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

Abstract

We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin ␥-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (Gla FII -PS) or in PS deleted of the thrombinsensitive region (TSR) (Gla FII -⌬TSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solventexposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into Gla FII -PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC. IntroductionProtein S (PS) is a vitamin K-dependent protein involved in regulating the anticoagulant activity of activated protein C (APC). In purified systems, PS functions as a nonenzymatic cofactor for APC in proteolytic inactivation of activated factor V (FVa) and activated factor VIII (FVIIIa), 1 reflecting the ability of PS to interact with APC. However, these effects are relatively weak and are inconsistent with the major physiologic role of PS. Indeed, the importance of PS as a natural anticoagulant is clearly demonstrated by the occurrence of severe thrombotic complications in infants with homozygous hereditary PS deficiency and by a mild thrombotic tendency in heterozygous subjects. 2 This suggests that additional components may determine the expression of the APC cofactor activity of PS in plasma. Thus, activated factor X (FXa) and activated factor IX (FIXa) have been shown to protect FVa and FVIIIa from inactivation by APC, and, in this system, PS is able to abolish these protective effects. [3][4][5] Another determinant is FV, which acts in synergy with PS in FVIIIa inactivation by APC in purified systems. 6 PS also demonstrates APC-independent anticoagulant activity by inhibiting prothrombinase and tenase activities, 7,8 and this may contribute to the overall anticoagulant activity of PS in plasma.PS is a single-chain, 635-amino acid glycoprotein composed of an N-terminal domain rich in ␥-carboxyglutamic acid (Gla domain; amino acids 1-45), a thrombin-sensitive region (TSR; amino acids 46-74), 4 epidermal growth factor (EGF)-like domains (amino acids 75-242), 9 and a large C-terminal sex hormone-binding globulin (SHBG)-like region (amino acids 243-635). 10 The SHBGlike domain of PS binds tightly to C4b...

Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain

“…Despite numerous studies describing the important protein S domains/residues for protein S cofactor function with APC (22,26,28,37), the mapping of APC residues involved in mediating the interaction with protein S has been limited. One study, however, broadly implicated residues 22-45 of the APC Gla domain in enabling protein S to act as an APC cofactor (18).…”

“…Protein S has also been reported to remove factor Xa (FXa) protection of FVa in the prothrombinase complex (21). Several protein S domains have been shown to contribute to APC cofactor activity, including the thrombin-sensitive region, EGF1, EGF2, and the Gla domain (21)(22)(23)(24)(25)(26)(27)(28). A direct role for the APC Gla domain in mediating protein S cofactor activity was suggested by a study in which a recombinant protein C chimera was generated and the protein C Gla domain (residues 1-45) was replaced by that of prothrombin (18).…”

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

“…Protein S enhancement of TFPI is known to be phospholipid dependent. To ensure that any decrease in TFPI cofactor function was not caused by a lack of binding to phospholipid membranes, we only studied protein S Gla variants already known to bind phospholipid membranes with the same affinity as WT protein S. 27,28 All protein S Gla variants enhanced TFPI to a similar or moderately decreased level as WT protein S, suggesting that the protein S Gla domain has no other function in the enhancement of TFPI than binding to phospholipids. For 2 composite Gla-variants, Face1 and GLA2, covering the majority of Gla substitutions, this was also confirmed in FXa inhibition assays (results not shown).…”

“…26,27 Only protein S Gla variants previously shown to bind normally to phospholipid membranes were evaluated for their TFPI cofactor function. 27,28 In addition, the cDNA for 3 previously described protein S/Gas6 chimeras were available. 24,25 In these, either the whole SHBG-like domain (Val243-Ser635; chimera III), or its individual LG1 (Ser283-Val459; chimera I) or LG2 (Ser460-Ser635; chimera II) subunits, were replaced by the corresponding domains of the homologous Gas6.…”

The vitamin K–dependent anticoagulant factor, protein S, inhibits multiple VEGF-A–induced angiogenesis events in a Mer- and SHP2-dependent manner

“…ProS plays a critical role as an anticoagulant, [1][2][3][4][5] whereas Gas6 has no anticoagulant function but plays an important role in platelet aggregation. 46 Human Gas6 is a potent, whereas human ProS is a weak, ligand for TAM tyrosine kinase receptors.…”