Abstract
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.
Highlights
Transforming growth factor b1 (TGF-b1) is a multifunctional cytokine that plays an important role in regulating immune response, cell proliferation, angiogenesis, wound healing, and tissue fibrosis [1,2,3]
Virtually all TGF-b1 released from platelets is in an inactive multicomponent complex [large latent complex (LLC)] in which TGF-b1 is noncovalently bound to latency-associated peptide (LAP), which, in turn, is disulfide bonded to latent TGF-b binding protein-1 (LTBP-1)[12,13]
To assess whether TSP-1 contributes to stirring-dependent activation of latent TGF-b1, serum samples were obtained from WT and Thbs12/2 mice on five days
Summary
Transforming growth factor b1 (TGF-b1) is a multifunctional cytokine that plays an important role in regulating immune response, cell proliferation, angiogenesis, wound healing, and tissue fibrosis [1,2,3]. In vitro studies have used multiple methods to activate latent TGF-b1, including exposure to proteases, thrombospondin-1 (TSP-1), reactive oxygen species, and binding to integrin receptors[7,8,10,13,14,15,16,17,18,19,20,21,22,23,24,25,26], but the mechanism of in vivo activation remains unclear. To gain additional insights into the effects of TSP-1 on TGF-b1 activation, we studied the effects of stirring and shear on latent TGF-b1 activation in WT and Thbs12/2 mice both in vitro and in vivo. We focused on the activation of TGF-b1 released from platelets because TGF-b1 and TSP-1 are both highly concentrated in platelet a-granules and released with platelet activation[19,31,32,33,34,35]
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