Complex formation with pentraxin-2 regulates factor X plasma levels and macrophage interactions.

Recently, we have identified scavenger receptor class A member I (SR-AI) as a receptor for coagulation factor X (FX), mediating the formation of an FX reservoir at the macrophage surface. Here, we demonstrate that the FX/SR-AI-complex comprises a third protein, pentraxin-2 (PTX2). The presence of PTX2 is essential to prevent internalization of FX by SR-AI, and the presence of FX is needed to interfere with internalization of PTX2. Binding studies showed that FX, SR-AI, and PTX2 independently bind to each other (KD,app: 0.2-0.7 µM). Surprisingly, immunoprecipitation experiments revealed that FX and PTX2 circulate as a complex in plasma, and complex formation involves the FX activation peptide. No binding of PTX2 to other vitamin K-dependent proteins was observed. Short hairpin RNA-mediated inhibition of PTX2 levels in mice resulted not only in reduced levels of PTX2, but also in similarly reduced FX levels. Moreover, PTX2 and FX levels were correspondingly reduced in SR-AI-deficient mice. Analysis of 71 human plasma samples uncovered a strong correlation between FX and PTX2 plasma levels. Furthermore, plasma samples of patients with reduced FX levels (congenital/acquired FX deficiency or after anti-vitamin K treatment) were characterized by concomitantly decreased PTX2 levels. In conclusion, we identified PTX2 as a novel partner for FX, and both proteins cooperate to prevent their SR-AI-mediated uptake by macrophages. Interestingly, their respective plasma levels are interdependent. These findings seem of relevance in perspective of ongoing clinical trials, in which plasma depletion of PTX2 is used as a therapeutical approach in the management of systemic amyloidosis.

Macrophage receptor SR-AI is crucial to maintain normal plasma levels of coagulation factor X.

Beside its classical role in the coagulation cascade, coagulation factor X (FX) is involved in several major biological processes including inflammation and enhancement of virus-induced immune responses. We recently reported that the long circulatory half-life of FX is linked to its interaction with liver-resident macrophages. Importantly, we now observed that macrophages, but not undifferentiated monocytes, support this interaction. Using cell biology approaches with primary and THP1-derived macrophages as well as transfected cells, we further identified the scavenger receptor type A member I (SR-AI) to be a macrophage-specific receptor for FX. This result was confirmed using SR-AI-deficient mice, which exhibit reduced circulating levels of FX in vivo and loss of FX-macrophage interactions in vitro. Binding studies using purified proteins revealed that FX binds specifically (half-maximal binding, 3 µg/mL) to the extracellular domain of SR-AI. Altogether, we demonstrate that macrophages regulate FX plasma levels in an SR-AI-dependent manner.

A liver-targeting Cu(i) chelator relocates Cu in hepatocytes and promotes Cu excretion in a murine model of Wilson's disease.

Copper chelation is the most commonly used therapeutic strategy nowadays to treat Wilson's disease, a genetic disorder primarily inducing a pathological accumulation of Cu in the liver. The mechanism of action of Chel2, a liver-targeting Cu(i) chelator known to promote intracellular Cu chelation, was studied in hepatic cells that reconstitute polarized epithelia with functional bile canaliculi, reminiscent of the excretion pathway in the liver. The interplay between Chel2 and Cu localization in these cells was demonstrated through confocal microscopy using a fluorescent derivative and nano X-ray fluorescence. The Cu(i) bound chelator was found in vesicles potentially excreted in the canaliculi. Moreover, injection of Chel2 either intravenously or subcutaneously to a murine model of Wilson's disease increased excretion of Cu in the faeces, confirming in vivo biliary excretion. Therefore, Chel2 turns out to be a possible means to collect and excrete hepatic Cu in the faeces, hence restoring the physiological pathway.

Evaluation of the accuracy of exchangeable copper and relative exchangeable copper (REC) in a mouse model of Wilson's disease.

Wilson's disease (WD) is caused by mutations in the ATP7B gene responsible for a toxic copper overload mainly in the liver and the central nervous system. Phenotypic heterogeneity may challenge the diagnostic confirmation. Exchangeable copper (CuEXC) has recently been proposed as a new marker of WD, and its ratio to the total serum copper (Cus), Relative Exchangeable Copper (REC = CuEXC/Cus), as a diagnostic marker. This study aimed to investigate whether this could be confirmed in Atp7b-/- mice, an engineered WD animal model. Atp7b-/- (n = 137) and wild type (WT; n = 101) mice were investigated under the same conditions at 6-8, 20, 39, or 50 weeks of age. Twenty-four Atp7b-/- mice received D-penicillamine treatment from 39 to 50 weeks of age. Serum and liver [histology and intrahepatic copper (IHCu)] data were evaluated. In the WT group, all serum and liver data were normal. Atp7b-/- livers developed a chronic injury from isolated moderate inflammation (6-8 weeks: 16/33 = 48%) to inflammatory fibrosis with cirrhosis (50 weeks: 25/25 = 100% and 16/25 = 64% respectively). Cus and CuEXC increased until week 39, whereas IHCu and REC were stable with increasing age and much higher than in WT mice (mean ±â€¯SD: 669 ±â€¯269 vs. 13 ±â€¯3 µg/g dry liver and 39 ±â€¯12 vs. 11 ±â€¯3%, respectively). A threshold value of 20% for REC provided a diagnostic sensitivity and specificity of 100%, regardless of sex, age, or the use of D-penicillamine. Eleven weeks of 100 mg/kg D-penicillamine reduced liver fibrosis (p = 0.001), IHCu (p = 0.026) and CuEXC (p = 0.175). In conclusion, this study confirms REC as a WD diagnostic marker in a mouse model of chronic liver disease caused by copper overload. Further studies are needed to assess the usefulness of CuEXC to monitor the evolution of WD, particularly during treatment.

Apoptotic Platelet Events Are Not Observed in Severe von Willebrand Disease-Type 2B Mutation p.V1316M.

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.

Functional consequences of the over-expression of TRPC6 channels in HEK cells: impact on the homeostasis of zinc.

The canonical transient receptor potential 6 (TRPC6) protein is a non-selective cation channel able to transport essential trace elements like iron (Fe) and zinc (Zn) through the plasma membrane. Its over-expression in HEK-293 cells causes an intracellular accumulation of Zn, indicating that it could be involved in Zn transport. This finding prompted us to better understand the role played by TRPC6 in Zn homeostasis. Experiments done using the fluorescent probe FluoZin-3 showed that HEK cells possess an intracellular pool of mobilisable Zn present in compartments sensitive to the vesicular proton pump inhibitor Baf-A, which affects endo/lysosomes. TRPC6 over-expression facilitates the basal uptake of Zn and enhances the size of the pool of Zn sensitive to Baf-A. Quantitative RT-PCR experiments showed that TRPC6 over-expression does not affect the mRNA expression of Zn transporters (ZnT-1, ZnT-5, ZnT-6, ZnT-7, ZnT-9, Zip1, Zip6, Zip7, and Zip14); however it up-regulates the mRNA expression of metallothionein-I and -II. This alters the Zn buffering capacities of the cells as illustrated by the experiments done using the Zn ionophore Na pyrithione. In addition, HEK cells over-expressing TRPC6 grow slower than their parental HEK cells. This feature can be mimicked by growing HEK cells in a culture medium supplemented with 5 µM of Zn acetate. Finally, a proteomic analysis revealed that TRPC6 up-regulates the expression of the actin-associated proteins ezrin and cofilin-1, and changes the organisation of the actin cytoskeleton without changing the cellular actin content. Altogether, these data indicate that TRPC6 is participating in the transport of Zn and influences the Zn storage and buffering capacities of the cells.