Molecular mechanism and RNAi correction of a dominantnegative von Willebrand Factor gene deletion.

Understanding molecular mechanisms leading to the dominant inheritance of von Willebrand disease (VWD) would improve our knowledge on pathophysiological aspects underlying its high prevalence. We produced a cellular model of severe type 2 VWD, caused by an heterozygous deletion in the VWF gene, to investigate the altered biosynthesis. Co-expression of the VWF inframe deleted cDNA (p.P1105_C1926delinsR) impaired wild type vector-driven protein secretion and function (VWF collagen-binding 1.9 ± 0,5% of wt), which mimicked the patient’s phenotype. Protein studies and cell immunostaining delineated the highly efficient dominant-negative mechanism. The deleted VWF was synthesized in large amounts and preferentially processed, and through a correctly encoded cysteine knot domain formed heterodimers and heterotetramers with wild type VWF. Impaired multimerization was associated with reduced amounts of VWF in late endosomes. The key role of heterodimers as multimer terminators was further supported by introduction of the dimerization mutation C2773R in the deleted construct, which resulted in a quantitative defect with normal multimer size. Targeting the mRNA breakpoint by siRNA selectively inhibited the in-frame deleted VWF expression, and restored secretion of functional VWF (28.0 ± 3.3% of wt). This provided a novel tool to explore mutation-specific gene therapy in a severe form of dominant VWD

Pigmented Nodular Basal Cell Carcinomas in Differential Diagnosis with Nodular Melanomas: Confocal Microscopy as a Reliable Tool for In Vivo Histologic Diagnosis

Nodular basal cell carcinoma, especially when pigmented, can be in differential diagnosis with nodular melanomas, clinically and dermoscopically. Reflectance confocal microscopy is a relatively new imaging technique that permits to evaluate in vivo skin tumors with a nearly histological resolution. Here, we present four cases of challenging nodular lesions where confocal microscopy was able to clarify the diagnosis

CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in MiceSignificance

Platelets are important to the development and progression of atherosclerotic lesions. However, relatively little is known about the contribution of platelet signaling to this pathological process. Our recent work identified two independent, yet synergistic signaling pathways that lead to the activation of the small GTPase Rap1; one mediated by the guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), the other by P2Y12, a platelet receptor for ADP and the target of anti-platelet drugs. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr−/−) mice lacking CDGI and/or P2Y12 in hematopoietic cells

Desialylation of <i>O</i>-glycans on glycoprotein Ibα drives receptor signaling and platelet clearance

During infection neuraminidase desialylates platelets and induces their rapid clearance from circulation. The underlying molecular basis, particularly the role of platelet glycoprotein (GP)Ibα therein, is not clear. Utilizing genetically altered mice we report that the extracellular domain of GPIbα, but neither von Willebrand factor nor ADAM17 (a disintegrin and metalloprotease 17), is required for platelet clearance induced by intravenous injection of neuraminidase. Lectin binding to platelets following neuraminidase injection over time revealed that the extent of desialylation of O-glycans correlates with the decrease of platelet count in mice. Injection of α2,3-neuraminidase reduces platelet counts in wild-type but not in transgenic mice expressing only a chimeric GPIbα that misses most of its extracellular domain. Neuraminidase treatment induces unfolding of the O-glycosylated mechanosensory domain in GPIbα as monitored by single-molecule force spectroscopy, increases the exposure of the ADAM17 shedding cleavage site in the mechanosensory domain on the platelet surface, and induces ligand-independent GPIb-IX signaling in human and murine platelets. These results suggest that desialylation of O-glycans of GPIbα induces unfolding of the mechanosensory domain, subsequent GPIb-IX signaling including amplified desialylation of N-glycans, and eventually rapid platelet clearance. This new molecular mechanism of GPIbα-facilitated clearance could potentially resolve many puzzling and seemingly contradicting observations associated with clearance of desialylated or hyposialylated platelets

RASA3 is a critical inhibitor of RAP1-dependent platelet activation

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders

In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments

VON WILLEBRAND FACTOR ABNORMALITIES STUDIED IN THE MOUSE MODEL: WHAT WE LEARNED ABOUT VWF FUNCTIONS

Up until recently, von Willebrand Factor (VWF) structure-function relationships have only been studied through in vitro approaches. A powerful technique known as hydrodynamic gene transfer, which allows transient expression of a transgene by mouse hepatocytes, has led to an important shift in VWF research. Indeed this approach has now enabled us to transiently express a number of VWF mutants in VWF-deficient mice in order to test the relative importance of specific residues in different aspects of VWF biology and functions in an in vivo setting. As a result, mice reproducing various types of von Willebrand disease have been generated, models that will be useful to test new therapies. This approach also allowed a more precise identification of the importance of VWF interaction with subendothelial collagens and with platelets receptors in hemostasis and thrombosis. The recent advances gathered from these studies as well as the pros and cons of the technique will be reviewed here