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. 2010 Sep 30;6(9):e1000950. doi: 10.1371/journal.pcbi.1000950

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Chatterjee et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) A titration of the fluorogenic substrates Boc-VPR-MCA (blue circle) and Z-GGR-MCA (green square) with 0 added TF showed a mild inhibitory influence of Z-GGR-MCA. *s indicates statistically significant difference (p<0.05) between initiation times detected with the two different substrates. (B) A titration of the fluorogenic substrates Boc-VPR-MCA and Z-GGR-MCA with 1 pM added TF showed inhibitory influence of both substrates where Boc-VPR-MCA was found to be the better substrate to detect initiation and exhibited little inhibition at 10 µM concentration. (C) TAT formation with 0 added TF, in the absence and presence of fluorogenic substrates showed less inhibitory influence of Boc-VPR-MCA on initiation defined by a burst in TAT compared to Z-GGR-MCA. Absolute [TAT] after initiation is decreased in the presence of either substrate. (D) TAT formation with 1 pM added TF, in the absence and presence of fluorogenic substrates showed decreased [TAT] during the propagation phase of coagulation in the presence of either substrate. Initiation detected by TAT correlated well with T_i determined by our fluorogenic assay. In panels C and D, * indicates [TAT] significantly greater than baseline levels (p<0.05) and # indicates statistically significant differences compared to no substrate (blue). Experiments in panels A, B, C and D were carried out with blood from the same phlebotomy. (E) TF titration done in blood anticoagulated with CTI alone (green), Citrate + CTI (red) and Citrate alone (blue). Dashed lines indicate controls with no added TF. No significant difference was detected in titrations done with and without citrate, showing no evidence of inhibition by the anticoagulant. Effects of the contact factor pathway were apparent only below 100 fM added TF.