![Fig. 2. TFPI blocks LBP- or plasma-mediated transfer of BODIPY-LPS to CD14. (A) rLBP (1 μg/mL), sCD14 (2.5 μg/mL), and the indicated amounts of rTFPI were mixed quickly with the BODIPY-LPS (0.2 μg/mL) and the fluorescence increase was recorded. From the recorded curve, the initial velocity (Vi) during the first 20 seconds was calculated and is shown on the Y axis. Error bars represent standard errors of linear regression of each data point. (▴) A control sample in which BODIPY-LPS was incubated with LBP alone in the absence of sCD14. TFPI alone did not show any effect on fluorescence compared with buffer controls (not shown). Experiments were repeated four times with similar results and shown is a representative experiment. Data are plotted on logarithmic X scale. (B) Normal human plasma (10%) was used instead of rLBP for the BODIPY-LPS transfer assay to see whether rTFPI could block the plasma-mediated LPS transfer to sCD14. BODIPY-LPS at 0.5 μg/mL and sCD14 at 5 μg/mL were mixed with the indicated amounts of rTFPI. The rest of the conditions were the same as above. Experiments were repeated twice with similar results. Data are plotted on logarithmic X scale. (C) Inhibition of LBP-mediated BODIPY-LPS transfer to sCD14 is overcome by high LPS concentrations. The experiment was performed as described above with fixed doses of TFPI (5 μg/mL), LBP (0.25 μg/mL), and sCD14 (5 μg/mL). A reverse linear relationship is shown between LPS doses and the percentage of inhibition. The percentage of inhibition was calculated as follows: (1 − [V − Vc]/[Vm − Vc]) × 100, where Vm is Vi of the sample without TFPI, V is Vi of the sample with TFPI, and Vc is Vi of the control sample with LBP alone in a given LPS dose. (D) The BODIPY-LPS transfer assay was performed as above with full-length rTFPI or rTFPI13-161 . BODIPY-LPS at 0.1 μg/mL, rLBP at 0.2 μg/mL, and sCD14 at 2 μg/mL were used. Control represents a sample with BODIPY-LPS plus 0.2 μg/mL of rLBP but without sCD14. Experiments were repeated twice with essentially the same results.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/89/12/10.1182_blood.v89.12.4268/3/bl_0049f2c.jpeg?Expires=1735514558&Signature=PS4FA8PGcTB7bs-clS-i8WNJ4CbHrX60sOez8s6FfEXppuCZ87xKAVnm3Nv6445V~X4gB4axx7GRlm0tD~U32m8HpmHeSj3zvMHES6vUfzYjs~Z0y7gHdBdDtsGVW~4cDlonqxccpCVbfPm6ij5pPUAkS-Fr1GqM07mYVjm9ubAHYvuQRwlnpKSu740Wcvhdu3M32AhdvzHe3WwZoeWw8CUUBF2wdHxsXc5efYJRAm46AMS8pxzXO~QCqpEW9lSdgHTZy7alCcwr8HmQgU7HmnXC1Hg46T2Oko1ozIccMXa322vgkrVAzVLmNDYcnZuieDLMrmM6-JIV2tl~zaSKKA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
TFPI blocks LBP- or plasma-mediated transfer of BODIPY-LPS to CD14. (A) rLBP (1 μg/mL), sCD14 (2.5 μg/mL), and the indicated amounts of rTFPI were mixed quickly with the BODIPY-LPS (0.2 μg/mL) and the fluorescence increase was recorded. From the recorded curve, the initial velocity (Vi) during the first 20 seconds was calculated and is shown on the Y axis. Error bars represent standard errors of linear regression of each data point. (▴) A control sample in which BODIPY-LPS was incubated with LBP alone in the absence of sCD14. TFPI alone did not show any effect on fluorescence compared with buffer controls (not shown). Experiments were repeated four times with similar results and shown is a representative experiment. Data are plotted on logarithmic X scale. (B) Normal human plasma (10%) was used instead of rLBP for the BODIPY-LPS transfer assay to see whether rTFPI could block the plasma-mediated LPS transfer to sCD14. BODIPY-LPS at 0.5 μg/mL and sCD14 at 5 μg/mL were mixed with the indicated amounts of rTFPI. The rest of the conditions were the same as above. Experiments were repeated twice with similar results. Data are plotted on logarithmic X scale. (C) Inhibition of LBP-mediated BODIPY-LPS transfer to sCD14 is overcome by high LPS concentrations. The experiment was performed as described above with fixed doses of TFPI (5 μg/mL), LBP (0.25 μg/mL), and sCD14 (5 μg/mL). A reverse linear relationship is shown between LPS doses and the percentage of inhibition. The percentage of inhibition was calculated as follows: (1 − [V − Vc]/[Vm − Vc]) × 100, where Vm is Vi of the sample without TFPI, V is Vi of the sample with TFPI, and Vc is Vi of the control sample with LBP alone in a given LPS dose. (D) The BODIPY-LPS transfer assay was performed as above with full-length rTFPI or rTFPI13-161 . BODIPY-LPS at 0.1 μg/mL, rLBP at 0.2 μg/mL, and sCD14 at 2 μg/mL were used. Control represents a sample with BODIPY-LPS plus 0.2 μg/mL of rLBP but without sCD14. Experiments were repeated twice with essentially the same results.
