CALCIUM-ION BINDING TO THROMBOSPONDIN-1

We have quantified the binding of Ca2+ to platelet thrombospondin 1 (TSP1) using equilibrium dialysis with (CaCl2)-Ca-45. Ca2+ binding to TSP1 was found to be cooperative with 10% occupancy at 15-20 mu M CaCl2, 90% occupancy at 100 mu M CaCl2, and a Hill coefficient of 2.4 +/- 0.2. The average apparent K-d was 52 +/- 5 mu M. Maximum binding, assuming M(r) = 450,000 and epsilon = 0.918 (A280/mg/ml), was 35 +/- 3 Ca2+/TSP1. This value is close to the 33 sites (11 per subunit) predicted based on homology of the epidermal growth factor (1 site) and aspartate rich (10 sites) regions to known Ca2+ binding sequences, Ca2+ protected the aspartate-rich region from trypsin proteolysis, but not until nearly all of the Ca2+ binding sites were filled. At lower occupancy of Ca2+ binding sites, several limited tryptic digest products were obtained, This finding and the previous demonstration of extensive thiol disulfide isomerization within the aspartate-rich regions suggest that subregions of the aspartate-rich region are stabilized in different conformers, Zn2+, Cu2+, Mn2+ Mg2+ Co2+, Cd2+, and Ba2+ were tested for their ability tb modulate Ca2+ binding and protease sensitivity of TSP1, Zn2+ inhibited 40% of the Ca2+ binding but neither protected TSP1 from trypsin proteolysis, nor labilized TSP1 toward trypsin proteolysis. These results provide direct evidence for high capacity, cooperative and specific binding of Ca2+ to conformationally labile aspartate-rich repeats of TSP1.