A novel crosslinking reagent and its application for the detection and isolation of heparin-binding protein(s) on the platelet surface

A new hetero-bifunctional photo crosslinking reagent, 2-(4-azidoanilyl) -4- (4-azabicyclo-[2,2,2] hexylammonio)-6-morpholino-1,3,5-triazine chloride, w as designed to detect and isolate heparin-binding protein(s) that may act a s heparin-receptor(s) on the platelet surface, In a preliminary study using ethanol as a model substrate, the reagent was shown to react with the alco holic hydroxy group under mild conditions and its crosslinking photoreactiv ity was high. The reagent effectively formed similar covalent bonds with he parin, while preserving its anticoagulant anti-Xa activity, [H-3] Heparin l abeled with this reagent crosslinked to antithrombin III very specifically but not to ovalbumin, as analyzed by the Bio-imaging Analyzer System (BAS(T M), Fuji Photo Film, Tokyo). Affinity crosslinking of [3H] heparin was then used to detect heparin-binding proteins on the surface of intact platelets . Several discrete protein bands were detected by the BAS-imaging of SDS-PA GE.