Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family.
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.
Tait
J F
JF
Department of Biochemistry, University of Washington, Seattle 98195.
Gibson
D
D
Fujikawa
K
K
D017302:Annexins; D001777:Blood Coagulation; D002118:Calcium / Q000494:pharmacology; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006023:Glycoproteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D017302:Annexins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011257:Pregnancy Proteins; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium