Protein disulfide isomerase has no stimulatory chaperone effect on factor X activation by factor VIIa-soluble tissue factor.
BACKGROUND
It was recently reported that protein disulfide isomerase (PDI) stimulates factor X (FX) activation by factor VIIa (FVIIa) bound to soluble tissue factor (sTF) in a purified system and that PDI may be responsible for activating cellular tissue factor (TF) and switching it between its roles in blood coagulation and cellular signalling. This study further investigates the former effect of PDI.


METHODS
FX activations by FVIIa-sTF(1-219) were carried out in the presence of different forms of PDI, with annexin V or detergent present in the system and using various forms of FVIIa and FX. In addition, FVIIa-lipidated TF was used as the FX activator.


RESULTS
Recombinant human PDI did not influence FX activation by FVIIa-sTF(1-219), whereas PDI purified from bovine liver enhanced the activation rate in a dose-dependent manner. The inclusion of annexin V or detergent abolished the stimulatory effect. Removal of the phospholipid-interactive gamma-carboxyglutamic acid (Gla)-containing domain from either FVIIa or FX obliterated the bovine PDI-induced enhancement of FX activation, as did the introduction of F4A or L8A mutation in FVIIa. The presence of 25 nM bovine PDI lowered the apparent K(m) for FX from far above 10 microM to 1-2 microM. No PDI effect was seen when FVIIa-lipidated TF was the FX activator.


CONCLUSIONS
FX activation is insensitive to PDI per se and a phospholipid contaminant in the bovine PDI preparation acts stimulatory when sTF, but not lipidated TF, is the cofactor. Strong support is provided by the lacking effect of bovine PDI after removal or modification of the Gla domain in either FVIIa or FX as well as by the effects of annexin V and detergents and the decreased K(m) value.
Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park (G8.2.76), DK-2760 Måløv, Denmark. egpe@novonordisk.com
Egon
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Persson
D000818:Animals; D002417:Cattle; D015942:Factor VIIa / Q000737:chemistry / Q000502:physiology*; D005170:Factor X / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D019704:Protein Disulfide-Isomerases / Q000502:physiology*; D017434:Protein Structure, Tertiary; D013925:Thromboplastin / Q000502:physiology*
D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D019704:Protein Disulfide-Isomerases; D008274:Magnesium