A CHIMERIC PROTEIN-C CONTAINING THE PROTHROMBIN GLA DOMAIN EXHIBITS INCREASED ANTICOAGULANT ACTIVITY AND ALTERED PHOSPHOLIPID SPECIFICITY

To determine the structural basis of phosphatidylethanolamine (PE)-dep endent activated protein C (APC) activity, we prepared a chimeric mole cule in which the Gla domain and hydrophobic stack of protein C were r eplaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE, Protein S enhanced inacti vation 2-fold and independently of PE, PE and protein S had little eff ect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE, The cleavage patterns of factor Va by APC and the chimera were similar, an d PE enhanced the rate of Arg(506) and Arg(306) cleavage by APC but no t the chimera, APC and the chimera bound to phosphatidylserine:phospha tidylcholine vesicles with similar affinity (K-d approximate to 500 nM ), and PE increased affinity 2-3-fold, Factor Va and protein S synergi stically increased the affinity of APC on vesicles without PE to 140 n M and with PE: to 14 nn, but they were less effective in enhancing chi mera binding to either vesicle, In a factor Xa one-stage plasma clotti ng assay, the chimera had similar to 5 times more anticoagulant activi ty than APC on PE-containing vesicles. Unlike APC, which showed a 10 f old dependence on protein S, the chimera was insensitive to protein S, To map the site of the PE and protein S dependence further, we prepar ed a chimera in which residues 1-22 were derived hom prothrombin and t he remainder were derived from protein C, This protein exhibited PE an d protein S dependence, Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction, We conclude that the protein C Gla domain possesses unique properties allowing synergistic interact ion with factor Va and protein S on PE-containing membranes.