title: Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII.
abstract: Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Haematology, University Hospital Utrecht, The Netherlands.
authors.forename: S J
authors.initials: SJ
authors.lastname: Koppelman
authors.forename: T M
authors.initials: TM
authors.lastname: Hackeng
authors.forename: J J
authors.initials: JJ
authors.lastname: Sixma
authors.forename: B N
authors.initials: BN
authors.lastname: Bouma
mesh_terms: D000925:Anticoagulants; D001777:Blood Coagulation; D002352:Carrier Proteins / Q000378:metabolism; D003169:Complement Inactivator Proteins; D004789:Enzyme Activation; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D006023:Glycoproteins; D006801:Humans; D066298:In Vitro Techniques; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D017293:Protein S / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000925:Anticoagulants; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D010743:Phospholipids; D017293:Protein S; D014841:von Willebrand Factor; D005169:Factor VIII; D005170:Factor X; D015949:Factor IXa; D013917:Thrombin