Factor H co-purifies with thrombospondin isolated from platelet secretate.
Thrombospondin is a trimeric glycoprotein that has several known functions, including roles in platelet aggregation, phagocytosis and an inhibitor of angiogenesis. Typically the molecule is isolated from platelet secretate by heparin affinity followed by sizing chromatography. In this study, purity is analysed by 7.5% SDS-PAGE under reducing conditions when thrombospondin monomers run as a band at around 180 kDa. Under nonreducing conditions of 7.5% SDS-PAGE, thrombospondin does not penetrate beyond the stacking gel; however, under these conditions a major contaminating band can be seen which, upon reduction, merges into the thrombospondin band. Further purification of this contaminating protein was achieved by DEAE chromatography and it was identified as Factor H by peptide sequencing and immunoblotting. Factor H function was demonstrated by the ability of the protein to function as a cofactor in the Factor-I-mediated cleavage of C3b. Since Factor H has several known functions, such contamination could confound functional studies of thrombospondin thus purified and a pre-elution step of the heparin affinity column is recommended.
Carron
J A
JA
Cancer Research Unit, Faculty of Medicine, University of Newcastle, Royal Newcastle Hospital, NSW, Australia.
Bates
R C
RC
Smith
A I
AI
Tetoz
T
T
Arellano
A
A
Gordon
D L
DL
Burns
G F
GF
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D017242:Complement Factor H / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006493:Heparin; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015539:Platelet Activation; D019699:Thrombospondins
C493495:CFH protein, human; D008562:Membrane Glycoproteins; D019699:Thrombospondins; D017242:Complement Factor H; D006493:Heparin