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

Thrombospondin 1 (TSP-1), which is contained in platelet α-granules and released with activation, has been shown to activate latent TGF-β1 in vitro, but its in vivo role is unclear as TSP-1-null (Thbs1(−/−)) mice have a much less severe phenotype than TGF-β1-null (Tgfb1(−/−)) mice. We recently demonstrated that stirring and/or shear could activate latent TGF-β1 released from platelets and have now studied these methods of TGF-β1 activation in samples from Thbs1(−/−) mice, which have higher platelet counts and higher levels of total TGF-β1 in their serum than wild type mice. After either two hours of stirring or shear, Thbs1(−/−) samples demonstrated less TGF-β1 activation (31% and 54% lower levels of active TGF-β1 in serum and platelet releasates, respectively). TGF-β1 activation in Thbs1(−/−) mice samples was normalized by adding recombinant human TSP-1 (rhTSP-1). Exposure of platelet releasates to shear for one hour led to near depletion of TSP-1, but this could be prevented by preincubating samples with thiol-reactive agents. Moreover, replenishing rhTSP-1 to human platelet releasates after one hour of stirring enhanced TGF-β1 activation. In vivo TGF-β1 activation in carotid artery thrombi was also partially impaired in Thbs1(−/−) mice. These data indicate that TSP-1 contributes to shear-dependent TGF-β1 activation, thus providing a potential explanation for the inconsistent in vitro data previously reported as well as for the differences in phenotypes of Thbs1(−/−) and Tgfb1(−/−) mice.