FACTOR-H COPURIFIES WITH THROMBOSPONDIN ISOLATED FROM PLATELET SECRETATE

Thrombospondin is a trimeric glycoprotein that has several known funct ions, including roles in platelet aggregation, phagocytosis and an inh ibitor of angiogenesis. Typically the molecule is isolated from platel et secretate by heparin affinity followed by sizing chromatography. In this study, purity is analysed by 7.5% SDS-PAGE under reducing condit ions when thrombospondin monomers run as a band at around 180 kDa. Und er nonreducing conditions of 7.5% SDS-PAGE, thrombospondin does not pe netrate beyond the stacking gel; however, under these conditions a maj or contaminating band can be seen which, upon reduction, merges into t he thrombospondin band. Further purification of this contaminating pro tein was achieved by DEAE chromatography and it was identified as Fact or H by peptide sequencing and immunoblotting. Factor H function was d emonstrated by the ability of the protein to function as a cofactor in the Factor-I-mediated cleavage of C3b. Since Factor H has several kno wn functions, such contamination could confound functional studies of thrombospondin thus purified and a pre-elution step of the heparin aff inity column is recommended.