Abstract
The interaction of human platelet thrombospondin (TSP) with human platelet glycoproteins GPIIb-IIIa was studied using a solid-phase binding assay. Polystyrene test tubes were coated with TSP, and 125I-labeled GPIIb-IIIa was added, allowed to bind, and the bound radioactivity was measured. After 90 min, the binding became time independent, and in most experiments, more than 10% of the exogenously added radioactivity was bound to the tube. Analysis of the bound radioactivity by polyacrylamide gel electrophoresis and autoradiography indicated that it was from labeled GPIIb-IIIa. Several lines of evidence indicate that the binding of GPIIb-IIIa to TSP was specific. (a) TSP immobilized on plastic or Sepharose bound 3-10-fold more GPIIb-IIIa than immobilized bovine serum albumin. (b) Addition of unlabeled excess GPIIb-IIIa reversed the binding of 125I-labeled GPIIb-IIIa to immobilized TSP. (c) Addition of EDTA inhibited the binding of GPIIb-IIIa to TSP by more than 90%, whereas addition of 1 mM CaCl2 and 1 mM MgCl2 potentiated the binding by more than 100%. (d) Monoclonal antibodies against TSP and GPIIb-IIIa inhibited the binding by 30-70% as compared with control and polyclonal anti-fibrinogen anti-serum. (e) A plot of GPIIb-IIIa bound versus GPIIb-IIIa added was best described as a rectangular hyperbola by regression analysis with half-saturation at 60 ng/ml GPIIb-IIIa. Similar results were obtained when labeled TSP was added to tubes coated with GPIIb-IIIa. These results show that TSP and GPIIb-IIIa can specifically interact in vitro and suggest that GPIIb-IIIa may function as a platelet TSP receptor during platelet aggregation.
Highlights
From the Departmentof Medicine, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129and the SLankenau Medical Research Center, Philadelphia, Pennsylvania 19151
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Serum. (e) A plot of GPIIb-111, bound versus GPIIb-111, The role of thrombospondin in the mechanism of platelet added was best described as a rectangular hyperbola aggregation and blood coagulation is poorly understood. It is by regression analysis with half-saturation at 60 ngl ml GPIIb-IIIa.Similar results were obtained when labeled TSP was added to tubescoated with GPIII,III,. These results show that TSP GaPnIdIb-111. can interact in vitro and suggest that GP11b-111, may function as a platelet TSP receptorduringplatelet aggregation
Summary
From the Departmentof Medicine, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129and the SLankenau Medical Research Center, Philadelphia, Pennsylvania 19151. AzAs is directed against the GPIIb-III, complex (lo), andSSAGis GPIII, specific (11).TSP was purified by fibrinogen-Sepharose chromatography from the released proteins of ionophore-stimulated platelets essentially as described previously (2). Binding Assay-The inside surfaces of 3-ml polystyrene test tubes reversible with excess unlabeled GPIIb-111, added 30 min after (Elkay) were coated with either GPIIb-III., bovine serum albumin initiation of binding (Fig. 1).Theseresults indicate that (BSA), fibrinogen, or TSP.
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