Characterization of the binding of thrombospondin to human platelets and its association with the platelet cytoskeleton.

To characterize the interaction between thrombospondin and human platelets, thrombospondin was purified from the supernatant of thrombin-activated human platelets, labeled with iodine 125, and allowed to interact with the washed platelets. With concentrations of 10 to 50 micrograms/ml, only minute amounts of 125I-labeled thrombospondin bound to resting platelets or to platelets activated by adenosine diphosphate. In contrast, when platelets were stimulated with thrombin, binding increased fivefold to sixfold in a time-dependent and 125I-labeled thrombospondin concentration-dependent manner. Binding of 125I-labeled thrombospondin to thrombin-activated platelets required the presence of divalent cations, proceeded concomitantly with platelet release, and at a concentration of 1 nmol/L thrombin, reached a maximum of 2200 +/- 260 molecules of 125I-labeled thrombospondin bound per platelet. After its binding to platelets, 125I-labeled thrombospondin was not internalized, because up to 85% of the 125I-labeled thrombospondin was dissociated from the cell surface by adding ethylenediaminetetraacetic acid. Using various experimental approaches, including studies with severe type I thrombasthenic platelets, we further demonstrated that the interaction of 125I-labeled thrombospondin with thrombin-stimulated platelets occurred as a fibrinogen- and fibrin-independent process, and that the glycoprotein IIb-IIIa complex did not function as a physiologic plasma membrane receptor for 125I-labeled thrombospondin. Last, about 60% of the 125I-labeled thrombospondin molecules bound to the platelet surface were found to be associated with the platelet cytoskeleton recovered from platelets solubilized with Triton X-100. On Western blot analysis, this cytoskeletal fraction lacked detectable glycoprotein IV, the putative platelet receptor for thrombospondin. These results suggest that on the surface of thrombin-activated platelets, a fraction of 125I-labeled thrombospondin does not associate with glycoprotein IV but instead with other plasma membrane components that have yet to be identified.