Overexpression of glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor-1 inhibits tissue factor activity.
The cellular initiation of coagulation by the tissue factor (TF)-activated factor VII complex is transiently inhibited by endogenous tissue factor pathway inhibitor-1 (TFPI-1), whereas exogenously added TFPI-1 is targeted to a degradation pathway. This study investigates the relevance of glycosyl phosphatidylinositol (GPI) anchoring for the anticoagulant properties of TFPI-1. Experiments were performed with the human cell line ECV304 using liposomal gene transfer. For GPI anchoring of TFPI-1 we used a fusion protein of TFPI-1 and the GPI attachment sequence of decay-accelerating factor (GPI-TFPI-1), and compared it with wild-type TFPI-1. We measured TF and TFPI-1 surface expression by flow cytometry and TF proteolytic activity by a chromogenic assay for activated factor X generation. After transfection of GPI-TFPI-1, surface expression of TFPI-1 increased to 134 +/- 9% of mock transfected cells (mean +/- SEM, P = 0.004), and transfection with wild-type TFPI-1 did not significantly alter TFPI-1 surface expression. After transfection with GPI-TFPI-1, TF activity was reduced by 18 +/- 9% compared with mock transfections (P = 0.003), whereas after transfection with TFPI-1 wild type no significant inhibition was observed. This effect was not due to altered TF expression. GPI anchoring is an essential prerequisite for surface expression of TFPI-1 and inhibition of TF activity. Gene transfer of GPI-anchored TFPI, therefore, may be an efficient tool to inhibit local TF-induced coagulation.
Ott
Ilka
I
Medizinische Klinik und Deutsches Herzzentrum, Technische Universität München, Lazarettstr. 36, 80636 München, Germany. ott@dhm.mhn.de
Vukovich
Ruth
R
Schömig
Albert
A
Neumann
Franz-Josef
FJ
D000925:Anticoagulants / Q000008:administration & dosage; D045744:Cell Line, Tumor; D002863:Chromogenic Compounds; D015951:Factor Xa / Q000096:biosynthesis; D015316:Genetic Therapy / Q000379:methods; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000494:pharmacology*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D013925:Thromboplastin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014162:Transfection / Q000379:methods
D000925:Anticoagulants; D002863:Chromogenic Compounds; D017261:Glycosylphosphatidylinositols; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin; D015951:Factor Xa