Dataset Information

G protein-dependent basal and evoked endothelial cell vWF secretion.

ABSTRACT: von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-G?13(-/-);G?12(-/-) mice that could be normalized by infusion of human vWF. Blood from G?12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that G?12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in G?12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-G?q(-/-);G?11(-/-) and G?12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of G?12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein ? (?-SNAP), but not G?13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated G?12 promoted vWF secretion. In G?q, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that G?12 N-terminal residues 10-15 mediated the binding of G?12 to ?-SNAP, and an engineered ?-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of G?12 and G?q/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

SUBMITTER: Rusu L

PROVIDER: S-EPMC3894496 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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G protein-dependent basal and evoked endothelial cell vWF secretion.

Rusu Luiza L Andreeva Alexandra A Visintine David J DJ Kim Kyungho K Vogel Stephen M SM Stojanovic-Terpo Aleksandra A Chernaya Olga O Liu Guoquan G Bakhshi Farnaz R FR Haberichter Sandra L SL Iwanari Hiroko H Kusano-Arai Osamu O Suzuki Nobuchika N Hamakubo Takao T Kozasa Tohru T Cho Jaehyung J Du Xiaoping X Minshall Richard D RD

Blood 20130930 3

von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion r ...[more]

PMID: 24081657

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Project description:Essentials CLEC4M is an endocytic receptor for factor FVIII. CLEC4M interacts with FVIII in a VWF-dependent and independent manner. CLEC4M binds to mannose-containing glycans on FVIII. CLEC4M internalization of FVIII involves clathrin coated pits. SUMMARY: Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate in the plasma as a non-covalent complex, and the majority of FVIII is likely to be cleared by VWF-dependent pathways. Clearance of VWF-free FVIII is rapid and underlies the pathological basis of some quantitative FVIII deficiencies. The receptor pathways that regulate the clearance of VWF-bound and VWF-free FVIII are incompletely uncharacterized. The human liver-expressed endothelial lectin CLEC4M has been previously characterized as a clearance receptor for VWF, although its influence on FVIII is unknown. Objective The interaction between FVIII and CLEC4M was characterized in the presence or absence of VWF. Methods FVIII interactions with CLEC4M were evaluated by in vitro cell-based and solid phase binding assays. Interactions between FVIII and CLEC4M or liver sinusoidal endothelial cells were evaluated in vivo by immunohistochemistry. Results CLEC4M-expressing HEK 293 cells bound and internalized recombinant and plasma-derived FVIII through VWF-dependent and independent mechanisms. CLEC4M binding to recombinant FVIII was dependent on mannose-exposed N-linked glycans. CLEC4M mediated FVIII internalization via a clathrin-coated pit-dependent mechanism, resulting in transport of FVIII from early and late endosomes for catabolism by lysosomes. In vivo hepatic expression of CLEC4M after hydrodynamic liver transfer was associated with a decrease in plasma levels of endogenous murine FVIII:C in normal mice, whereas infused recombinant human FVIII was associated with sinusoidal endothelial cells in the presence or absence of VWF. Conclusions These findings suggest that CLEC4M is a novel clearance receptor that interacts with mannose-exposed glycans on FVIII in the presence or absence of VWF.

Project description:ObjectivesProuroguanylin (ProUGN) in the intestine is cleaved to form uroguanylin (UGN), which stimulates guanylate cyclase C (GUCY2C), inducing cyclic guanosine monophosphate signaling. Paracrine release regulates fluid secretion, contributing to bowel function, whereas endocrine secretion evoked by eating forms a gut-brain axis, controlling appetite. Whereas hormone insufficiency contributes to hyperphagia in obesity, its contribution to the pathophysiology of constipation syndromes remains unexplored. Here, we compared circulating ProUGN and UGN in healthy subjects and in patients with chronic idiopathic constipation (CIC) and patients with irritable bowel syndrome with constipation (IBS-C).MethodsCirculating ProUGN and UGN levels were measured in 60 healthy subjects, 53 patients with CIC, and 54 patients with IBS-C. After an overnight fast, the participants ingested a standardized meal; blood samples were drawn at fasting and at 30, 60, and 90 minutes thereafter, and hormone levels were quantified by enzyme-linked immunosorbent assay.ResultsFasting ProUGN levels were >30% lower in patients with CIC and those with IBS-C compared with healthy subjects regardless of age, sex, or disease state. After eating, ProUGN levels increased compared with fasting levels, although the rate of change was slower and maximum levels were lower in patients with CIC and those with IBS-C. Similarly, fasting UGN levels were lower in patients with CIC and those with IBS-C compared with healthy subjects. However, unlike ProUGN levels, UGN levels did not increase after eating.DiscussionThese observations support a novel pathophysiologic model in which CIC and IBS-C reflect a contribution of ProUGN insufficiency dysregulating intestinal fluid and electrolyte secretion.Translational impactThis study suggests that CIC and IBS-C can be treated by oral GUCY2C hormone replacement. Indeed, these observations provide a mechanistic framework for the clinical utility of oral GUCY2C ligands like plecanatide (Trulance) and linaclotide (Linzess) to treat CIC and IBS-C.

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G protein-dependent basal and evoked endothelial cell vWF secretion.

Publications

G protein-dependent basal and evoked endothelial cell vWF secretion.

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