047 and RN9893. TRPV4 activation also triggers the actin cytoskeleton and adherens junction (VE‐Cadherin) rearrangement in both arterial and venous endothelial cells and leads to rapid decreases of trans‐endothelial electrical resistance. In addition to its effect on endothelial barrier integrity, TRPV4 activation selectively increases ICAM‐1 surface expression in arterial and venous endothelial cells, due to the stimulation of ICAM‐1 gene expression through the NF‐κB transcription factor. TRPV4 channel activation also induced apoptosis of venous and arterial endothelial cells, while TRPV4 blockade reduced apoptosis, even in the absence of TRPV4 activation. As altered barrier integrity, increased adhesion molecule expression and apoptosis are hallmarks of the pro‐inflammatory state of endothelial cells, our results indicate that TRPV4 channel activity can induce the transition of both venous and arterial endothelial cells toward a pro‐inflammatory phenotype., Using both arterial and venous endothelial cells, this paper shows that TRPV4 channel activity can induce the transition of both venous and arterial endothelial cells toward a pro‐inflammatory phenotype, as evidenced by altered barrier integrity, increased adhesion molecule expression, and apoptosis.

International audience; In this work, a series of cyclic bridged analogs of isocombretastatin A-4 (isoCA-4) with phenyl or pyridine linkers were designed and synthesized. The synthesis of the desired analogs was performed by the formation of nitro-vinyl intermediates, followed by a Cadogan cyclization. Structure activity relationship (SAR) study demonstrates the critical role of the combination of quinaldine as ring A, pyridine as the linker, and indole as ring B in the same molecule, for the cytotoxic activity. Among all tested compounds, compound 42 showed the highest antiproliferative activity against a panel of cancer cell lines with average IC50 values of 5.6 nM. Also, compound 42 showed high antiproliferative activity against the MDR1-overexpressing K562R cell line; thus, it was 1.5-and 12-fold more active than the reference compounds, isoCA-4 and CA-4, respectively. Moreover, 42 displayed a strong antiproliferative activity against the colon-carcinoma cells (HT-29), which are resistant to combretastatin A-4 and isoCA-4, and it was found to be 8000-fold more active than natural CA-4. Compound 42 also effectively inhibited tubulin polymerization both in vitro and in cells, and induced cell cycle arrest in G2/M phase. Next, we demonstrated that compound 42 dose-dependently caused caspase-induced apoptosis of K562 cells through mitochondrial dysfunction. Finally, we evaluated the effect of compound 42 in human no cancer cells compared to the reference compound. We demonstrated that 42 was 73 times less cytotoxic than isoCA-4 in quiescent peripheral blood lymphocytes (PBLs). In summary, these results suggest that compound 42 represents a promising tubulin inhibitor worthy of further investigation.

Deep vein thrombosis is a common disease associated with a variety of complications including post-thrombotic syndrome as a late complication. It is now clear that in addition to classical deep vein thrombosis triggers such as blood flow disturbance, hypercoagulability, and vessel wall changes, inflammation has a key role in the pathophysiology of deep vein thrombosis, and there is a close relationship between inflammation and coagulation. As attested by changes in several plasma biomarkers, inflammation may have a significant role in the development of post-thrombotic syndrome. Here, we review the link between inflammation and deep vein thrombosis and thus the potential value of anti-inflammatory and/or anticoagulant drugs in the treatment of deep vein thrombosis and the prevention of post-thrombotic syndrome.

Designing multitarget drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC activity. On the basis of 1,1-diarylethylenes ( isoCA-4) and belinostat, a series of hybrid molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent antiproliferative activity.

SummaryIn the absence of specific antidote to fondaparinux, two modified forms of antithrombin (AT), one recombinant inactive (ri-AT) and the other chemically inactivated (chi-AT), were designed to antagonise AT-mediated anticoagulants, e. g. heparins or fondaparinux. These inactive ATs were previously proven to effectively neutralise anticoagulant activity associated with heparin derivatives in vitro and in vivo, as assessed by direct measurement of anti-FXa activity. This study was undertaken to evaluate in vitro the effectivity of inactive ATs to reverse anticoagulation by heparin derivatives and to compare them with non-specific fondaparinux reversal agents, like recombinant-activated factor VII (rFVIIa) or activated prothrombin-complex concentrate (aPCC), in a thrombin-generation assay (TGA). Addition of fondaparinux (3 μg/ml) to normal plasma inhibited thrombin generation by prolonging lag time (LT) as much as 244 % and lowering endogenous thrombin potential (ETP) to 17 % of their control (normal plasma) values. Fondaparinux-anticoagulant activity was reversed by ri-AT and chi-AT, as reflected by the corrections of LT up to 117 % and 114 % of its control value, and ETP recovery to 78 % and 63 %, respectively. Unlike ri-AT that had no effect on thrombin generation in normal plasma, chi-AT retained anticoagulant activity that minimises its reversal capacity. However, both ATs were more effective than rFVIIa or aPCC at neutralising fondaparinux and, unlike non-specific antidotes, inactive ATs specifically reversed AT-mediated anticoagulant activities, as suggested by their absence of procoagulant activity in anticoagulant-free plasma.

With the aim to determine the binding affinity of a new generation of recombinant antithrombin (AT) toward heparin, we developed a dynamic equilibrium-affinity capillary electrophoresis (DE-ACE) method. This method allows the determination of an AT-heparin binding constant (Kd) directly from the cell culture supernatant used to produce the AT variants. Eight measurements per AT variant are sufficient to determine an accurate Kd (uncertainty ≤ 22%, regression coefficient ≥ 0.97), which is not significantly different from the value obtained from a higher number of measurements. Due to the relatively short time required to determine the Kd of one AT variant (2h), this method has the potential for being a low throughput screening method. The method was validated by analyzing five AT variants, whose Kd have been reported in the literature using fluorescence spectroscopy. Finally, the method was applied to estimate the Kd of one new AT variant and one AT conformer, a latent form, that exhibits a significant loss of affinity.

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400,000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by re-balancing coagulation. Protein S is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting protein S rebalances coagulation in hemophilia. Protein S gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking protein S in plasma of hemophilia patients normalized in vitro thrombin generation. Both protein S and TFPIα were detected in hemophilic mice joints. Protein S and TFPI expression was stronger in joints of hemophilia A than hemophilia B patients when receiving on demand therapy, e.g., during a bleeding episode. In contrast, protein S and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, and comparable to osteoarthritis patients. These results establish protein S inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine protein S silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

International audience; A novel series of tubulin polymerization inhibitors, based on fluorinated derivatives of isocombretastatin A-4 was synthesized with the goal of evaluating the effect of these compounds on the proliferative activity. The introduction of fluorine atom was performed on the phenyl ring or at the linker between the two aromatic rings. The modification of isoCA-4 by introduction of difluoromethoxy group at the para-position (3i) and substitution of the two protons of the linker by two fluorine atoms (3m), produced the most active compounds in the series, with IC50 values of 0.15 -2.2 nM (3i) and 0.1-2 nM (3m) respectively, against a panel of six cancer cell lines. Compounds 3i and 3m had greater antiproliferative activity in comparison with references CA-4 or isoCA-4, the presence of fluorine group leads to a significant enhancement of the antiproliferative activity. Molecular docking studies indicated that compounds 3i and 3m occupy the colchicine binding site of tubulin. Evaluation of cytotoxicity in Human noncancer cells indicated that the compounds 3i and 3m were practically ineffective in quiescent peripheral blood lymphocytes, and may have a selective antiproliferative activity against cancer cells. Analyses of cell cycle distribution, and morphological microtubules organization showed that compound 3m induced G(2)/M phase arrest and, dramatically disrupted the microtubule network.

Purpura fulminans is a deadly complication of Neisseria meningitidis infections due to extensive thrombosis of microvessels. Although a Disseminated Intra-vascular Coagulation syndrome (DIC) is frequently observed during Gram negative sepsis, it is rarely associated with extensive thrombosis like those observed during meningococcemia, suggesting that the meningococcus induces a specific dysregulation of coagulation. Another specific feature of N. meningitidis pathogenesis is its ability to colonize microvessels endothelial cells via type IV pili. Importantly, endothelial cells are key in controlling the coagulation cascade through the activation of the potent anticoagulant Protein C (PC) thanks to two endothelial cell receptors among which the Endothelial Protein C Receptor (EPCR). Considering that congenital or acquired deficiencies of PC are associated with purpura fulminans, we hypothesized that a defect in the activation of PC following meningococcal adhesion to microvessels is responsible for the thrombotic events observed during meningococcemia. Here we showed that the adhesion of N. meningitidis on endothelial cells results in a rapid and intense decrease of EPCR expression by inducing its cleavage in a process know as shedding. Using siRNA experiments and CRISPR/Cas9 genome edition we identified ADAM10 (A Disintegrin And Metalloproteinase-10) as the protease responsible for this shedding. Surprisingly, ADAM17, the only EPCR sheddase described so far, was not involved in this process. Finally, we showed that this ADAM10-mediated shedding of EPCR induced by the meningococcal interaction with endothelial cells was responsible for an impaired activation of Protein C. This work unveils for the first time a direct link between meningococcal adhesion to endothelial cells and a severe dysregulation of coagulation, and potentially identifies new therapeutic targets for meningococcal purpura fulminans., Author summary Neisseria meningitidis (meningococcus) is responsible for a severe syndrome called purpura fulminans in which the coagulation system is totally dysregulated, leading to an extensive occlusion of blood microvessels. The pathogenesis of this syndrome is still not understood. Here we show that the meningococcus, when adhering on the apical surface of endothelial cells, induces the activation of membranous protease named ADAM-10, which in turn hydrolyses a cellular receptor called EPCR. The latter is key for the activation of a circulating potent anticoagulant, the Protein C (PC). PC activation is then impaired following meningococcal adhesion on endothelial cells. This work unveils for the first time a specific dysregulation of coagulation induced by the meningococcus and potentially identifies new therapeutic targets for meningococcal purpura fulminans.

Background: Protein S (PS) and growth arrest-specific gene 6 (Gas6) are vitamin K-dependent proteins. PS is an anticoagulant whose pivotal role is illustrated by purpura fulminans (PF), a life-threatening condition characterized by purpuric skin lesions, disseminated intravascular coagulation and thrombosis. Unlike PS, Gas6 displays a procoagulant effect. Its complete deficiency has anti-hemostatic, pro-inflammatory and anti-erythropoietic effects. In tissues, PS and Gas6 have an impact on cellular functions by binding to and activating receptor tyrosine kinases of the TAM family. PS complete deficiency-induced PF is thought to result from the imbalance between procoagulant and anticoagulant forces. However, its pathophysiological mechanism has not yet been fully elucidated. Previous studies showed that Pros1-/-mice die in utero with a PF phenotype similar to the one observed in human. We therefore considered Pros1-/- mice as a suitable model of the human PF. The aim of this work was to investigate if Gas6 deficiency could rebalance hemostasis in PS deficient mice and rescue them from PF. Methods & results: Pros1 +/- Gas6 -/- mice were intercrossed to obtain Pros1-/-Gas6-/- mice. Embryonic mortality was higher in Pros1+/-Gas6-/- than in Pros1+/- mattings (6% vs 3 % at E14, 21% vs 18 % at E16, 30% vs 20 % at E17, respectively). About 7.5% of dead embryos from Pros1+/-Gas6-/- mattings were found necrotic and macerated versus only 3.8% inPros1+/- mattings. Macroscopically, 74% of Pros1-/-Gas6-/- embryos displayed maximal bleeding scores (3) corresponding to intracranial and major body bleedings compared to 47% in Pros1-/- embryos (p More immature RBC were found in Pros1-/- than in Pros1+/+Gas6+/+ circulating blood (17% vs 12%, p Conclusion: Gas6 deficiency did not rebalance hemostasis in Pros1-/-embryos and prevent PF. Notably, Gas6 and PS combined deficiency leads to a more dramatic phenotype than PS deficiency alone with a higher mortality rate, more major bleedings, phagocytosis impairment, more inflammation and an erythropoietic defect compatible with anemia of inflammation. Disclosures No relevant conflicts of interest to declare.

Summary Background Heparin and its analogs, mediating their anticoagulant activity through antithrombin (AT) activation, remain largely used for the preventive and curative treatment of thrombosis. The major adverse reaction of these drugs is the bleeding risk associated with overdose. Unfractionnated heparin (UFH) can be efficiently and rapidly neutralized by protamine sulfate, but this reversal partially neutralizes low-molecular-weight heparin (LMWH) and is inefficient in reversing fondaparinux. To secure administration of AT-mediated anticoagulants and counteract bleeding disorders, we previously designed a recombinant inactive AT as an antidote to heparin derivatives. Objectives To get around the limited production level of recombinant AT, we propose in this study an alternative strategy to produce a chemically modified inactive AT, exhibiting increased heparin affinity, as an antagonist of heparin analogs. Methods Plasma-derived AT was chemically modified with 2,3 butanedione, a diketone known to specifically react with the arginine side chain. The chemical reaction was conducted in the presence of heparin to preserve basic residues within the heparin binding site from modifications. Results AT treated by butanedione and selected for its high heparin affinity (AT-BD) was indeed modified on reactive Arg393 and thus exhibited decreased anticoagulant activity and increased heparin affinity. AT-BD was able to neutralize anticoagulant activity of heparin derivatives in vitro and in vivo and was devoid of intrinsic anticoagulant activity, as assessed by activated partial thromboplastin time assay. Conclusions AT-BD appears to be as efficient as protamine to neutralize UFH in vivo but could be more largely used because it also reverses fondaparinux and LMWH.

Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros+/− heterozygous mice. In the null (−) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros+/− mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros+/− mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor–induced thromboembolism model. No viable Pros−/− mice were obtained through mating of Pros+/− parents. Most E17.5 Pros−/− embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros−/− embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.

Antithrombin (AT) is a plasma glycoprotein which possesses anticoagulant and anti-inflammatory properties. AT exhibits various forms, among which are native, latent and heterodimeric ones. We studied the potential of capillary electrophoresis-mass spectrometry (CE-MS) using a sheath liquid interface, electrospray ionization (ESI), and a quadrupole-time-of-flight (Q-TOF) mass spectrometer to separate and quantify the different AT forms. For CE separation, a neutral polyvinyl alcohol (PVA) coated capillary was employed. The protein conformation was preserved by using a background electrolyte (BGE) at physiological pH. A sheath liquid of isopropanol-water 50:50 (v/v) with 14 mM ammonium acetate delivered at a flow rate of 120 μL h −1 resulted in optimal signal intensities. Each AT form exhibited a specific mass spectrum, allowing unambiguous distinction. Several co-injection experiments proved that latent AT had a higher electrophoretic mobility (μ ep ) than native AT, and that these conformers could associate to form a heterodimer during the CE analysis. The developed CE-MS method enabled the detection and quantitation of latent and heterodimeric forms in a commercial AT preparation stored at room temperature for three weeks.

Calcium ions (Ca2+) are versatile messengers that need to be tidily regulated in time and space in order to create a large number of signals. The coupling between Ca2+ entry and Ca2+ refilling is playing a central role in this Ca2+ homeostasis. Since the capacitative Ca2+ entry has been described, different mechanisms have been proposed in order to explain how the Ca2+ entry could be under control of intracellular store Ca2+ depletion. Today, in addition of STIM1 and Orai1, the two major elements of SOCe, increasing attention is put on the role of the transient receptor potential canonical (TRPC), that can form protein clusters with Orai1, and Sarco/endoplasmic reticulum Ca2+ATPases (SERCAs), that refill the stores and are also located in the same environment than SOC clusters. Altogether, these proteins elaborate either Ca2+ microdomains in the vicinity of the membrane or larger Ca2+ increases overtaking the whole cell. The coupling between Ca2+ entry and Ca2+ refilling can possibly act much further away from the plasma membrane. Ca2+, uptaken by SERCAs, have been described to move faster and further in the ER than in the cytosol and to create specific signal that depends on Ca2+ entry but at longer distance from it. The complexity of such created Ca2+ currents resides in the heteromeric nature of channels as well as the presence of different intracellular stores controlled by SERCA2b and SERCA3, respectively. A role for mitochondria has also been explored. To date, mitochondria are other crucial compartments that play an important role in Ca2+ homeostasis. Although mitochondria mostly interact with intracellular stores, coupling of Ca2+ entry and mitochondria cannot be completely rule out.

Antithrombin (AT) is a human plasma glycoprotein that possesses anticoagulant and anti-inflammatory properties. However, the native (active) form of AT is unstable and undergoes conformational changes, leading to latent, cleaved, and heterodimeric forms. The presence of these alternative forms mostly inactive can highly impact the quality and therapeutic activity of pharmaceutical AT preparations. We developed a capillary zone electrophoresis method, based on a neutral polyethylene oxide-coated capillary and a buffer close to physiological conditions, enabling the separation of more than eight forms of AT. Several peaks were identified as native, latent, and heterodimeric forms. The CZE method was reproducible with intraday relative standard deviations less than 0.5 and 2% for migration times and peak areas, respectively. The method was applied to the comparison of AT preparations produced by five competitive pharmaceutical companies, and statistical tests were performed. Important differences in the proportion of each form were highlighted. In particular, one AT preparation was shown to contain a high quantity of heterodimer, and two preparations contained high quantities of latent form. In addition, one AT preparation exhibited additional forms, not yet identified.

We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin γ-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (GlaFII-PS) or in PS deleted of the thrombin-sensitive region (TSR) (GlaFII-ΔTSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solvent-exposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into GlaFII-PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC.

Introduction&Aim Hemophilia A and B are X-linked disorders caused by an absence or a reduction in coagulation FVIII and respectively, FIX. Patients with hemophilia often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis. Here, we investigated whether targeting protein S (PS) could promote hemostasis in hemophilia by re-balancing coagulation. PS is a natural anticoagulant, acting as non-enzymatic cofactor for activated protein C (APC) in the inactivation of FVa and FVIIIa, and for tissue factor pathway inhibitor (TFPI) in the inhibition of FXa. This dual role makes PS a key regulator of the inhibition of thrombin generation (TG). Methods & Results Hemophilic (F8-/- or F9-/-) Pros1+/- mice were intercrossed. F8-/-Pros1-/- and F9-/- Pros1-/- mice were viable and found at the expected Mendelian frequency with no increased mortality compared to hemophilic mice. F8-/-Pros1-/- mice did not show DIC onset neither an increased mortality once challenge with tissue factor (TF). Ex vivo evaluation of TG potential showed that F8-/-Pros1-/- mice were APC-resistant, they had an improved low TF induced-TG and a 20% more dense fibrin network with larger fibrin fibers diameter as compared to F8-/-mice. Comparable results were found in human HA plasma where blocking PS raised TG even in the presence of high inhibitor titer. To assess the in vivo effect of PS inhibition on HA mice hemostasis, two tail- clipping (TC) assays were used. In both, mild TC model (2 mm cut) and severe TC (4 mm cut), blood loss significantly decreased in F8-/-Pros1-/- compared to F8- /- mice (mild TC: 407±21 vs 661±29ul, p As recurrent joint bleeding is the most common manifestation of HA, we challenged F8-/-Pros1-/- mice in an acute hemarthrosis (AH) model. Joint swelling 72 hours after injury was reduced in F8-/-Pros1-/- compared to F8-/- (0.11±0.03 vs 1.02±0.07mm, p To understand the improved phenotype of F8-/-Pros1-/- after AH, the function of macrophages were investigated. At the steady state, F8-/-Pros1-/- presented significantly 2-fold increased levels of inflammatory macrophages (M1) than in F8-/- mice. In addition bone marrow derived macrophages from F8-/-Pros1-/- exhibit 10-fold higher RBC phagocytic activity than F8-/- . Preliminary results indicate an increase of a monocyte attractant MCP-1 level, in knee lavage after AH in F8-/-Pros1-/- than F8-/- mice. Conclusion These data provide the first evidence that blocking PS has the ability to ameliorate hemophilia as judged by in vivo improvement of bleeding phenotype in the TC assay as well as in the AH model, suggesting a new valuable tool for hemophilia therapy. In addition, the modulable presence of PS and TFPI in human and mice joints is a novel pathophysiological aspect of hemarthrosis and constitutes a potential therapeutical target. Disclosures Kremer Hovinga: NovoNordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL-Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Growth arrest-specific gene 6 (Gas6) is expressed in antigen-presenting cells and endothelial cells (ECs) but not in T cells. When wild-type (WT) or Gas6−/− mice received allogeneic non–T cell–depleted bone marrow cells, hepatic graft-versus-host disease (GVHD) was alleviated in Gas6−/− recipients regardless of donor genotype, but not in WT recipients. T-cell infiltration was more prominent and diffuse in WT than in Gas6−/− recipients' liver. When mice received 0.5 × 106 allogeneic T cells with T cell–depleted allogeneic bone marrow, clinical signs indicated that GVHD was less severe in Gas6−/− than in WT recipients, as shown by a significant improvement of the survival and reduced liver GVHD. These data demonstrate that donor cells were not involved in the protection mechanism. In addition, lack of Gas6 in antigen-presenting cells did not affect WT or Gas6−/− T-cell proliferation. We therefore assessed the response of WT or Gas6−/− ECs to tumor necrosis factor-α. Lymphocyte transmigration was less extensive through Gas6−/− than WT ECs and was not accompanied by increases in adhesion molecule levels. Thus, the lack of Gas6 in ECs impaired donor T-cell transmigration into the liver, providing a rationale for considering Gas6 pathway as a potential nonimmunosuppressive target to minimize GVHD in patients receiving allogeneic hematopoietic stem cell transplantation.

Protein S (PS) homozygous deficiency causes purpura fulminans (PF) thought to result from the imbalance between procoagulant and anticoagulant factors.PS shares similarities with Growth Arrest Specific gene 6 (Gas6) but Gas6-/- mice display an antithrombotic phenotype whereas complete PS deficient (ProS1-/-) mice have a thrombotic phenotype. Beyond their role in coagulation both Gas6 and PS have cellular functions through tyrosine kinase receptors TAM. Gas6 plays also a role in erythropoiesis and previous data suggest that ProS1-/- embryos are anemic. To mimic severe acquired PS deficiency and thus monitor PF development in adult mice, we induced Pros1 gene silencing via poly I:C-inducible Mx1-Cre+ mice. Once induced, ProS1lox/loxMx1 Cre+ and ProS1lox/- Mx1Cre+ mice showed a marked reduction of plasma PS levels compared with ProS1lox/lox mice (47.7±6% ProS1lox/loxMx1 Cre+; 14.5±7% ProS1lox/-Mx1 Cre+, p As a second strategy to reduce PS, we treated ProS1+/- and ProS1+/+ adult mice with warfarin (0.8mg/day, during 5 days), and the treatment induced 75% mortality in ProS1+/- and 5% in WT mice (p Because Gas6-/- mice are protected against thrombosis, our third strategy was to generate a mouse model combining ProS1 and Gas6 deficiency to overcome the ProS1-/- lethal phenotype. Surprisingly, embryonic mortality, due to massive thrombosis with fibrin deposition, occurred earlier in ProS1-/- Gas6-/- embryosthan in ProS1-/- embryos. RBC extravasation observed during warfarin treatment pointed to a vascular wall involvement during PF. Consequently, we investigated the vasculature using E15 embryonic dorsal skin. Staining with anti-VE-Cadherin and anti-Ter119 antibodies (Ab) confirmed RBC extravasation while anti-CD31 Ab showed areas with underdeveloped and less dense vascular networks, collapsed vessels and less vessels branch points in ProS1-/- andProS1-/-Gas6-/- than in WT mice. Massively enlarged lymphatic vessels and increased macrophages infiltration were observed in ProS1-/- andProS1-/-Gas6 -/- embryos by anti-Lyve1 and F4/80 Ab. Histology of blood vessels and liver from ProS1-/- and ProS1-/- Gas6-/- embryos revealed a high number of circulating immature RBC compatible with increased erythropoiesis because of severe bleeding due to consumption coagulopathy and vascular damage. In addition, numerous isolated erythroblast nuclei were found in these embryos indicating altered phagocytosis. Moreover, preliminary data showed higher cytokine levels in ProS1-/- than in WT embryos. Exclusively in ProS1-/- Gas6-/-, we found a very high level of hypochromic erythrocytes suggesting troubled iron metabolism linked to inflammation and iron recycling. To investigate potential erythropoietic defects, fetal liver (E14.5) single-cell suspensions were cultured for colony forming assays. No difference was found in BFU-E colonies from ProS1-/- and WT embryos. In contrast, BFU-E colonies were 2-fold reduced in ProS1-/-Gas6-/- compared toProS1+/+ Gas6-/- embryos. These data were consistent with our previous observations on the role of Gas6 in erythropoiesis. Both ProS1-/- and ProS1-/-Gas6-/- displayed 50% less CFU-E colonies than WT embryos, pointing to a blunted response to erythropoietin (EPO) in the context of inflammation promoted by PF. These results were confirmed by flow cytometry. In conclusion, with 14.5% plasmaticPS, no PF lesions were observed in adult mice. Gas6 deficiency did not overcome lethality in mice homozygous for PS deficiency. PF developed during warfarin treatment and in ProS1-/- andProS1-/-Gas6-/- embryos which showed thrombosis occurring together with vascular wall damage and inflammatory processes that appeared to impact iron metabolism and response to EPO. Disclosures No relevant conflicts of interest to declare.

Growth arrest-specific gene 6 (gas6) product enhances the formation of stable platelet macroaggregates in response to various agonists. To determine whether Gas6 amplifies the response to known platelet agonists through one or more of its receptor tyrosine kinases of the Tyro3 family, mice deficient in any one of the Gas6 receptors (Gas6-Rs: Tyro3, Axl, or Mer) were submitted to thrombosis challenge and their platelet function. The loss of any one of the Gas6-Rs protects mice against thromboembolism induced by collagen-epinephrine and stasis-induced thrombosis. Importantly, these mice do not suffer spontaneous bleeding and have a normal bleeding time but a tendency to repetitively re-bleed after transient hemostasis. Re-bleeding in mice lacking any one of the Gas6-Rs is not due to thrombocytopenia or coagulopathy but to a platelet dysfunction characterized by a lack of the second wave of platelet aggregation and an impaired clot retraction, at least in part by reducing outside-in alpha(IIb)beta(3) signaling and platelet granule secretion. The early release of Gas6 by agonists perpetuates platelet activation through its three receptors, reinforcing outside-in alpha(IIb)beta(3) signaling by activation of PI3K and Akt signaling and stimulation of tyrosine phosphorylation of the beta(3) integrin. Furthermore, "trapping" Gas6 prevents pathological thrombosis, which indicates that blocking this novel cross-talk between the Gas6-Rs and alpha(IIb)beta(3) integrin may constitute a novel target for antithrombotic therapy.

Introduction Protein S (ProS) is a natural anticoagulant, acting as non-enzymatic cofactor for activated Protein C (APC) in the inactivation of activated factor V (FV) and factor VIII (FVIII), and for Tissue Factor Pathway Inhibitor (TFPI) in the inhibition of activated factor X (FXa). This dual role makes ProS a key regulator of the inhibition of thrombin generation as suggested by the phenotypes of ProS-deficient patients: venous thromboembolism at adult age for heterozygous patients and massive disseminated intravascular coagulation (DIC) incompatible with life for homozygous. Hemophilia A (HA) is an X-linked disorder caused by mutations in the F8gene, leading to deficiency or absence of FVIII, an essential component of the intrinsic pathway of blood coagulation. HA patients suffer from spontaneous excessive bleeding episodes affecting soft tissue, joints and muscles. Currently, recombinant factor FVIII (rFVIII) is the first line of HA therapy. Unfortunately, a major drawback observed in 30% of HA patients, is the generation of inhibitory alloantibodies that abrogate rFVIII activity, rendering the therapy ineffective. Another disadvantage is the short half-life of rFVIII. Aims To enhance thrombin production in HA by specific blockage of ProS, thus overcoming the problems of rFVIII therapy. Lack of ProS cofactor activities on APC and TFPI might increase the generation of FXa, thus compensating for FVIII deficiency. Methods & Results HA mice are useful models to probe the effectiveness of procoagulant therapeutics as they recapitulate aspects of the human disease. Taking advantage from the availability of HA and ProS+/- mice, we aimed at generating ProS and FVIII double knock-out mice (HA-ProS-/-). To date ProS-/- mice were not viable and died in utero between E15.5-17.5 due to severe intracranial hemorrhages and consumptive coagulopathy. Surprisingly, by mating HA-ProS+/- mice we were able to rescue the lethal ProS-/- embryonic phenotype: HA-ProS-/-mice were viable and were found at the expected Mendelian frequency (45/192 neonates, 25%) with no increased mortality compared to HA mice. Plasma levels of FVIII and ProS further confirmed that HA-ProS-/- mice had comparable low FVIII and extremely reduced ProS compared to HA mice (1.8±0.5 vs 93.8±15.7 % respectively P=0.001) (mean±SEM). Analysis of complete blood counts established that these animals had a normal blood cell profile. Moreover, no differences were found in FV or FIX activity and in APTT and PT between HA and HA-ProS-/- mice. A decrease in fibrinogen levels in HA-ProS-/-when compared to HA mice (1.3±0.1 vs 1.7±0.1 g/L respectively, P< 0.001) was observed. To assess the in vivo effect of ProS-/- on HA mice hemostasis, a tail-clipping assay was used. In particular, blood loss assayed as blood volume lost over 10 min following a 4-mm tail transection significantly decreased to 158±11 uL (n=6) in HA-ProS-/- mice compared to HA and HA-ProS+/- (247 ±19 uL, n=10 and 239±11 uL, n=8 respectively, P As recurrent joint bleeding is the most common manifestation of HA resulting in arthropathy, we challenged HA-ProS-/- in an acute hemarthrosis model. Joint swelling assessed as the ratio between punctured and non-punctured knees showed that HA-ProS-/- reached the biggest swelling at 24h (1.09±0.03, n=7 vs 1.28±0.08, n=7 HA) but remained stable until 72h in contrast to HA mice (1.11±0.02, n=7 vs 1.48±0.13, n=7 HA; P=0.018). Moreover, a relevant decrease of joint bleeding in HA-ProS-/- mice (0.5±0.2 vs 2.67±0.17; P Conclusion These data provide the first evidence that blocking ProS has the ability to ameliorate HA as judged by in vivo improvement of bleeding phenotype in the tail clipping assay as well as in the acute hemarthrosis model. Strikingly, total FVIII deficiency does rescue the embryonic lethal phenotype of ProS-/- embryos and allows their survival without overt DIC. Together these results indicate that specific targeting of ProS favors the extrinsic pathway overcoming the intrinsic pathway deficiency, suggesting a new valuable tool for hemophilia therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 529 Protein S (ProS) is an important negative regulator of blood coagulation. Its physiological importance is evident in purpura fulminans and other life-threatening thrombotic disorders typical of ProS deficient patients. Our previous characterization of ProS deficiency in mouse models has shown similarities with the human phenotypes: heterozygous ProS-deficient mice (Pros+/−) had increased thrombotic risk whereas homozygous deficiency in ProS (Pros−/−) was incompatible with life (Blood 2009; 114:2307-2314). In tissues, ProS exerts cellular functions by binding to and activating tyrosine kinase receptors of the Tyro3 family (TAM) on the cell surface. To extend the analysis of coagulation defects beyond the Pros−/− phenotype and add new insights into the sites of synthesis ProS and its action, we generated mice with inactivated ProS in hepatocytes (Proslox/loxAlbCre+) as well as in endothelial and hematopoietic cells (Proslox/loxTie2Cre+). Both models resulted in significant reduction of circulating ProS levels and in a remarkable increased thrombotic risk in vivo. In a model of tissue factor (TF)-induced venous thromboembolism (VTE), only 17% of Proslox/loxAlbCre+ mice (n=12) and only 13% of Proslox/loxTie2Cre+ mice (n=14) survived, compared with 86% of Proslox/lox mice (n=14; P To mimic a severe acquired ProS deficiency, ProS gene was inactivated at the adult stage using the polyI:C-inducible Mx1-Cre system (Proslox/loxMx1Cre+). Ten days after polyI:C treatment, Proslox/loxMx1Cre+ mice developed disseminated intravascular coagulation with extensive lung and liver thrombosis. It is worth noting that no skin lesions compatible with purpura fulminans were observed in any of the above-described models of partial ProS deficiency. In order to shed light on the pathogenesis of purpura fulminans, we exposed the different ProS-deficient mice to warfarin (0.2 mg/day). We observed that Pros+/−, Proslox/loxAlbCre+ and Proslox/loxTie2Cre+ mice developed retiform purpura (characterized by erythematous and necrotic lesions of the genital region and extremities) and died after 3 to 5 days after the first warfarin administration. In human, ProS is also synthesized by megakaryocytes and hence stored at high concentrations in circulating platelets (pProS). The role of pProS has been investigated by generating megakaryocyte ProS-deficient model using the PF4 promoter as Cre driver (Proslox/loxPf4Cre+). In the TF-induced VTE model, Proslox/loxPf4Cre+ (n=15) mice showed a significant increased risk of thrombosis compared to Proslox/lox controls (n=14; survival rate 47% and 86%, respectively; P We further investigated the ability of ProS to function as a ligand of TAM receptors, by using homozygous and heterozygous deficient mice for both the TAM ligands ProS and Gas6. Gas6−/−Pros−/− mice died in utero and showed comparable dramatic bleeding and thrombotic phenotype as described for Pros−/− embryos. In conclusion, like complete ProS deficiency, double deficiency in ProS and Gas6 was lethal, whereas partial ProS deficiency was not. Mice partially deficient in ProS displayed a prothrombotic phenotype, including those with only deficiency in pProS. Purpura fulminans did not occur spontaneously in mice with partial Pros deficiency but developed upon warfarin administration. Thus, the use of different mice models of ProS deficiency can be instrumental in the study of its highly variable thrombotic phenotype and in the investigation of additional roles of ProS in inflammation and autoimmunity through TAM signaling. Disclosures: No relevant conflicts of interest to declare.

Gas6 binding to its cognate receptor tyrosine kinases (Tyro3, Axl and Mer) down-regulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. Mer activation inhibits TNF-α production by macrophages and alleviates endotoxic shock in mice. Gas6 receptors are cleaved in the extracellular domain to generate soluble receptors. Soluble receptors function as decoy receptors, thereby inhibiting the Gas6 interaction with cell-associated receptors. The aim of the study was to determine whether Gas6 pathway plays a role in sepsis in human and mice. In the first part of the study, we measured plasma levels of Gas6 and its soluble receptors sTyro3 and sAxl in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with acute non-infectious inflammatory diseases. Gas6 and sAxl concentrations were higher in septic patients than in healthy subjects or in patients with non-infectious inflammatory disease (P≤ 0.0001 for Gas6 and