Abstract
Thrombospondin 1 (TSP1) is a 450 kDa homotrimeric multidomain glycoprotein
with fundamental roles in many cell-cell and cell-matrix interactions. These varied, and
sometimes conflicting, functions are mediated by specific domains in TSP1. One
region with diverse biological roles is the Ca2+ binding loops (or type 3 repeats). The
biological activity of this region is determined through a complex assembly of disulfide
bonds linking structure and function.
Disulfide interchange in a protein is usually very specific and quite slow, unless
catalysed. I have found that protein disulfide isomerase (PDI) is expressed on the
surface of platelets and endothelial cells in a reduced active conformation. The
presence of enzymatically active PDI on the surface of TSP1-secreting cells suggests
PDI is well positioned to catalyse disulfide interchange in, and regulate the
structure/function relationships of, TSP1. PDI was observed to form disulfide-linked
complexes with TSP1. Moreover, incubation of platelet or fibroblast TSP1 with PDI
enhanced binding of an isomer-specific anti-TSP1 antibody whose epitope is in the
Ca2+ binding loops. These findings suggest that PDI may mediate disulfide bond
rearrangement in both the soluble and extracellular matrix-bound forms of TSP1.
TSP1 is a tight-binding competitive inhibitor of neutrophil cathepsin G; however,
incubation with PDI increased the Ki for the interaction ≥10-14-fold. TSP1 bound
platelet-derived growth factor (PDGF) tightly in the region of the Ca2+ binding loops and
supported binding of PDGF to its receptor. PDI-mediated disulfide interchange in
TSP1 ablated PDGF binding, indicating that PDI-catalysed disulfide interchange in
TSP1 may modulate PDGF-TSP1 complex formation and the biological activity of
PDGF. Finally, PDI-catalysed isomerization of TSP1 potently affected its cell adhesive
properties. Treatment of TSP1 with PDI enhanced adhesion and spreading of
endothelial cells through the αvβ3 integrin receptor to TSP1, by exposure of a cryptic
RGD sequence. Thus, endothelial cell surface PDI may be a physiological regulator of
RGD-dependent binding to TSP1.
These data suggest that cell-surface PDI may regulate the disulfide-bonded
structure and certain biological functions of TSP1. In conclusion, I propose a novel
mechanism for the post-translational regulation of TSP1 structure/function, which in
turn may regulate certain aspects of TSP1 in vascular biology.