Thrombospondin's activity has been mapped to several domains, in particular the
amino-terminal heparin-binding domain, the procollagen domain, the properdin-like type I repeats, and the globular
carboxy-terminal domain. The protein also contains type II repeats with epidermal growth factor-like homology and type III repeats that contain an
RGD sequence.
N-terminus The
N-terminal heparin-binding domain of TSP1, when isolated as a 25
kDa fragment, has been shown to be a potent inducer of cell migration at high concentrations. However, when the heparin-binding domain of TSP1 is cleaved, the remaining anti-angiogenic domains have been shown to have decreased anti-angiogenic activity at low concentrations where increased
endothelial cell (EC) migration occurs. This may be explained in part by the ability of the heparin-binding domain to mediate attachment of TSP1 to cells, allowing the other domains to exert their effects. The separate roles that the heparin-binding region of TSP1 plays at high versus low concentrations may be in part responsible for regulating the two-faced nature of TSP1 and giving it a reputation of being both a positive and negative regulator of angiogenesis.
Procollagen domain Both the procollagen domain and the type I repeats of TSP1 have been shown to inhibit neovascularization and
EC migration. However, it is unlikely that the mechanisms of action of these fragments are the same. The type I repeats of TSP1 are capable of inhibiting
EC migration in a
Boyden chamber assay after a 3-4 hour exposure, whereas a 36- to 48-hour exposure period is necessary for inhibition of
EC migration with the procollagen domain. TSP1 contains three type I repeats, only the second two of which have been found to inhibit
angiogenesis. The type I repeat motif is more effective than the entire protein at inhibiting angiogenesis and contains not one but two regions of activity. The amino-terminal end contains a tryptophan-rich motif that blocks
fibroblast growth factor (FGF-2 or bFGF) driven angiogenesis. This region has also been found to prevent FGF-2 binding
ECs, suggesting that its mechanism of action may be to sequester FGF-2. The second region of activity, the CD36 binding region of TSP1, can be found on the carboxy-terminal half of the type I repeats. Type I repeats have also been shown to bind to
heparin,
fibronectin,
TGF-β, and others, potentially antagonizing the effects of these molecules on ECs. However, CD36 is generally considered to be the dominant inhibitory signaling receptor for TSP1, and
EC expression of CD36 is restricted to microvascular ECs. Soluble type I repeats have been shown to decrease EC numbers by inhibiting proliferation and promoting apoptosis. Attachment of
endothelial cells to
fibronectin partially reverses this phenomenon. However this domain is not without a two-faced nature of its own. Bound protein fragments of the type I repeats have been shown to serve as attachment factors for both
ECs and melanoma cells.
C-terminus The
carboxy-terminal domain of TSP1 is believed to mediate cellular attachment and has been found to bind to another important receptor for TSP1, IAP (or
CD47). This receptor is considered necessary for
nitric oxide-stimulated TSP1-mediated vascular cell responses and
cGMP signaling. Various domains of and receptors for TSP1 have been shown to have pro-adhesive and chemotactic activities for cancer cells, suggesting that this molecule may have a direct effect on cancer cell biology independent of its anti-angiogenic properties. == Cancer treatment ==