Characterisation of insulin-like growth factor-binding protein-3 binding to a novel receptor on human platelet membranes

Insulin-like growth factor-binding protein-3 (IGFBP-3) is an important regulator of insulin-like growth factor (IGF) bioavailability and IGF-independent growth responses. IGFBP-3 is stored within the alpha granules of platelets, permitting its rapid and concentrated delivery at sites of platelet lysis. Previous studies have demonstrated a lack of mRNA for IGFBP-3 in platelets and in the megakaryocytes from which platelets are formed, indicating that IGFBP-3 is endocytosed from the extracellular milieu. In this study, the binding of IGFBP-3 to platelet membranes was characterised to determine whether specific cell-surface IGFBP-3 receptors exist that might account for IGFBP-3 uptake into the alpha granules by megakaryocytes. IGFBP-3 binding to platelets was saturable, requiring at lease 4.6 nM I-125-labelled IGFBP-5 to occupy all binding sites present oil 100 mug of platelet membranes. Non-linear regression analysis revealed the presence of a single class of high-affinity binding sites fur IGFBP-3 on platelets, with a K-d between 2.6 x 10(-10) and 8.0 x 10(-10) M and 1.51-4.89 x 10(11) binding sites/mg of platelet membrane. Kinetic analysis of I-125-IGFBP-3 binding to platelet membranes demonstrated. a forward rate (k(on)) of 8.1 x 10(8) per M per min, The reverse rate constant (k(off)) was calculated to be 1.6 x 10(-1) per min (t(1/2) = 4.2 min) and confirmed experimentally to be 3.3 x 10(-1) per min (t(1/2) = 2.1 min). Binding of I-125-IGFBP-3 to platelet membranes was inhibited in a dose-dependent manner by recombinant Escherichia coli IGFBP-3. In contrast, rat IGFBP-4 was not able to compete with I-125-IGFBP-3 for platelet binding sites. Additionally, concentrations of IGF-I ranging from a 15-fold to a 40 000-fold molar excess caused a consistent 20% reduction in I-125-IGFBP-3 binding. The mechanism of this slight reduction is unknown, but suggests that IGF-I does not compete directly with IGFBP-3 for receptor binding sites. However, it does not preclude the possibility that IGF-I may be endocytosed into the alpha granules as part of an IGF-I-IGFBP-3 complex. These results demonstrate the presence of high-affinity binding sites for IGFBP-3 on human platelet membranes. The nature and kinetics of the binding reaction are characteristic of a receptor-ligand interaction. This receptor may be involved in the endocytosis of circulating IGFBP-3 by megakaryocytes for packaging within the alpha granules of platelets. It is unknown if it is present in other tissues.