Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin

V. M. Dixit, G. A. Grant, S. A. Santoro, W. A. Frazier

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Abstract

Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn(1)-Arg-Ile-Pro-Glu(5)-Ser-Gly-Gly-Asp-Asn(10)-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an M(r) 25,000 fragment from the thermolysin digest and an M(r) 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the M(r) 180,000 TSP peptide chain.

Original languageEnglish
Pages (from-to)10100-10105
Number of pages6
JournalJournal of Biological Chemistry
Volume259
Issue number16
StatePublished - Jan 1 1984

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