SYNERGISTIC INHIBITION OF THE INTRINSIC-FACTOR-X ACTIVATION BY PROTEIN-S AND C4B-BINDING PROTEIN

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding pro tein can bind to protein S, thereby inhibiting the cofactor activity o f protein S for activated protein C, In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition o f the intrinsic factor X activating reaction by the complex of C4b-bin ding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-bind ing protein led to a potentiation of the inhibition to almost 90%, Bec ause C4b-binding protein atone had no effect on the activation of fact or X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b -binding protein lacking the beta-chain, which is unable to bind to pr otein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the bind ing affinity of protein S for factor VIII. Therefore, a possible inter action of C4b-binding protein with factor VIII was investigated. C4b-b inding protein bound to factor VIII and to thrombin activated factor V III in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta- chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also b ound to factor VIII. Monoclonal antibodies directed against the alpha- chain of C4b-binding protein inhibited the binding to factor VIII, whe reas monoclonal anti bodies directed against the beta-chain had no eff ect on the binding to factor VIII. This finding indicates that the bin ding site for factor VIII on C4b-binding protein is localized on the a lpha-chains of C4b-binding protein. The potentiation by C4b-binding pr otein of the inhibition of the factor X activation by protein S was bl ocked by a monoclonal antibody directed against the alpha-chain of C4b -binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b- binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of prote in S on prothrombinase activity. We conclude that C4b-binding protein can potentiate the inhibitory effect of protein S on the intrinsic fac tor X activation via interactions with both protein S and factor VIII and that the binding site for factor VIII is localized on the alpha-ch ains of C4b-binding protein. (C) 1995 by The American Society of Hemat ology.