Isolation and identification of a 23,000-Dalton heparin binding fragment from the amino terminus of bovine thrombospondin

Abstract

Bovine freeze-thaw lysed platelets were fractionated by dextran sulfate affinity chromatography and a purified protein of 23,000 Da was subsequently obtained by G-75 gel filtration of the 0.5 m NaCl fraction. This protein had an amino terminal sequence of AsnArgIleProGluSerGlyGlyAspAsnSerValPhe AspIlePheGluLeuThrGlyAlaAlaTrpLys, a sequence identical to that reported for human thrombospondin. Thrombin-released platelets, fractionated in an identical manner, yielded a protein of 30,000 Da. Immunoblotting of purified bovine platelet thrombospondin and the 150,000- and 30,000-Da plasmin-generated thrombospondin fragments indicated that polyclonal antisera raised against the 23,000-Da protein cross-reacted with intact thrombospondin and the 30,000-Da fragment but not the 150,000-Da fragment. The 23,000-Da protein possessed weak heparin neutralization activity.