Präparation von Thrombospondin aus Thrombozytenkonzentraten

Abstract

Thrombospondin (TSP), a 420-kDa glycoprotein found in human platelet alpha gra-nula, is thought to play an important role in platelet haemostatic 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 ×1010 platelets. Washed platelets were stimulated with thrombin (1.5 U/ml), protease activity was inhibited by hirudin (5 U/ml) and DIFP (1 mM). Cellular fragments and micro-somes were separated by centrifugation (700g, 5min; 48,000g, 30min). The clear protein solution was further purified by HiTrap Heparin affinity chromatography and a following MonoQ ion exchange chromatography using a NaCl gradient (0-2 M) on an FPLC System. 800 µg/U TSP were isolated within a total separation time of 60min. Purity of the isolated fraction was proved by SDS PAGE showing no additional proteins. TSP-binding studies are now made feasible without long-term planning and set-up. This method may be a helpful tool for further characterisation of platelet TSP receptor.