Figure 2.
Figure 2. Arg41Gln and Arg46Gln PAR1 point mutations cause the predicted cellular responses to thrombin, TRAP, and APC as determined using microvascular BECs. (A) After BECs from QQ41-PAR1 mice or Wt mice were seeded in 384 well plates and the calcium dye Fluo-8 AM was added and incubated for 30 minutes, calcium release was triggered by addition of 20 nM thrombin (final concentration) (blue lines), ionomycin (purple lines), or control buffer (black lines), and calcium flux was recorded over 240 seconds. Solid lines represent results for BECs from Wt mice and dashed lines represent data for BECs from QQ41-PAR1–homozygous mice. (B) Area under curves (AUC) for calcium release (seen in panel A) were plotted for different doses of thrombin, which showed that thrombin gave the predicted dose-dependent increase in calcium ion release in Wt BECs whereas thrombin failed to initiate ion flux in BECs from QQ41-PAR1 mice. (C) Calcium flux was determined as described in “Materials and methods” following addition of different doses of a 10-mer TRAP10 and of mouse (m) or human (h) thrombin at varying doses to BECs from QQ41-PAR1 mice or Wt mice. Data points represent the normalized relative integrated total ion flux (RFU) increase that was recorded over 4 minutes compared with controls. Error bars represent standard error of the mean (SEM) for at least 3 separate experiments. Significance was analyzed using 2-way ANOVA. *P < .05. (D) Time-dependent changes in TER caused by thrombin (2.5, 5, and 10 nM) were recorded for BECs from Wt mice and QQ41-PAR1 mice. (E) The changes in TER caused by 20 μM TRAP10 for cultured BECs obtained from Wt mice and QQ-PAR1 mice were recorded. (F-G) Phosphorylation of Ser473 in Akt in BECs obtained from Wt mice and QQ41-PAR1 mice caused by murine APC (mAPC; 90 nM final in panel F) or BECs obtained from Wt mice and QQ46-PAR1 mice was determined. Total Akt antigen levels were used as loading controls to determine the ratio of phosphorylated Akt (pAkt)/Akt, which was normalized to 1.0 for no APC at zero time. Blots were scanned on LICOR and quantified error bars represent SEM for at least 3 separate experiments. Significance was analyzed using 2-way ANOVA. CI, confidence interval.

Arg41Gln and Arg46Gln PAR1 point mutations cause the predicted cellular responses to thrombin, TRAP, and APC as determined using microvascular BECs. (A) After BECs from QQ41-PAR1 mice or Wt mice were seeded in 384 well plates and the calcium dye Fluo-8 AM was added and incubated for 30 minutes, calcium release was triggered by addition of 20 nM thrombin (final concentration) (blue lines), ionomycin (purple lines), or control buffer (black lines), and calcium flux was recorded over 240 seconds. Solid lines represent results for BECs from Wt mice and dashed lines represent data for BECs from QQ41-PAR1–homozygous mice. (B) Area under curves (AUC) for calcium release (seen in panel A) were plotted for different doses of thrombin, which showed that thrombin gave the predicted dose-dependent increase in calcium ion release in Wt BECs whereas thrombin failed to initiate ion flux in BECs from QQ41-PAR1 mice. (C) Calcium flux was determined as described in “Materials and methods” following addition of different doses of a 10-mer TRAP10 and of mouse (m) or human (h) thrombin at varying doses to BECs from QQ41-PAR1 mice or Wt mice. Data points represent the normalized relative integrated total ion flux (RFU) increase that was recorded over 4 minutes compared with controls. Error bars represent standard error of the mean (SEM) for at least 3 separate experiments. Significance was analyzed using 2-way ANOVA. *P < .05. (D) Time-dependent changes in TER caused by thrombin (2.5, 5, and 10 nM) were recorded for BECs from Wt mice and QQ41-PAR1 mice. (E) The changes in TER caused by 20 μM TRAP10 for cultured BECs obtained from Wt mice and QQ-PAR1 mice were recorded. (F-G) Phosphorylation of Ser473 in Akt in BECs obtained from Wt mice and QQ41-PAR1 mice caused by murine APC (mAPC; 90 nM final in panel F) or BECs obtained from Wt mice and QQ46-PAR1 mice was determined. Total Akt antigen levels were used as loading controls to determine the ratio of phosphorylated Akt (pAkt)/Akt, which was normalized to 1.0 for no APC at zero time. Blots were scanned on LICOR and quantified error bars represent SEM for at least 3 separate experiments. Significance was analyzed using 2-way ANOVA. CI, confidence interval.

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