Isolation of thrombospondin released from thrombin-stimulated human platelets by fast protein liquid chromatography...

Thrombospondin, a glycoprotein found in human platelet alpha granules, is thought to play a major role in platelet haemostatic functions. A rapid method to isolate thrombospondin for functional and structural studies was developed. Freshly prepared supernatants from thrombin-stimulated platelets were separated on an anion-exchange Mono-Q column on a fast protein liquid chromatography system. Detection of thrombospondin in the eluted peaks was performed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis combined with silver staining and a solid-phase radioimmunoassay with monoclonal antibodies directed against thrombospondin and other platelet granule glycoproteins. Thrombospondin was isolated rapidly to a high degree of purity using the fast protein liquid chromatography Mono-Q system (20 min), compared with the time taken with other techniques.