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.
