A 37-KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP), has been shown to be present in a subset of TTP patients. The platelet agglutination induced by PAP p37 has been demonstrated to be inhibited by igG from normal adults. To elucidate the mechanism of inhibition of IgG, the interaction between PAP p37 and IgG was studied. The complex formation was demonstrated by the binding of fluid-phase IgG to adsorbed PAP using enzyme-linked immunosorbent assay. The binding was specific, concentration dependent and saturable. The IgG covalently cross-linked to Sepharose 4B bound 125I-PAP but not to 125I-fibrinogen. Sucrose density gradient ultracentrifugation of a mixture of 125I-PAP and IgG also revealed the fluid phase complex formation with a sedimentation value of 19S. Complexes of molecular weight ranging from 180,000 to over 350,000 daltons were also detected by molecular sieve chromatography. The specific complex formation between PAP p37 and IgG is likely to account for the in vitro inhibition of TTP plasma-induced agglutination and , at least partly, the in vivo successful treatment with IgG-containing normal plasma.
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. using 125I-labeled p37, the properties of its binding to platelets were studied. The binding of p37 to washed human platelets from 4 normal subjects and two TTP patients after recovery was specific, concentration dependent and saturable. The Scatchard analysis revealed that the binding sites for p37 was about 100,000 per platelet with a dissociation constant of 48 × 10−9 M. The binding of p37 to erythrocytes was very little and non-specific. Stimulation of platelets by thrombin or ADP did not have any effect on the binding of p37 to platelets. The monoclonal antibodies to platelet GP lb (6D1) and GP Ilb-llla (10E5)(A gift of Dr. Barry coller) did not inhibit the binding of p37 to platelets. Fibrinogen (1 mg/ml) and FVIII/vWF (250 ug/ml) reduced the binding slightly. The polyclonal antibodies to p37 as well as concanavalin-A inhibited the binding of p37 to platelets through their direct interaction with p37. Other lectins such as phytohemagglutinin, potato lectin and helix pomatia lectin did not have any effect. At 40 mM, sialic acid, α-D-(+)-glucose, D-(+)-mannose and D-fructose caused 91%,44%,79%, and 63% inhibition of p37 binding respectively. D-(+)-galactose did not interfere with the binding. It is concluded that p37 binds to platelets on the sites other than GP lb and Gp IIb-IIIa and its binding to platelets is inhibited by certain sugars.
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