Murine tissue factor coagulant activity is critically dependent on the presence of an intact allosteric disulfide.
Tissue factor activation (decryption) has been proposed to be dependent on the cysteine 186-cysteine 209 allosteric disulfide in the tissue factor extracellular domain. Tissue factor procoagulant activity is under the control of protein disulfide isomerase-dependent modulation and nitrosylation of this disulfide. Human tissue factor disulfide mutants have been proposed as a model for encrypted tissue factor, but poor expression of these mutants hampers research into tissue factor decryption. We, therefore, investigated whether mouse tissue factor cysteine 186-cysteine 209 disulfide bond mutants form a better suited model for tissue factor decryption. Stable mouse wild-type tissue factor, tissue factor(C190A), tissue factor(C213A) and tissue factor(C190/213A) disulfide mutant-expressing baby hamster kidney cells with equal levels of surface tissue factor were established. Tissue factor coagulant activity on these cells was determined using an active factor Xa-dependent chromogenic assay. The effect of nitrosylation on tissue factor function was also assessed. A tissue factor(C190/213A) mutant exerted marginal procoagulant activity, also after addition of supraphysiological concentration of factor VIIa. Tissue factor(C190A) and tissue factor(C213A) mutants showed reduced activity and the presence of tissue factor dimers. Nitrosylation of wild-type tissue factor cells decreased procoagulant function, an effect which was reversed by incubation with bacitracin, an inhibitor of protein disulfide isomerase, suggesting that this isomerase promotes de-nitrosylation of tissue factor. Mouse tissue factor procoagulant function is dependent on the Cys190-Cys213 disulfide bond and is modulated by nitrosylation. The murine model of disulfide-mutated tissue factor is more suitable for studying tissue factor decryption than are human tissue factor mutants.
Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands.
Lisa G
LG
van den Hengel
Susanne
S
Osanto
Pieter H
PH
Reitsma
Henri H
HH
Versteeg
D000494:Allosteric Regulation; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D004220:Disulfides / Q000097:blood / Q000378:metabolism*; D006801:Humans; D051379:Mice; D013925:Thromboplastin / Q000235:genetics* / Q000378:metabolism*
D004220:Disulfides; D013925:Thromboplastin; D003545:Cysteine