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The C-type lectin receptor CLEC4M binds, internalizes, and clears von Willebrand factor and contributes to the variation in plasma von Willebrand factor levels.

ABSTRACT: Genetic variation in or near the C-type lectin domain family 4 member M (CLEC4M) has been associated with plasma levels of von Willebrand factor (VWF) in healthy individuals. CLEC4M is a lectin receptor with a polymorphic extracellular neck region possessing a variable number of tandem repeats (VNTR). A total of 491 participants (318 patients with type 1 von Willebrand disease [VWD] and 173 unaffected family members) were genotyped for the CLEC4M VNTR polymorphism. Family-based association analysis on kindreds with type 1 VWD demonstrated an excess transmission of VNTR 6 to unaffected individuals (P = .0096) and an association of this allele with increased VWF:RCo (P = .029). CLEC4M-Fc bound to VWF. Immunofluorescence and enzyme-linked immunosorbent assay demonstrated that HEK 293 cells transfected with CLEC4M bound and internalized VWF. Cells expressing 4 or 9 copies of the CLEC4M neck region VNTR showed reduced interaction with VWF relative to CLEC4M with 7 VNTR (CLEC4M 4%-60% reduction, P < .001; CLEC4M 9%-45% reduction, P = .006). Mice expressing CLEC4M after hydrodynamic liver transfer have a 46% decrease in plasma levels of VWF (P = .0094). CLEC4M binds to and internalizes VWF, and polymorphisms in the CLEC4M gene contribute to variable plasma levels of VWF.

SUBMITTER: Rydz N

PROVIDER: S-EPMC3722568 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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The C-type lectin receptor CLEC4M binds, internalizes, and clears von Willebrand factor and contributes to the variation in plasma von Willebrand factor levels.

Rydz Natalia N Swystun Laura L LL Notley Colleen C Paterson Andrew D AD Riches J Jacob JJ Sponagle Kate K Boonyawat Boonchai B Montgomery Robert R RR James Paula D PD Lillicrap David D

Blood 20130325 26

Genetic variation in or near the C-type lectin domain family 4 member M (CLEC4M) has been associated with plasma levels of von Willebrand factor (VWF) in healthy individuals. CLEC4M is a lectin receptor with a polymorphic extracellular neck region possessing a variable number of tandem repeats (VNTR). A total of 491 participants (318 patients with type 1 von Willebrand disease [VWD] and 173 unaffected family members) were genotyped for the CLEC4M VNTR polymorphism. Family-based association analy ...[more]

PMID: 23529928

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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:Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY:Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.

Dataset Information

The C-type lectin receptor CLEC4M binds, internalizes, and clears von Willebrand factor and contributes to the variation in plasma von Willebrand factor levels.

Publications

The C-type lectin receptor CLEC4M binds, internalizes, and clears von Willebrand factor and contributes to the variation in plasma von Willebrand factor levels.

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