RISTOCETIN-INDUCED ENDOTHELIAL CELL BINDING OF PLASMA VON WILLEBRAND FACTOR

Our laboratory previously used a technique of labeling plasma von Willebrand factor (vWf) with radiolabeled AVW1 - a “ neutral” monoclonal antibody to vWf. This technique has been used to study the binding of plasma vWf to platelets in the plasma milieu. Studies by several laboratories including ours have demonstrated structural glycoproteins on endothelial cells that are analogous to platelet GP Ilb/IIIa and we have shown that the platelet alloantigen Pl-Al is expressed on the surface of cultured endothelial cells. We undertook this study to evaluate the binding of plasma vWf to cultured endothelial cells in confluent monolayer cultures using the “ neutral” monoclonal antibody technique. Plasma vWf was “ labeled” using trace quantities of radiolabeled AVW-1. We then added 80,000 cpm of monoclonal-labeled plasma to 48 well culture plates containing confluent secondary cultures of human umbilical vein endothelial cells. Following the addition of ristocetin, the plates were incubated for 1 hour at room temperature, centrifuged, and the count8 bound and the counts remaining in the supernate were determined. In the presence of ristocetin, 67.5% of the labeled vWf bound to the endothelial cells. When “ labeled“ severe von Willebrand plasma was used or when ristocetin was omitted, less than 5% of the counts bound. Controls using mouse serum or excess mouse IgG to rule out Fc receptor binding and controls to evaluate binding to the subcellular matrix were performed and demonstrated this binding to be vWf and cell surface dependent. Unlike platelet vWf binding, this binding was not inhibited by monoclonal or polyclonal antibodies to platelet GPIb. We studied plasma from patients with type I, a variant of type I, and type Ila vWd and found normal binding with the type I plasma, but reduced binding with the type I variant plasma (14.5%) and the type Ila plasma (7.1%). AVW3, a monoclonal antibody to vWf that blocks vWf binding to platelet GPIb, blocked vWf binding to endothelial cells. Endothelial cells, like platelets, have the ability to bind plasma von Willebrand factor in the presence of ristocetin. This phenomenon occurs on the surface of endothelial cells in culture. Qualitative and quantitative reductions of this vWf binding are found with the plasma of patients with von Willebrand’s disease.