The endothelial cell receptor stabilin-2 regulates VWF-FVIII complex half-life and immunogenicity
Laura L. Swystun, Jesse D. Lai, Colleen Notley, Ilinca Georgescu, A. Simonne Paine, Jeff Mewburn, Kate Nesbitt, Kai Schledzewski, Cyrill Géraud, Julia Kzhyshkowska, Sergij Goerdt, Wilma Hopman, Robert R. Montgomery, Paula D. James, David Lillicrap
Laura L. Swystun, Jesse D. Lai, Colleen Notley, Ilinca Georgescu, A. Simonne Paine, Jeff Mewburn, Kate Nesbitt, Kai Schledzewski, Cyrill Géraud, Julia Kzhyshkowska, Sergij Goerdt, Wilma Hopman, Robert R. Montgomery, Paula D. James, David Lillicrap
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Research Article Genetics Hematology

The endothelial cell receptor stabilin-2 regulates VWF-FVIII complex half-life and immunogenicity

Abstract

Quantitative abnormalities of the von Willebrand factor–factor VIII (VWF-FVIII) complex associate with inherited bleeding or thrombotic disorders. Receptor-mediated interactions between plasma VWF-FVIII and phagocytic or immune cells can influence their hemostatic and immunogenic activities. Genetic association studies have demonstrated that variants in the STAB2 gene, which encodes the scavenger receptor stabilin-2, associate with plasma levels of VWF-FVIII. However, the mechanistic basis and pathophysiological consequences of this association are unknown. We have demonstrated that stabilin-2–expressing cells bind and internalize human VWF and FVIII in a VWF-dependent manner, and stabilin-2–deficient mice displayed prolonged human VWF-FVIII half-life compared with controls. The stabilin-2 variant p.E2377K significantly decreased stabilin-2 expression and impaired VWF endocytosis in a heterologous expression system, and common STAB2 variants associated with plasma VWF levels in type 1 von Willebrand disease patients. STAB2-deficient mice displayed a decreased immunogenic response to human VWF-FVIII complex, while coinfusion of human VWF-FVIII with the stabilin-2 ligand hyaluronic acid attenuated the immune response to exogenous FVIII. Collectively, these data suggest that stabilin-2 functions as both a clearance and an immunoregulatory receptor for VWF-FVIII, making stabilin-2 a novel molecular target for modification of the half-life of VWF-FVIII and the immune response to VWF-FVIII concentrates.

Authors

Laura L. Swystun, Jesse D. Lai, Colleen Notley, Ilinca Georgescu, A. Simonne Paine, Jeff Mewburn, Kate Nesbitt, Kai Schledzewski, Cyrill Géraud, Julia Kzhyshkowska, Sergij Goerdt, Wilma Hopman, Robert R. Montgomery, Paula D. James, David Lillicrap

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Figure 6

Genetic and biochemical regulation of VWF interactions with stabilin-2.

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Genetic and biochemical regulation of VWF interactions with stabilin-2. ...
HEK 293 cells were transiently transfected with the human stabilin-2 cDNA and incubated with 2 U/ml human pdVWF or 2 U/ml human pdVWF pretreated with PNGase F to remove its N-linked glycans (de-N VWF). (A) Quantification of de-N VWF binding to stabilin-2–expressing cells. (B and C) Human pdVWF and de–N-glycosylated VWF (green) binding to human stabilin-2–expressing (red) cells. Images are representative of n = 6 independent experiments. Scale bars: 40 μm. (D) The influence of VWF N-linked glycans on the clearance of VWF by stabilin-2 was characterized in vivo (n = 8 animals per condition). See statistical summary of half-life studies in Table 1. The stabilin-2 variant p.E2377K was introduced into the human stabilin-2 cDNA by site-directed mutagenesis. (E and F) HEK 293 cells were transiently transfected with the stabilin-2 cDNA p.E2377K 1:1 with WT (E, red) or p.E2377K stabilin-2 alone (F, red) and incubated with 2 U/ml human pdVWF (green). Images are representative of n = 4 independent experiments; scale bars: 40 μm. For all experiments, blue indicates DAPI; and yellow, colocalization. (G) Quantification of VWF binding to HEK 293 cells expressing the p.E2377K stabilin-2 variant. (H) Flow cytometric analysis of p.E2377K stabilin-2 expression. Pathogenicity assessment of the stabilin-2 p.E2377K variant is described in Table 2. To demonstrate competitive binding to stabilin-2, known stabilin-2 ligands were preincubated with HEK 293 cells expressing murine stabilin-2 for 15 minutes and then incubated with 2 U/ml human pdVWF for 1 hour. (I) Quantitative IF was performed to compare untreated VWF binding to cells pretreated with stabilin-2 ligands (n = 3–4 independent experiments). HA, hyaluronic acid; DS, dermatan sulfate; UFH, unfractionated heparin. Throughout figure, *P < 0.05, **P < 0.001 as determined by t test.