PREPARATION OF THROMBOSPONDIN FROM PLATEL ET CONCENTRATES

Thrombospondin (TSP), a 420-kDa glycoprotein found in human platelet a lpha granula, is thought to play an important role in platelet haemost atic function. The aim of this study was to develop a rapid method to isolate TSP for functional and structural studies. For preparation we used two PRPs, each unit containing 2.6 x10(10) platelets. Washed plat elets were stimulated with thrombin (1.5 U/ml), protease activity was inhibited by hirudin (5 U/ml) and DIFP (1 mM). Cellular fragments and microsomes were separated by centrifugation (700g, 5 min; 48,000g, 30m in). The clear protein solution was further purified by HiTrap Keparin affinity chromatography and a following MonoQ ion exchange chromatogr aphy using a NaCl gradient (0-2 M) on an FPLC System. 800 mu g/LT TSP were isolated within a total separation time of 60 min. Purity of the isolated fraction was proved by SDS PAGE showing no additional protein s. TSP-binding studies are now made feasible without long-term plannin g and set-up. This method may be a helpful tool for further characteri sation of platelet TSP receptor.