Localization of the hemagglutinating activity of platelet thrombospondin to a 140 000-dalton thermolytic fragment.
The platelet protein thrombospondin (TSP) which is secreted from alpha-granules upon platelet activation agglutinates trypsinized, glutaraldehyde-fixed human erythrocytes. Optimal conditions for the hemagglutinating activity require that both Ca2+ and Mg2+ be present in final concentrations of 2 mM. In the presence of dithiothreitol (i.e., reduction of disulfide bonds), the lectin-like activity decreases in a manner proportional to the extent of reduction of the molecule from its native trimeric configuration into its Mr 180 000 subunits. Proteolysis of purified TSP with thermolysin, which produces discrete domains with the capacity to bind fibrinogen and heparin, also diminishes, but does not abolish, the hemagglutinating activity. Fibrinogen was without effect on hemagglutinating activity while heparin was found to be a potent inhibitor. Other proteoglycans such as hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, and heparan sulfate had no effect. That portion of the TSP molecule apparently responsible for the hemagglutinating activity was identified by incubating a thermolytic digest of TSP with red blood cells and then determining which fragment was bound to the cell surface. The binding site resides within a peptide fragment of 140 000 daltons but is absent from an Mr 120 000 fragment derived from the Mr 140 000 fragment. Under the conditions for optimal expression of hemagglutinating activity (i.e., 2 mM MgCl2 and 2 mM CaCl2), this Mr 140 000 fragment was also shown to have heparin binding activity.
D001792:Blood Platelets / Q000032:analysis*; D002118:Calcium / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000097:blood*; D006384:Hemagglutination / Q000187:drug effects; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000097:blood*; D013820:Thermolysin / Q000494:pharmacology; D019699:Thrombospondins
D006023:Glycoproteins; D010446:Peptide Fragments; D019699:Thrombospondins; D006493:Heparin; D013820:Thermolysin; D008274:Magnesium; D002118:Calcium; D004229:Dithiothreitol