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

Stirring increases TGF-β1 activation in platelet releasates from WT mice more than from <i>Thbs1</i><i>⁻</i>^{<i>/</i><i>−</i>} mice and recombinant human TSP-1 corrects the defect.

<p>(A) Thrombin-induced aggregation of washed platelets from WT and <i>Thbs1^−/−</i> mice. (B, C, D) Platelet releasates were either left unstirred (US) at 37°C or stirred (S) at 1,200 rpm for 120 min and then total (B) and active (C, D) TGF-β1 were measured. (B) The decline of total TGF-β1 in stirred versus unstirred platelet releasates was similar in WT and <i>Thbs1^−/−</i> mice. (C, D) Active TGF-β1 increased more in WT than <i>Thbs1^−/−</i> mice [p = 0.004 (absolute values) and p = 0.005 (percentages of total TGF-β1) for interaction by ANOVA]. The final values were also higher in WT vs <i>Thbs1^−/−</i> mice (p = 0.005 and p = 0.008 by t-test). (E) Immunoblotting of platelet releasates obtained from WT or TSP-1 null mice demonstrated absence of TSP-1 protein in a <i>Thbs1^−/−</i> mouse (lane 4); addition of 20 µg/mL of rhTSP-1 protein to the <i>Thbs1^−/−</i> mouse sample (lane 2) achieved a TSP-1 level similar to those in a WT mouse sample (lane 1) and a human platelet releasate (lane 3). (F) Adding rhTSP-1 (+) versus BSA (−) corrected the defect in stirring-induced activation of TGF-β1 in <i>Thbs1^−/−</i> mice.</p

TSP-1 is required for <i>in vivo</i> time-dependent activation of TGF-β1 in platelet-rich thrombi.

<p>Thrombosis was induced in the carotid arteries of WT or <i>Thbs1^−/−</i> mice by exposure to either 8% FeCl₃ (filled triangles, n = 20) or 20% FeCl₃ (open circles, n = 36) for 3 min. At either 5 min or 120 min after the vessel became completely occluded, approximately 4 mm carotid arterial segments were excised. The thrombi from the vessels were removed and extracts from them were prepared for analysis of active and total TGF-β1. Since the effects of 20% and 8% FeCl₃ were similar, the data were pooled. Active TGF-β1 levels expressed as a percentage of total TGF-β1 were similar after 5 min. The values of active TGF-β1 increased with time after occlusion in WT but not in <i>Thbs1^−/−</i> mice (p = 0.0024 for interaction by ANOVA). Moreover, the values in the 120 min samples of WT mice were higher than those in <i>Thbs1^−/−</i> mice (p = 0.041 by t-test).</p

Sera from <i>Thbs1^−/−</i> mice have reduced ability to undergo activation of TGF-β1 by stirring or shear.

<p>(A) Immunoblots of WT and <i>Thbs1^−/−</i> mice sera demonstrate absence of TSP-1 in the <i>Thbs1^−/−</i> mice. (B, C, D) Sera from WT (n = 23) and <i>Thbs1^−/−</i> (n = 23) mice were stirred (S) at 1,200 rpm or left unstirred (US) for 120 min at 37°C and then total (B) and active (C, D) TGF-β1 were measured; the latter was expressed either as an absolute value (ng/mL) (C) or as a percentage of total TGF-β1 (D). Levels of active TGF-β1 increased less in <i>Thbs1^−/−</i> than WT mice with stirring [p = 0.057 (absolute values) and p = 0.016 (percentages of total TGF-β1) for interaction by ANOVA]. The post-stirring values were also higher in WT than <i>Thbs1^−/−</i> mice [p = 0.19 (absolute values) and p = 0.001 (percentages of total TGF-β1) by t-test]. (E, F) Sera from WT (n = 10) or <i>Thbs1^−/−</i> (n = 10) mice were either incubated at 37°C (−) or subjected to shear (+) at 1,800 s⁻¹ at 37°C for 120 min. Active TGF-β1 increased more in WT mice, both in terms of absolute values (p = 0.18 by t-test) (E) and as percentages of total TGF-β1 (p = 0.039 by t-test) (F).</p

WT, wild type; <i>Thbs1^−/−</i>, TSP-1-null; WBC, white blood count; RBC, red blood cell count; HCT, hematocrit; MCV, mean corpuscular volume; PLT, platelets; MPV, mean platelet volume; fL, femtoliters.

*<p>p<0.005 vs WT.</p

Shear depletes TSP-1 via a thiol-dependent mechanism.

<p>(A) The proteins in human platelet releasates were labeled with MPB (100 µM) for 30 min either before (−) or after (+) shear for 2 hours. The labeled proteins were either analyzed directly (left two lanes) or after affinity-purification using Streptavidin-coupled beads (right two lanes). Shearing led to a dramatic decrease in intensity of the HRP reaction in select regions. (B) One of the MPB-labeled proteins (boxed) that was most affected by shearing was identified as TSP-1 by LC-MS/MS analysis. (C) Platelet releasates were passed through either a control-Sepharose column (Con) or a thiol-Sepharose column (Thiol) and then labeled with MPB. Depletion of thiol-reactive proteins by the column was analyzed by reaction of the separated proteins with Streptavidin (left panel) and depletion of TSP-1 protein was measured by immunoblotting with an anti-TSP-1 antibody (right panel). Nearly all of the proteins that labeled with MPB from the control column were not labeled after passage through the thiol-Sepharose column. (D) Effect of increasing time of exposure to shear on depletion of TSP-1 from platelet releasates. MPB labeling of TSP-1 was concordantly reduced with the loss to TSP-1 protein during shear as judged by reaction with Streptavidin-HRP (left panel) and immunoblotting with an anti-TSP-1 antibody (middle panel). TGF-β1 depletion was much less pronounced as judged by immunoblotting with an anti-TGF-β1 antibody (right panel). (E) Addition of MPB (100 µM) before shear partially prevented the loss of TSP-1 protein as shown by immunoblotting with an anti-TSP-1 antibody. Addition of the other thiol-reactive reagents, BMCC (F) or NEM (G), similarly protected against loss of TSP-1. Vertical lines in (F) indicate deletion of intermediate lanes from the same gel.</p