The structure of the Gla‐domainless form of the human anticoagulant enzyme activated protein C has been solved at 2.8 A resolution. The light chain is composed of two domains: an epidermal growth factor (EGF)‐like domain modified by a large insert containing an additional disulfide, followed by a typical EGF‐like domain. The arrangement of the long axis of these domains describes an angle of approximately 80 degrees. Disulfide linked to the light chain is the catalytic domain, which is generally trypsin‐like but contains a large insertion loop at the edge of the active site, a third helical segment, a prominent cationic patch analogous to the anion binding exosite I of thrombin and a trypsin‐like Ca[II] binding site. The arrangement of loops around the active site partially restricts access to the cleft. The S2 and S4 subsites are much more polar than in factor Xa and thrombin, and the S2 site is unrestricted. While quite open and exposed, the active site contains a prominent groove, the surface of which is very polar with evidence for binding sites on the primed side, in addition to those typical of the trypsin class found on the non‐primed side.

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To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10 -20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect 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, and PE enhanced the rate of Arg 506 and Arg 306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (K d Ϸ 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had ϳ5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and 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 interaction with factor Va and protein S on PE-containing membranes.