Shear stress‐independent binding of von Willebrand factor‐type 2B mutants p.R1306Q & p.V1316M to LRP1 explains their increased clearance

Abstract

Summary. Background: von Willebrand factor (VWF) is cleared in a shear stress-and macrophage-dependent manner by LRP1. von Willebrand disease (VWD)-type 2B mutants are endocytosed more efficiently than wildtype (wt)-VWF by macrophages. Objective: To investigate if VWD-type 2B mutations in the VWF A1-domain affect LRP1 binding and LRP1-dependent clearance. Methods: Recombinant Fc-tagged A1 domain (A1-Fc, A2-Fc, A3-Fc) and full-length VWF (wt or mutants thereof) were tested for binding to LRP1 or a recombinant fragment thereof in a static immunosorbent assay. Mutant and wt-VWF were also compared for clearance in mice lacking macrophage LRP1 (macLRP1 À ) and control mice (macLRP1 + ). Results: We found that A1-Fc but not A2-Fc or A3-Fc binds dose-dependently to LRP1. Binding of A1-Fc to LRP1 was markedly enhanced by the VWD-type 2B mutation p.V1316M. As expected, full-length wt-VWF was unable to bind LRP1 under static conditions unless ristocetin was added. In contrast, the presence of the p.V1316M or p.R1306Q mutation induced spontaneous binding to LRP1 without the need for ristocetin or shear stress. Both mutants were cleared more rapidly than wt-VWF in control macLRP1 + mice. Surprisingly, deletion of macrophage LRP1 abrogated the increased clearance of the VWF/p.R1306Q and VWF/p.V1316M mutant. Conclusion: The VWF A1-domain contains a binding site for LRP1. Certain VWD-type 2B mutations relieve the need for shear stress to induce LRP1 binding. Enhanced LRP1 binding coincides with a reduced survival of VWF/p.R1306Q and VWF/p.V1316M. Our data provide a rationale for reduced VWF levels in at least some VWD-type 2B patients.

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

“…Recent studies have shown that hepatic macrophages contribute to the clearance of full-length VWF. [21][22][23][24][25][26] To determine whether macrophages modulate A1-A2-A3 clearance in vivo, clearance studies were repeated following clodronate-induced macrophage depletion. In keeping with previous studies, clearance of full-length rVWF was significantly reduced following macrophage depletion ( Figure 1B).…”

“…2,14 However, accumulating data have demonstrated that macrophages play important roles in regulating VWF clearance in vivo, [21][22][23][24][25][26] and a number of putative macrophage receptors for VWF have been identified. 23,26,38 Critically, however, the specific regions of the VWF glycoprotein involved in modulating interactions with these different macrophage receptors have not been determined. In this study, using a series of in vivo and in vitro methodologies, we demonstrate that the A1-A2-A3 domains of VWF contain a receptor-recognition site important in mediating VWF binding to macrophages in vitro and in regulating VWF clearance by macrophages in vivo.…”

“…23,26,53 In addition, LRP1 has been shown to bind to VWF in a shear-dependent manner. Importantly, Rastegarlari et al previously demonstrated that the inhibitory effects of RAP on VWF clearance were predominantly modulated through macrophage LRP1 rather than LRP1 (A) A model of the VWF A2 domain was prepared as previously described.…”

“…For example, Wohner et al recently showed that specific type 2B VWD variants (R1306Q and V1316M within the A1 domain) result in increased VWF clearance that is predominantly modulated through the macrophage LRP1 receptor. 26 Nevertheless, the molecular mechanism through which so many different amino acid substitutions clustered within the A1 domain of VWF result in enhanced clearance remains poorly understood.…”

“…[15][16][17][18][19][20] Recent evidence further suggests that hepatic and splenic macrophages may play key roles in mediating VWF clearance. [21][22][23][24][25][26] For example, differentiated primary human macrophages can bind and endocytose purified VWF in vitro. 21 Furthermore, macrophage depletion significantly prolonged the in vivo survival of VWF infused into VWF 2/2 mice.…”