Thrombospondin (TSP) is responsible for the secretion-dependent phase of platelet aggregation. The mechanism of this action is believed to be through the binding of TSP to fibrinogen, resulting in the stabilization of the platelet aggregate. It has been established that the binding of fibrinogen to the platelet surface is dependent upon peptide sequences present, respectively, in the Aa- and y-chains. We have hypothesized that the binding of TSP to fibrinogen is also dependent upon unique fibrinogen peptide sequences. To test this hypothesis we have examined the interaction of TSP and f.ih.r.inogen. using..a.-blat-b.inding assaLy of reduced fibrinogen, the separated fibrinogen chains, selected fibrinogen domains or peptides generated from cyanogen bromide cleaved chains. Iodinated TSP bound specifically to the Aα - and Bβ - chains. Binding to these chains was calcium independent, mutually exclusive and could be blocked either by preincubation of TSP with 9.4 μ M fibrinogen or by preincubation of fibrinogen with 1.1 nM thrombospondin. TSP bound to the D and DD plasmin fragment of fibrinogen; TSP interacted exclusively with the B-chain component of the DD fragment. The cyanogen bromide fragments of the separated Aα - and Bβ -chains were resolved through a combination of gel filtration and reverse-phase chromatography. TSP was found to bind to a single peptide within these fibrinogen chains. These studies demonstrate that thrombospondin interacts with at least two distinct sites on fibrinogen, and these sites differ from those already described for fibrinogen binding to platelets.
Cyanogen bromide cleavage of bovine fibrinogen. Identification of a dimeric N-terminal peptide and two other disulfide containing fragments
