Binding and degradation of platelet thrombospondin by cultured fibroblasts.

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Abstract
Thrombospondin was purified from human platelets and labeled with 125I, and its metabolism was quantified in cell cultures of human embryonic lung fibroblasts. 125I-Thrombospondin bound to the cell layer. The binding reached an apparent steady state within 45 min. Trichloroacetic acid-soluble radioactivity was detectd in the medium after 30 min of incubation; the rate of degradation of 125I-thrombospondin was linear for several hours thereafter. Degradation of 125I-thrombospondin was saturable. The apparent Km and Vmax for degradation at 37.degree. C were 6 .times. 10-8 M and 1.4 .times. 105 molecules/cell per min, respectively. Degradation was inhibited by chloroquine or by lowering the temperature to 4.degree. C. Experiments in which cultures were incubated with thrombospondin for 45 min and then incubated in medium containing no thrombospondin revealed 2 fractions of bound thrombospondin. One fraction was localized by indirect immunofluorescence to punctate structures; these structures were lost coincident with the rapid degradation of 50-80% of bound 125I-thrombospondin. The 2nd fraction was localized to a trypsin-sensitive, fibrillar, extracelluar matrix. 125I-Thrombospondin in the matrix was slowly degraded over a period of hours. Binding of 125I-thrombospondin to the extracellular matrix was not saturable and indeed was enhanced at thrombospondin concentrations > 3 .times. 10-8 M. The ability of 125I-thrombospondin to bind to extracelluar matrix was diminished 10-fold by limited proteolytic cleavage with trypsin. Degradation of trypsinized 125I-thrombospondin was also diminished, although to a lesser extent than matrix binding. Heparin inhibited both degradation and matrix binding. Thrombospondin may play a transitory role in matrix formation and/or organization. Specific receptors on the cell surface are responsible for the selective removal of thrombospondin from the extracellular fluid and matrix.