von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP.
von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin-induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.
Adelman
B
B
Department of Medicine, Medical College of Virginia, Richmond.
Carlson
P
P
Powers
P
P
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D012310:Ristocetin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism*
D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D012310:Ristocetin; D000244:Adenosine Diphosphate