Cystine 186-cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption.
Tissue factor (TF) on cell surfaces resides mostly in a cryptic state. It is not entirely clear how cryptic TF differs from procoagulantly active TF and how deencryption occurs. Here, we critically evaluated the importance of cystine 186-cystine 209 (Cys186-Cys209) bond formation for TF procoagulant activity and its de-encryption. Chinese hamster ovary cells transfected with TF(C186S), TF(C209S), or TF(C186S/C209S) expressed little procoagulant activity at the cell surface. TF monoclonal antibody and activated factor VII (FVIIa) binding studies showed that little TF protein was present at the cell surface in cells expressing mutant TF. Similar data were obtained in human umbilical vein endothelial cells (HUVECs) transduced to express TF(C186S), TF(C209S), or TF(C186S/C209S). Analysis of TF activity in HUVECs expressing similar levels of wild-type TF and TF(C186S/C209S) showed that TF mutant in the presence of saturating concentrations of FVIIa exhibited similar coagulant activity as that of wild-type TF. More importantly, treatment of HUVECs expressing TF(C186S/C209S) with HgCl(2) or ionomycin increased the cell-surface TF activity to the same extent as that of the wild-type TF. Our data provide clear evidence that TF lacking the Cys186-Cys209 bond is coagulantly active once it is complexed with FVIIa, and TF de-encryption does not require Cys186-Cys209 disulfide bond formation.
Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, TX 75708, USA.
Hema
H
Kothari
Ramesh C
RC
Nayak
L Vijaya Mohan
LV
Rao
Usha R
UR
Pendurthi
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003553:Cystine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D042783:Endothelial Cells / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D008627:Mercuric Chloride / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D004220:Disulfides; D050505:Mutant Proteins; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D003553:Cystine; D008627:Mercuric Chloride; D013925:Thromboplastin; D015942:Factor VIIa