Abstract
Human placental anticoagulant protein-I (PAP-I) is a member of the lipocortin/calpactin/annexin family of Ca2+-dependent phospholipid binding proteins. PAP-I was labeled with fluorescein 5-isothiocyanate (1 mol/mol); this derivative had anticoagulant activity identical to the unlabeled protein and could be used to measure Ca2+-dependent binding to phospholipid vesicles through changes in fluorescence quenching. At 1.2 mM Ca2+, 0.50 M ionic strength, pH 7.4, 25 degrees C, fluorescein-labeled PAP-I bound to phospholipid vesicles containing 80% phosphatidylcholine, 20% phosphatidylserine with a Kd of 1.2 +/- 0.2 nM (mean +/- S.D.). At an ionic strength of 0.15 M, the Kd decreased to less than 0.1 nM. Prothrombin and factor Xa both competed with fluorescein-labeled PAP-I for binding to anionic phospholipid vesicles, but with affinities at least 1000-fold weaker than PAP-I. PAP-I bound only weakly (Kd greater than 2 x 10(-5) M) to neutral or anionic phospholipid monomers, and this binding was not calcium-dependent. These results show that the affinity of PAP-I for anionic phospholipid surfaces is sufficient to explain its potency as an in vitro anticoagulant.
Highlights
Human placental anticoagulant protein-I (PAP-I) is tion at sites of trauma
We show that PAP-I binds to anionic phospholipid vesicles with high affinity, which is quantitatively sufficient to explain illumination; the excitation shutter was kept closed during incubations lasting greater than 2 min
Fluorescence Quenching Assay for Phospholipid Binding-We have previously demonstrated that PAP-I binds to anionic phospholipid vesicles as assessed by gel filtration [11].In the present study, a fluorometric assay was developed for further study of this interaction
Summary
Human placental anticoagulant protein-I (PAP-I) is tion at sites of trauma. The factor VIIa-tissue factor complex a member ofthe lipocortin/calpactin/annexinfamily of can be inhibited by plasma high-density lipoproteins [9]. X lo-‘ M) to neutral or anionic phospholipid monomers, two bovineaortic anticoagulant proteins [16].PAP-I inhibited and this binding was not calcium-dependent. Such studies a stable fluorescence intensity during at least 10 min of continuous are aIso important because phospholipid binding is involved in all the proposed functions of the lipocortin-like proteins.
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