Thiol-Disulfide Exchange by Thrombospondin

Abstract

Thrombospondin (TSP) forms sodium dodecyl sulfate (SDS)-stable complexes with thrombin and other proteases, apparently by thiol-disulfide ex change. It also forms complexes with other mole cules of TSP by a similar mechanism. TSP has a conformation-sensitive thiol and intrachain disul fide bond. Thus, TSP has the potential for covalently cross-linking itself and other proteins.In studies with labeled thrombin derivatized with a photoactivatable cross-linking reagent, Danishefsky and Detwiler 1 observed that after photoactivation a labeled complex with a mass greater than 500 kd was recovered in the supernatant solution. When the derivatized thrombin was added to the supernatant solution of platelets activated with the ionophore A23187 and photoactivated, there was incorporation of label into a similar complex, indi cating that the complex included a protein released from activated platelets. The complex formed within 20 sec, the period of photoactivation. It did not form with fibrinogen or α 2 -macroglobulin, but it did form with purified TSP. The significance of this study was the demonstration that thrombin and TSP formed a complex with sufficient affinity (ap parently greater than that with fibrinogen or α 2macroglobulin) to permit cross-linking by the photoactivatable reagent.

In Vitro and In Vivo Evidence that Thrombospondin-1 (TSP-1) Contributes to Stirring- and Shear-Dependent Activation of Platelet-Derived TGF-β1

“…The first is that only a subpopulation of TSP-1 molecules in the correct conformation(s) (regardless of post-translational modifications) can participate in the activation process. In fact, it has been demonstrated that TSP-1 adopts markedly different conformations, depending on the temperature, pH, cation concentrations, and degree of purification[42]–[45]. Support for this hypothesis comes from evidence that the conformation of TSP-1 affects its efficiency in mediating thiol-disulfide exchange with other molecules[42]–[44], [46].…”

Thrombospondins: from structure to therapeutics

“…The C-terminal proximal TSR3 module contains an RGDA sequence, potentially allowing interactions of TSP1 with platelet integrins a IIb b 3 , a v b 3 and a 5 b 1 . The accessibility of the RGDA sequence and, therefore, the adhesive properties of TSP1 are thus tightly related to the Ca 2+ -ion concentration and disulfide bond matching [36,37]. Sun et al [38] have shown that controlled reduction of disulfide bonds in TSP1 by dithiothreitol increases its RGDA-dependent cell-adhesive properties.…”

“…Free thiols and a Ca 2+ -modulated C-terminal region confer to TSP1 the capacity to form covalent bonds with other proteins, such as thrombin. TSP1 multimerization, due to disulfide bond exchange, has also been described [36]. More recently, protein disulfide isomerase (PDI) secreted by activated platelets was found to catalyze disulfide exchange reactions on the platelet surface [39].…”

“…TSP1 polymerization is independent of bivalent cations [94]; polymers do not pre-exist in a-granules, but are found in the supernatant of activated platelets and during TSP1 purification [36,94]. TSP1 could be a substrate for PDI, potentially triggering TSP1 multimerization.…”

Thrombospondin‐1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor

“…Upon release from activated platelets, TSP-1 binds back to the platelet surface in a Ca 2+ -dependent manner and is required for the secondary, secretion-dependent, phase of platelet aggregation (10). TSP-1 exists in multiple conformational states, some of which can interconvert reversibly, while others are restricted by covalent disulphide bonds (11). TSP-1 conformation varies in solution or is matrix-bound, reflecting the relative availability of TSP-1 domains to interact with different cellsurface receptors, which result in different functional properties (12).…”