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

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.

Platelet TGF-β1 contributions to plasma TGF-β1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload

“…19 Platelets (0.5 ϫ 10 9 ) were stimulated with thrombin (0.125 U/mL) for 10 minutes at 37°C, and the releasates were collected as the supernatant after centrifuging the samples at 13 000g for 15 minutes at 4°C. Platelet pellets were lysed in lysis buffer (10mM Tris/HCl containing 1% Triton X-100) on ice for 30 minutes, and lysates were collected as the supernatant after centrifuging the sample at 13 000g for 20 minutes at 4°C as previously described.…”

Blockade of TSP1-Dependent TGF-β Activity Reduces Renal Injury and Proteinuria in a Murine Model of Diabetic Nephropathy

“…20 Activation can be achieved by multiple tissue-and disease-specific mechanisms, including modification of the latent complex by reactive oxygen species, proteolysis, integrin binding, cellular contractility, and shear forces, or through binding to thrombospondin 1 (TSP1). [21][22][23][24] We identified the role of TSP1 as a major physiologic activator of latent TGF-␤. 25,26 Activation occurs by a nonproteolytic mechanism through binding of the type 1 repeats of TSP1 to a conserved sequence [leucine serine lysine leucine (LSKL)] in the latency associated peptide (LAP) region of the latent complex: LAP binding to the mature domain is required to confer latency, and the RFK sequence of TSP1 disrupts this interaction.…”

Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation

“…Latent TGF-␤1 activation can occur either through protease-dependent cleavage of LAP that releases the C-terminal active TGF-␤1 domain from the latent complex or by induction of a conformational change in the latent complex that exposes the active TGF-␤1 domain to allow binding to its cell surface receptors (19 -21). Recent studies suggest that mechanical force is a factor that regulates latent TGF-␤1 activation (22,23). Induction of lung myofibroblast contraction or application of isometric stretch on cultured lung myofibroblasts results in latent TGF-␤1 activation from the ECM (22).…”