title: Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex.
abstract: Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme-cofactor-substrate fIXa-fVIIIa-fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membrane-bound binary complexes of fIXa, fVIIIa, and fX. Studies of the coordinate binding of coagulation factors to 0.8-microm phospholipid vesicles (25/75 phosphatidylserine/phosphatidylcholine) showed that fVIII (fVIIIa), fIXa, and fX bind to 32 700 +/- 5000 (33 200 +/- 14 100), 20 000 +/- 4500, and 30 500 +/- 1300 binding sites per vesicle with apparent K(d) values of 76 +/- 23 (71 +/- 5), 1510 +/- 430, and 223 +/- 79 nm, respectively. FVIII at 10 nm induced the appearance of additional high-affinity sites for fIXa (1810 +/- 370, 20 +/- 5 nm) and fX (12 630 +/- 690, 14 +/- 4 nm), whereas fX at 100 nm induced high-affinity sites for fIXa (541 +/- 67, 23 +/- 5 nm). The effects of fVIII and fVIIIa on the binding of fIXa or fX were similar. The apparent Michaelis constant of the fX activation by fIXa was a linear function of the fVIIIa concentration with a slope of 1.00 +/- 0.12 and an intrinsic K(m) value of 8.0 +/- 1.5 nm, in agreement with the hypothesis that the reaction rate is limited by the fVIIIa-fX complex formation. In addition, direct correlation was observed between the fX activation rate and formation of the fVIIIa-fX complex. Titration of fX, fVIIIa, phospholipid concentration and phosphatidylserine content suggested that at high fVIIIa concentration the reaction rate is regulated by the concentration of free fX rather than of membrane-bound fX. The obtained results reveal formation of high-affinity fVIIIa-fX complexes on phospholipid membranes and suggest their role in regulating fX activation by anchoring and delivering fX to the enzymatic complex.
authors.affiliation: Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland, USA. mapanteleev@yandex.ru
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Panteleev
authors.forename: Natalya M
authors.initials: NM
authors.lastname: Ananyeva
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Greco
authors.forename: Fazoil I
authors.initials: FI
authors.lastname: Ataullakhanov
authors.forename: Evgueni L
authors.initials: EL
authors.lastname: Saenko
mesh_terms: D004789:Enzyme Activation; D015944:Factor VIIIa / Q000378:metabolism / Q000502:physiology*; D005170:Factor X / Q000378:metabolism; D006801:Humans; D007437:Intrinsic Factor / Q000378:metabolism*; D007700:Kinetics; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding
chemical_list: D010743:Phospholipids; D015944:Factor VIIIa; D005170:Factor X; D007437:Intrinsic Factor