et Biophysics &ta ELSEVIER Biochimica et Biophysics Acta 1289 (1996) 305-311 Factor H co-purifies with thrombospondin isolated from platelet secretate J.A. Carron a, R.C. Bates a, A.I. Smith b, T. Tetoz b, A. Arellano ‘, D.L. Gordon ‘, G.F. Burns a, * a Cancer Research Unit, Faculty of Medicine, The University of Newcastle, Level 5, David Maddison Building, Royal Newcastle Hospital, Newcastle, NSW 2300, Australia b Peptide Biology Laboratory, Baker Medical Research Institute, Prahan, VIC, Australia ’ Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, SA, Australia Received 27 January 1995; accepted 9 May 1995 Abstract 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. Keywords: Factor H; Thrombospondin; Heparin affinity; Contamination; Glycoprotein 1. Introduction Thrombospondin is a large multidomain glycoprotein that exhibits remarkable functional diversity. There are at least five known discrete genes encoding different throm- bospondins as well as different isoforms generated by alternative mRNA splicing; however, the great majority of experimental work has been carried out with throm- bospondin isolated from the supematant of stimulated platelets which contain and secrete only thrombospondin- I (herein TSP). The primary function of TSP has not been clearly established. It appears to play an important role in the organization of the early mouse embryo [l]; in the phago- cytosis of apoptotic neutrophils by macrophages [2]; it is an essential component of platelet aggregation [3]; an * Corresponding author. Fax: + 61 49 236984. inhibitor of angiogenesis [4]; and it is haptotactic and chemotactic for tumour cells [5]. TSP has also been de- scribed as both a cell adhesion molecule [6] and as an anti-adhesion molecule [7]; it has also been shown to function as an essential co-mitogen for the proliferation of smooth muscle cells [8] and as an inhibitor of the mito- genie response of endothelial cells to foetal calf serum [9]. In vitro, purified TSP has been demonstrated to bind the extracellular matrix components fibronectin, collagens, glycosaminoglycans, fibrinogen, laminin, histidine-rich glycoprotein and calcium [eg. [lo]]. In addition, several domains of TSP bind to discrete cell-surface receptors (reviewed in Ref. [ll]) and the molecule binds both cell membrane sulfatides [12] and the malaria parasite, PZus- modium fulciparum [13]. TSP can form complexes with serine proteinases, other enzymes and substrates of the clotting cascade including plasmin, elastase, plasminogen, plasminogen activator (urokinase), and form covalent com- plexes with thrombin as well as with fibrin [14-181. 0304.4165/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0304-4165(95)00095-X