We have previously reported the purification of a 37 kDa platelet agglutinating
protein (PAP p37) from the plasma of a patient with
thrombotic thrombocytopenic purpura (
TTP) that was shown to be present in a subset of
TTP patients. To gain further insight into the interaction between PAP p37 and platelets, we have studied the properties of PAP p37 binding to platelets. Washed human platelets from two normal donors and two
TTP patients after recovery were used for the experiments. The PAP p37 binding to platelets was specific, concentration dependent and saturable. Scatchard analysis demonstrated about 20,564-27,090 PAP p37 binding sites per platelet. Stimulation of platelets with
thrombin or
ADP did not have any significant effect on its binding.
Thiol- and
serine-specific
protease inhibitors did not inhibit PAP p37 binding to the platelets.
Sugars such as
glucose,
fructose,
mannose, and
sialic acid, at 40 mM, inhibited its binding to platelets by 44%, 73%, 79%, and 91% respectively, but
galactose and
amino sugars did not have any significant effect. At 250 micrograms/ml,
Concanavalin-A inhibited 42% of binding, but other
lectins, such as
phytohemagglutinin-P,
potato lectin and helix pomatia
lectin (snail), did not. Pre-incubation of 125I-PAP p37 with the adult human
IgG, decreased its binding to the platelets. The
monoclonal antibodies to GP Ib (6D1) and
GP IIb-IIIa complex (10E5) did not inhibit the binding of 125I-PAP p37 to platelets.
Fibrinogen and
von Willebrand factor did not affect the binding either. These results suggest that PAP p37 binds to platelets on the sites other than GP Ib or
GP IIb-IIIa complex.