Platelets Are the Major Source of Circulating TGF-β1 in Mice. (2009)

Abstract 4021 Poster Board III-957 TGF-β1 is a multifunctional cytokine with profound effects on several biological processes, including malignancy, immunity, wound repair, and tissue fibrosis. In addition to local generation of TGF-β1, it also circulates in plasma where it has the potential to produce systemic effects. The source(s) of plasma TGF-β1 have not, however, been defined, and a very wide range of normal values have been reported (∼1.0 to 50.0 ng/mL). Since platelets contain 40-100 times more TGF-β1 than other cells, release of platelet TGF-β1 during blood drawing and/or sample preparation can influence the results. Citrated plasma prepared from blood samples obtained from C57Bl/6 mice by the retrobulbar capillary technique showed evidence of variable release of the platelet-specific α granule proteins platelet factor 4 (PF4) and thrombospondin-1 (TSP-1), and the degree of release correlated with the total TGF-β1 levels measured by an ELISA (Table 1). Adding PGE1 to the anticoagulant reduced but did not completely eliminate the release of platelet granule proteins. In contrast, plasma prepared from free-flowing blood obtained using ultrasound guidance to percutaneously enter the left ventricle inflow tract with a 27 gauge needle showed minimal or no evidence of release of platelet granule proteins and had a total TGF-β1 level of 2.1 ± 1.2 ng/mL. To more firmly assess the role of platelets in circulating TGF-β1, we measured the platelet, plasma, and serum TGF-β1 levels in mice with a megakaryocyte-specific conditional knockout of TGF-β1 (achieved by crossing mice carrying a floxed TGF-β1 allele with transgenic mice expressing a Cre-recombinase under the control of a PF4 promoter) and littermate control mice (WT; WT/ PF4Cre+; TGF-β1flox/flox/PF4Cre-). Compared to the controls, TGF-β1flox/flox/PF4Cre+ mice exhibited an ∼80 to 90% reduction of TGF-β1 in both platelets and serum as well as an ∼45% reduction in plasma TGF-β1 (Table 2). As a complementary test of the contribution of platelets to plasma TGF-β1, we generated profoundly thrombocytopenic mice by i.v. injection of the hamster anti-mouse αIIbβ3 mAb 1B5 (1.0 mg/kg). 24 hours later the platelet count decreased by >98% and plasma TGF-β1 levels in these thrombocytopenic animals were reduced by more than 70% (0.6 ± 0.2 ng/mL) compared to pretreatment values (3.2 ± 0.4 ng/mL; p=0.002) or values after giving saline or control polyclonal hamster IgG (3.2 ± 2.0 ng/mL; p=0.002). We conclude that platelets are the major source of plasma TGF-β1 in mice. In addition, based on the rapid decrease of plasma TGF-β1 levels after the induction of thrombocytopenia, we infer that plasma TGF-β1 has a relatively short survival time. Our data have potential implications for the systemic contribution of platelet-derived TGF-β1 in regulating various normal and disease states, including wound healing and organ fibrosis. Table 1 Effect of Blood Drawing Technique on Plasma Levels of TGF-β1, PF4, and TSP-1 Protein Retrobulbar Retrobulbar + PGE1 Left Ventricular TGF-β1 (ng/mL) n=25, 10, 15 6.0 ± 3.8 2.2 ± 0.8* 2.1 ± 1.2* PF4 Antigen (AU), n=22, 7, 12 41.0 ± 30 8.0 ± 5.0* 5.6 ± 3.0* TSP-1 Antigen (AU), n=22, 7, 12 71 ± 34 28 ± 14* 21. ± 5.0* AU: arbitrary unit; *P<0.005 vs. retrobulbar Table 2 TGF-β1 Levels in Control and Transgenic Mice Mice TGF-β1 Platelet (ng/109 platelets) Serum (ng/mL) Plasma (ng/mL) Controls [(WT; WT/ PF4Cre+; TGF-β1flox/flox/PF4Cre-) (n=9, 21, 10)] 120 ± 43 91.0 ± 23 4.5 ± 1.5 PF4Cre+/ TGF-β1flox/flox (n=4, 9, 10) 26 ± 8** 12.2 ± 1.5** 2.4 ± 0.6* * P<0.005 vs. WT; ** P<0.001 vs. WT Disclosures: No relevant conflicts of interest to declare.