OC 75.1 - Interplay between Circulating VWF and polymorphonuclear leukocytes: Implications on Inflammation, Leukocytes Function, and VWF Clearance

Background: Von Willebrand factor (VWF) plays a pivotal role in hemostasis, and emerging evidence suggests its involvement in inflammation, yet its multifaceted role in this context requires further investigation.

Aims: This study aimed to explore the interaction between circulating plasma VWF and neutrophils (polymorphonuclear cells, PMNs), elucidate VWF's fate post-binding, and investigate its impact on neutrophil behavior.

Methods: PMNs were isolated from healthy volunteers using the EasySep Kit. VWF interaction studies involved activation of cells by inflammatory molecules such as PMA, TNFα, IL-8, or shear flow. To investigate intracellular trafficking of VWF, immuno-staining of the PMNs with VWF and CD11b antibodies, along with endocytosis markers EEA1 and Rab7 was conducted. The images were acquired by using the Zeiss Apotome.2 microscope. VWF uptake was further investigated by measuring antigen levels in the supernatant pre- and post-stimulation. FACS analysis assessed CD66b and CD45 expression in stimulated PMNs with and without VWF.

Results: Immunofluorescence imaging revealed an enhanced binding of VWF to neutrophils following stimulation with PMA, TNFɑ, IL-8, and exposure to shear forces (Figure 1). Furthermore, immunofluorescence images demonstrated increased co-localization of VWF with the early endosome marker EEA1 and the late endosome marker Rab7 over time, indicating the uptake of VWF by neutrophils subsequent to the binding. This was supported by a decrease in VWF antigen levels in the supernatant of cells after stimulation (Figure 2). Moreover, stimulated neutrophils exposed to purified plasma-derived VWF exhibited elevated expression of neutrophil surface markers CD45 and CD66b, indicative of altered neutrophil function related to cell adhesion, migration, and phagocytosis.

Conclusion(s): In summary, our study provides novel insights into the complex interplay between VWF and neutrophils, shedding light on the multifaceted roles of VWF in inflammation. Importantly, neutrophils emerge as potential mediators in the clearance of VWF from circulation, introducing a novel mechanism for VWF removal.