title: Inhibition of factor IXa and factor Xa by antithrombin III/heparin during factor X activation.
abstract: We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation and inactivation curves were fitted to a three-parameter two-exponentional model to determine the pseudo first-order rate constants of inhibition of factor IXa and factor Xa by antithrombin III/heparin. In the absence of heparin, the second-order rate constant of inhibition of factor Xa generated by factor IXa was 2.5-fold lower than the rate constant of inhibition of exogenous factor Xa. It appeared that phospholipid-bound factor X protected factor Xa from inactivation by antithrombin III. It is, as yet, unclear whether an active site or a nonactive site interaction between factor Xa and factor X at the phospholipid surface is involved. The inactivation of factor IXa by antithrombin III was found to be very slow and was not affected by phospholipid, calcium, and/or factor X. With unfractionated heparin above 40 ng/ml and antithrombin III at 200 nM, the apparent second-order rate constant of inhibition of exogenous and generated factor Xa were the same. Thus, in this case phospholipid-bound factor X did not protect factor Xa from inhibition. In the presence of synthetic pentasaccharide heparin, however, phospholipid-bound factor X reduced the rate constant about 5-fold. Pentasaccharide had no effect on the factor IXa/antithrombin III reaction. Unfractionated heparin (1 micrograms/ml) stimulated the antithrombin III-dependent inhibition of factor IXa during factor X activation 400-fold. In the absence of reaction components this stimulated was 65-fold. We established that calcium stimulated the heparin-dependent inhibition of factor IXa.
authors.affiliation: Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Pieters
authors.forename: G
authors.initials: G
authors.lastname: Willems
authors.forename: H C
authors.initials: HC
authors.lastname: Hemker
authors.forename: T
authors.initials: T
authors.lastname: Lindhout
mesh_terms: D000465:Algorithms; D000990:Antithrombin III / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D015949:Factor IXa; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D010743:Phospholipids / Q000378:metabolism; D015842:Serine Proteinase Inhibitors
chemical_list: D010743:Phospholipids; D015842:Serine Proteinase Inhibitors; D000990:Antithrombin III; D005170:Factor X; D006493:Heparin; D015949:Factor IXa; D015951:Factor Xa; D002118:Calcium