Loss of KRIT1 increases TM in human brain endothelial cells. hCMEC/D3 cells were transduced with short hairpin KRIT1 (shKRIT1) or short hairpin scrambled control (shControl) using lentivirus. (A) shKRIT1 induced an ∼30% decrease in KRIT1 mRNA in hCMEC/D3 cells, as determined by real-time quantitative PCR (RT-qPCR). (B) KRIT1-depleted hCMEC/D3 cells expressed ∼2-fold as much TM mRNA as did ShControl cells (SEM, n = 4). (C) Silencing of KRIT1, using shKRIT1, in hCMEC/D3 cells resulted in an ∼2.9-fold increase in TM protein levels (SEM, n = 3). (D) Representative confocal images of TM (red) staining in hCMEC/D3 cells transduced with shKRIT1 or shControl. Nuclei were counterstained with DAPI (blue). Scale bar, 200 μm (n = 2). (E) Relative abundance of TM in hCMEC/D3 cells transduced with shKRIT1 or shControl, as assessed by flow cytometry. (F) Mean fluorescence intensity (MFI) of membrane-bound TM in shKRIT1 compared with shControl-transduced hCMEC/D3 cells in (B) (SEM, n = 3). (G) Assessment of protein C activation on hCMEC/D3 cells transduced with shKRIT1 or shControl. Thrombin (IIa) and protein C (PC) were added to shKRIT1 or ShControl brain endothelial cells in the presence or absence of blocking antibodies anti-TM (aTM) or anti-EPCR (rcr252). The nonblocking anti-EPCR (rcr2) antibody was used as control (SEM, n = 3). *P < .05, **P < .01, ***P < .001, Student t test.