Plasma transforming growth factor beta(1) and platelet activation: implications for studies in transplant recipients

Background. Evidence from animal models supports the hypothesis that dysreg ulated transforming growth factor beta (1) (TGF beta (1)) expression plays a role in chronic allograft rejection, the progression of diabetic nephropa thy and fibrotic glomerulopathies. However, more evidence is required to su pport this hypothesis in man, and the current literature concerning blood T GF beta (1) levels in clinical studies is highly confused. We have investig ated: (i) the hypothesis that the widespread practice of activating clinica l samples Drier to measurement of TGF beta (1) is detecting the platelet-re leased pool of TGF beta (1), artefactually generated on venepuncture and un representative of the real circulating in vivo TGF beta (1) pool; and (ii) the effect of different immunosuppressive drugs on apparent TGF beta (1) pl asma levels. Methods. The effect of two different venepuncture procedures on plasma TGF beta (1) was compared in 10 healthy volunteers, one procedure designed to m inimize platelet activation and the other representing standard venepunctur e practice in a clinic situation. Blood samples from 52 renal transplant re cipients on either cyclosporine or tacrolimus immunosuppression were taken by standard venepuncture to investigate the effect of immunosuppressive dru gs on plasma TGF beta (1). Plasma TGF beta (1) and beta thromboglobulin wer e measured by ELISA. Results, Among 10 healthy volunteers who underwent two different methods of venepuncture, eight of 10 had undetectable levels of TGF beta (1) (<100 pg /ml) under conditions that: minimize platelet activation. In contrast, all 10 paired plasma samples collected by vacutainer had measurable TGF<beta>(1 ) (median 7.70 ng/ml, interquartile range 5.87-13.64 ng/ml) following acid/ urea activation. The median beta TG level(a measure of platelet degranulati on) was 0.71 mug/ml (interquartile range 0.53-1.19 mug/ml) in the special c ollections compared with 3.39 mug/ml (interquartile range 2.27-4.33 mug/ml) in the vacutainer samples (P = 0.0029). Among 52 allograft recipients ther e was a significantly higher mean TGF beta (1) level in plasma from patient s on cyclosporine therapy compared with patients on tacrolimus (28090 +/- 2 6860 pg/ml vs 7173 +/- 10610 pg/ml, respectively; P < 0.002). Mean plasma < beta>TG levels were also significantly higher during cyclosporine therapy c ompared with tacrolimus (8.14 +/- 5.54 mug/ml vs 3.66 +/- 3.32 mug/ml, resp ectively; P < 0.002). However, when TGF<beta>(1) values were corrected for the degree of platelet activation (by factoring with beta TG) there was no significant difference between TGF beta (1) levels on cyclosporine or tacro limus (4117 +/- 2993 pg/mug beta TG vs 2971 +/- 658 pg/mug beta TG respecti vely; P = 0.294). Conclusions, To avoid erroneous hypotheses concerning TGF beta (1) and perp etuating confusion in the literature over levels in health and disease, it is imperative that proper internal controls for platelet activation are use d. The effects of experimental treatments and drugs on platelet biology mus t be rigorously controlled when attempting to measure and interpret plasma levels of TGF beta (1) in clinical practice.