A Chimeric Protein C Containing the Prothrombin Gla Domain Exhibits Increased Anticoagulant Activity and Altered Phospholipid Specificity

Abstract

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.

The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding

“…It has been shown that replacing the protein C Gla domain with that of prothrombin in a protein C/prothrombin chimera 45 causes a translocation of the APC active site toward the phospholipid surface from 9.4 to 8.9 nm compared with wild-type APC. 46 As demonstrated for this chimera, it is possible that the prothrombin Gla domain of Gla FII -PS may similarly translocate PS toward the phospholipid surface, resulting in impaired alignment of the TSR and EGF1 domains with APC.…”

“…on April 8, 2019. by guest www.bloodjournal.org From replaced by the prothrombin Gla domain has been found to be insensitive to PS in a plasma clotting assay 45 ; in addition, PS dependence is associated with the C-terminal part of the protein C Gla domain. 45 Thus, it was suggested that PS functions in plasma are mainly dependent on interaction with APC and that this interaction is disrupted by replacing the Gla domain of protein C with that of prothrombin. 45 Our findings suggest that replacing the PS Gla domain with the prothrombin Gla domain results in similar disruption of the PS-APC interaction.…”

“…16 As demonstrated here for PS, the Gla domains of other vitamin K-dependent proteins have been implicated in proteinprotein and protein-membrane interactions. 30,[47][48][49][50][51] Interestingly, a chimeric protein C species in which the Gla domain was replaced by the prothrombin Gla domain has been found to be insensitive to PS in a plasma clotting assay 45 ; in addition, PS dependence is associated with the C-terminal part of the protein C Gla domain. 45 Thus, it was suggested that PS functions in plasma are mainly dependent on interaction with APC and that this interaction is disrupted by replacing the Gla domain of protein C with that of prothrombin.…”

“…45 Thus, it was suggested that PS functions in plasma are mainly dependent on interaction with APC and that this interaction is disrupted by replacing the Gla domain of protein C with that of prothrombin. 45 Our findings suggest that replacing the PS Gla domain with the prothrombin Gla domain results in similar disruption of the PS-APC interaction. Taken together, these results may indicate that the Gla domains of PS and APC interact at the membrane surface.…”

Effects of Prothrombin on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

“…It was originally suggested that PT inhibited the APC cleavage by serving as a competitive inhibitor to protein S (34). However, more recent studies demonstrated inhibition of APC-mediated FVa inactivation also in the absence of protein S (35,36). The mechanism of the PT-dependent inhibition of APC and/or protein S is not known.…”