OC 22.1 - Angiopoietin-2 binds to Von Willebrand Factor within Endothelial Cells and after Release from Weibel Palade Bodies and mediates Von Willebrand Factor dependent angiogenesis

Background: Von Willebrand Factor (VWF) is a multimeric plasma glycoprotein essential for haemostasis, inflammation and angiogenesis. The majority of VWF is synthesised by endothelial cells (EC) and stored in Weibel-Palade bodies (WPB) with other proteins including angiopoietin-2 (Angpt-2), a pro-inflammatory and pro-angiogenic mediator, which interacts with the endothelial receptor tyrosine kinase Tie-2. We have previously shown that VWF regulates angiogenesis, raising the hypothesis that the angiogenic activity of VWF may be mediated by Angpt-2.

Aims: In the present study we characterise the biochemical parameters defining the interaction between VWF and Angpt-2 and investigate the role of Angpt-2 in VWF-dependent platelet capture and angiogenesis

Methods: We use static binding assays, VWF-platelet capture assay and in vitro fibrin bead angiogenesis assay.

Results: We demonstrate that Angpt-2 binds to VWF with high affinity (KD,app ~3nM) in a pH and calcium dependent manner. The interaction was predominantly localised to the VWF A1 domain; co-immunoprecipitation experiments demonstrate that the complex persisted following stimulated secretion from EC and was present in plasma. Angpt-2 was visible on VWF strings on stimulated EC. The VWF-Angpt-2 complex did not inhibit binding of Angpt-2 to Tie-2 and did not interfere with VWF platelet capture. However, Angpt-2 is involved in VWF-mediated control of angiogenesis, since two inhibitors of Angpt-2 activity, namely a recombinant Tie2-Fc chimeric protein and the anti-Angpt-2 function blocking antibody (AZ 3.19.3), were able to normalise sprouting angiogenesis in VWF-deficient HUVEC. The AZ 3.19.3 Ab was able to inhibit the increased angiogenesis of endothelial colony forming cells (ECFC) from a type 3 VWD patient.

Conclusion(s): These data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. These results also identify Angpt-2 as a mediator of VWF-dependent angiogenesis, suggesting a mechanism through which VWF regulates angiogenesis in patients with VWD.