The interaction of thrombospondin with platelet glycoprotein GPIIb-IIIa.
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.
Karczewski
J
J
Department of Medicine, Medical College of Pennsylvania, Philadelphia 19129.
Knudsen
K A
KA
Smith
L
L
Murphy
A
A
Rothman
V L
VL
Tuszynski
G P
GP
D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008970:Molecular Weight; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D019699:Thrombospondins
D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D008274:Magnesium; D002118:Calcium