The interaction of thrombospondin with platelet glycoprotein GPIIb-IIIa

• Published in: The Journal of biological chemistry 1989-12, Vol.264 (35), p.21322-21326
• Main Authors: Karczewski, J, Knudsen, K A, Smith, L, Murphy, A, Rothman, V L, Tuszynski, G P
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Calcium - pharmacology; Cell receptors; Cell structures and functions; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Humans; Kinetics; Magnesium - pharmacology; Membrane Glycoproteins - metabolism; Molecular and cellular biology; Molecular Weight; Platelet Membrane Glycoproteins - isolation & purification; Platelet Membrane Glycoproteins - metabolism; Protein Binding; Thrombospondins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)30082-1

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.