Neutralization and Binding of Heparin by S Protein / Vitronectin in the Inhibition of Factor Xa by Antithrombin I 11

The interference of the heparin-neutralizing plasma component S protein (vitronectin) (Mr = 78,000) with heparin-catalyzed inhibition of coagulation factor Xa by antithrombin I11 was investigated in plasma and in a purified system. In plasma, S protein effectively counteracted the anticoagulant activity of heparin, since factor Xa inhibition was markedly reduced in comparison to heparinized plasma deficient in S protein. Using purified components in the presence of heparin, S protein induced a concentration-dependent reduction of the inhibition rate of factor Xa by antithrombin 111. This resulted in a decrease of the apparent pseudo-first order rate constant by more than 10fold at a physiological ratio of antithrombin I11 to S protein. S protein not only counteracted the anticoagulant activity of commercial heparin but also of low molecular weight forms of heparin (mean M, of 4,500). The heparin-neutralizing activity of S protein was found to be mainly expressed in the range 0.2-10 pgl ml of high M, as well as low M, heparin. S protein and high affinity heparin reacted with apparent 1:l stoichiometry to form a complex with a dissociation constant KO = 1 X lo-’ M as determined by a functional assay. As deduced from dot-blot analysis, direct interaction of radiolabeled heparin with S protein revealed a dissociation constant KO = 4 X 10”’ M. Heparin binding as well as heparin neutralization by S protein increased significantly when reducedlcarboxymethylated or guanidine-treated S protein was employed indicating the existence of a partly buried heparin-binding domain in native S protein. Radiolabeled heparin bound to the native protein molecule as well as to a BrCN fragment (M, = 12,000) containing the heparinbinding domain as demonstrated by direct binding on nitrocellulose replicas of sodium dodecyl sulfate-polyacrylamide gels. Kinetic analysis revealed that the heparin neutralization activity of S protein in the inhibition of factor Xa by antithrombin I11 could be mimicked by a synthetic tridecapeptide from the aminoterminal portion of the heparin-binding domain. These data provide evidence that the heparin-binding domain of S protein appears to be unique in binding to heparin and thereby neutralizing its anticoagulant activity in the inhibition of coagulation factors by antithrombin 111. The induction of heparin binding and neutralization may be considered a possible physiological mech-