The latter two patients had the highest pre-LVAD levels of active TGF-1 and developed thrombus in the LVAD. LVAD implantation is associated with a decrease in HMW-vWf multimers, and there is a negative correlation between total TGF-1 and HMW-vWf multimer levels In-gel immunoblots showed a representative image of vWf multimer sizes and HMW-vWf multimers, defined as those above the 11th identifiable band, starting from the cathodal position (Physique 5A). Total plasma TGF-1 was 1.00.60 ng/ml in controls and 3.761.55 ng/ml in HF subjects (p 0.001), rising to 5.22.3 ng/ml following LVAD placement (p=0.006). These results were paralleled by the active TGF-1 values; controls had 3C16 pg/ml active TGF-1, while levels were 2.7-fold higher in HF patients before, and 4.2-fold higher after, LVAD implantation. Total TGF-1 correlated with levels of the platelet-derived protein thrombospondin-1 (r = 0.87; p 0.001), suggesting that plasma TGF-1 may serve as a surrogate indicator R788 (Fostamatinib) of in vivo platelet activation. von Willebrand factor high molecular weight multimers correlated inversely with TGF-1 levels (r =?0.63; p=0.023), suggesting a role for shear forces in loss of these multimers and platelet activation. In conclusion, accurate assessment of circulating TGF-1 may provide a valuable biomarker for in vivo platelet activation and thrombotic disorders. binds to endothelial cell glycosaminoglycans.20, 21 Effect of shear on release and activation of platelet TGF-1 in human plasma samples We previously demonstrated that shear can activate latent TGF-1 in thrombin-stimulated platelet releasates and in serum,16 and now tested whether shear can both initiate release of latent TGF-1 from platelets and then activate the released latent TGF-1. Subjecting human whole blood collected in 0.38% sodium citrate to a shear R788 (Fostamatinib) rate of 1 1,800 s?1 for 10 min at room temperature led to an increase in total TGF-1 from 1.4 0.5 ng/ml, to 3.9 0.9 ng/ml, (n=4; p=0.02) (Physique 1E). TSP-1 and PF4 plasma levels also increased after exposure to shear (data not shown), consistent with an effect of shear around the platelet release reaction as the mechanism of the increased plasma TGF-1 levels. We then measured active TGF-1 in the plasma before or after subjecting the plasma to additional shear at 1,800 s?1 for 2 h at room temperature. We WNT3 found that shear led to variable, but significant, increases in active TGF-1 (Physique 1F) (n=4; p=0.05, paired t-test). Active TGF-1 in shear-activated platelet-rich plasma samples was also measured by a cell-based MLEC assay and exhibited higher PAI-1 luciferase activity in sheared samples compared to unsheared controls (Physique 1G; n=4; p 0.001, paired t-test). The specificity of the assay for active TGF-1 was assessed with a TGF-1 neutralizing antibody (AF-101; R&D Systems) and 90% of PAI-1 luciferase activity was found to be neutralized by antibody (Physique 1G). The percentage of total TGF-1 that was activated in the samples in Physique 1G was determined by measuring both active and total TGF-1 using the ELISA and found to be much less than 1% in the unsheared sample and between 3C4% in the sheared sample (Physique 1H). Active TGF-1 measured by the ELISA was strongly correlated with PAI-1 luciferase activity measured by MLEC-based assay (r=0.877; p 0.001). Development of a high sensitivity single molecule counting assay (SMCA) to measure active TGF-1 in human plasma samples We found two mAbs (MAB240 and MAB2401) that bound to active TGF-1, but MAB2401 bound with much higher affinity than MAB240 as R788 (Fostamatinib) measured by their dissociation constants (KD) (5.19 10?11 vs. 1.59 10?9 M). The polyclonal chicken IgY also bound active TGF-1 (KD=1.14 10?9 M), but the two polyclonal rabbit antibodies showed minimal binding. MAB240, MAB2401, and the IgY antibody failed to bind to latent TGF-1 released from platelets after thrombin stimulation. Pairing MAB2401 as capture antibody and the chicken polyclonal IgY as detection antibody gave the best signal-to-noise ratio for detecting active recombinant mature TGF-1, with lower limits of quantification (LOQ) ranging from 0.5 to 3.9 pg/ml (n=18) in HBMT buffer (Figure 2A). In parallel assays, the LOQ for the ELISA method was much higher, ranging from 5 to 31.25 pg/ml (Figure 2B). The standard curves of active TGF-1 were linear with both assays at higher concentrations, but the SMCA showed better sensitivity and linearity at the lower levels of TGF-1 (Physique 2C). The inter-assay coefficient of variation (CV) was 7.6% and the intra-assay CV was calculated to be 5.8% and 7.6% for two experimental repeats. The specificity of the active TGF-1 was validated by incubating samples with a TGF-1 neutralizing antibody or Fc-TGFR, both of which inhibited the measured activity. We also performed a series of quality control assays and found no significant differences.