Morphological and Functional Studies of Ultra Long Von Willebrand Factor (ULVWF) Anchored on Endothelial Surface.

Von Willebrand factor multimers that are tethered on endothelial cells are ultra large, hyperactive and susceptible to ADAMTS13 cleavage. However, the morphology of acutely secreted ULVWF that are deposited on endothelial surface and the mechanism by which they persist under fluid shear stress remain elusive. In this study, we use immunofluorescence and electron microscopy to characterized HUVEC surface associated VWF. By conducting perfusion assays in a parallel plate flow chamber in the presence of an agonist and a fluorescent anti-VWF antibody, we directly visualized VWF secretion and dynamics in real time. Upon histamine or forskolin stimulation, VWF multimers secreted by confluent HUVEC formed extended strings parallel to flow direction. While strings formed independent of platelet adhesion, they bound platelets without requiring platelet activation. Quick-freeze, deep-etch electron microscopy of immuno-gold labeled VWF showed that multiple single strings can merge and form parallel or twisted bundles, suggesting lateral association between individual VWF multimers. These “strings” or “bundles of strings” were tethered to the plasma membrane by a limited number of anchoring points, remained stable for over 30 minutes of continuous laminar flow at the shear stress of 2.5dyn/cm², and about 50% of these strings were disrupted (bent or washed away) by a reversal of flow direction. Static adhesion assays indicated that both “RGDS” binding integrin and P-selectin were involved in binding of CHO-P cells to ULVWF containing HUVEC supernatant. However, “RGDS” peptide, but not soluble P-selectin or a polyclonal P-selectin antibody by itself, could interfere with the formation of fluorescent VWF strings under flow. These observations suggest that “RGDS” binding integrins are implicated in the presentation of ultra-long VWF strings on endothelial lumen, and that multiple protein-protein interactions could contribute to this process.