Shear stress-regulated interactions between von Willebrand factor and lipoprotein-receptor LRP1: basic aspects and pathophysiological consequences – ShearWil

In this project, the interaction between LRP1 and VWF is being addressed using different approaches. First, by using purified recombinant proteins, direct binding assays are being performed (immunosorbent assays, biolayer interferometry analysis). Second, by using cells expressing LRP1 (or their LRP1-deficient counterparts), binding of VWF or its variants to these cells is visualized via immune-fluorescent techniques (both wide-field microscopy and confocal microscopy).
Finally, dedicated animal models with deficiencies for the LRP1 are being used to investigate the in vivo relevance of VWF-LRP1 interactions.

We have explored the interaction between VWF mutants and LRP1, with the focus on VWD-type 2B mutants. Since most 2B mutations are located in the VWF A1 domain, we tested whether this domain binds to LRP1. Indeed, the A1 but not the A2 and A3 domains bind to LRP1. Binding is enhanced upon the introduction of the type 2B mutation p.V1316M. In full-length VWF, the presence of type 2B mutations renders the interaction between VWF and LRP1 independent of shear stress. By using mice with a deficiency of macrophage LRP1, we observed that clearance was delayed for the type 2B mutants. Thus, LRP1 contributes to the increased clearance of these mutants.

In a second part of the study, we observed that also other domains are involved in LRP1 binding (D'D3 region and D4 domain) and that certain mutations in these domains enhance LRP1 binding. However, while clearance was reduced in mice lacking macrophage-LRP1, this was not reduced to the level observed for wt-VWF. This suggests that also other receptors contribute to increased clearance. In search for additional receptors, we have recently identified a novel receptor that not only binds wt-VWF, but displays enhanced binding of VWF clearance mutants both in the context of purified proteins as well as in cell-binding experiments. Moreover, mice lacking this novel receptor displayed reduced VWF clearance, which is more pronounced for the VWF clearance mutants compared to the wt-protein. This receptor, identified as being Scavenger-receptor A1 (SR-A1; also known as CD204) is also relevant in vivo, as VWF clearance is markedly reduced in mice lacking SR-A1. This receptor is hence a novel player in the clearance process of VWF.

We have been able to identify mutants with increased LRP1 and SR-A1 binding, which may explain why these mutants are cleared more rapidly. This could be the rationale of the reduced VWF antigen levels in patients that carry these mutations. Our findings further show that the clearance mechanism of VWF is complex and involves multiple receptors.

- Wohner N, Legendre P, Casari C, Christophe OD, Lenting PJ, Denis CV. Shear stress-independent binding of von Willebrand factor-type 2B mutants p.R1306Q and p.V1316M to LRP1 explains their increased clearance.
J Thromb Haemostas (2015) 13:815-820
- Lenting PJ, Christophe OD, Denis CV. Von Willebrand factor biosynthesis, secretion & clearance: connecting the far ends.
Blood (2015) 125:2019-2028

The hemostatic system is designed to keep blood in a fluidic state under normal conditions and to minimize blood loss via the arrest of bleeding at sites of vascular injury. However, this system is closely intertwined with other physiological systems as well, such as the inflammatory- and immune-response pathways and the angiogenic system. One example of a hemostatic protein that is linked to other processes is von Willebrand factor (VWF). VWF is critical to hemostasis as is illustrated by the severe bleeding tendency that is associated with the functional deficiency of this protein, a disorder known as von Willebrand disease (VWD) affecting up to 1% of the general population. In recent years, it has become clear that VWF is also linked to other patho-physiological processes, including inflammation, tumor cell apoptosis, vessel wall thickening and angiogenesis. However, little is known about the molecular pathways that are being used by VWF.

Recently, we have identified a novel clearance receptor for VWF, named LRP1 (Rastegarlari et al. (2012) Blood 119:2126-34). This receptor is expressed in a wide variety of cells, including macrophages, endothelial cells, smooth muscle cells and megakaryocytes. Besides being known for its capacity as clearance receptor, LRP1 also acts as a signaling receptor and has been associated with similar pathological processes as VWF. It is tempting to speculate that LRP1 could link VWF to these processes.
Interestingly, VWF is unable to interact with LRP1 under static conditions, but needs to be exposed to shear stress. This peculiar type of interaction mimics that of the interaction between VWF and its platelet-receptor Glycoprotein-Iba and is related to the complex multimeric structure of VWF. Under static and low shear stress conditions, VWF is in a closed globular conformation with the respective binding sites being inaccessible. Exposure to increased shear forces changes the VWF conformation into an open elongated form, able to interact with its receptors.

The aim of the current application is to further explore the shear stress-dependent interactions between VWF and LRP1. Three specific objectives can be distinguished:
1: To identify LRP1 interactive site(s) within VWF
2: To investigate how LRP1 contributes to the pathogenesis of VWD
3: To determine how VWF-LRP1 interactions affect cellular function, viability and morphology of LRP1-expressing endothelial cells.

To address the various research questions, an interdisciplinary approach will be applied, combining biochemistry, cell-biology and the implementation of dedicated animal models. Importantly, the study will have a clinical link in that the results will contribute to a better understanding of VWD and probably also other pathological conditions. A major strength of this application is the extensive infrastructure where all necessary in vitro and in vivo tools on VWF and LRP1 are available. Moreover, the coordinator of this project has been studying both VWF and LRP1 for more than 15 years and expertise on both proteins is present.

Our project is timely and innovative from a scientific point of view, as it applies to a highly unusual interaction between two multifunctional proteins that has only recently been identified. Therefore, many uncharted territories of the patho-physiological consequences of this interaction are there to be explored. Also do we believe that our study may provide the basis for the development of new and improved therapeutic strategies in the treatment of VWD, hemophilia A and VWF-related thrombotic disorders.