Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

Yegneswaran, Subramanian; Smirnov, Mikhail D.; Safa, Omid; Esmon, Naomi L.; Esmon, Charles T.; Johnson, Arthur E.

doi:10.1074/jbc.274.9.5462

Cited by 52 publications

(94 citation statements)

References 40 publications

(49 reference statements)

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“…Second, the protein S-APC interaction affects the location of the active site of APC, reducing the distance of the active site from the membrane by approximately 1.0 nm. 14,15 From the degradation curves of normal FVa and FVa Leiden obtained in the absence of protein S, it is evident that the QGNSEDY-APC is highly efficient in cleaving FVa and that both the Arg506 and the Arg306 sites are cleaved at an enhanced rate by this APC variant. The main conclusion from the FVa-degradation system is that the enhanced membrane-binding ability of QGNSEDY-APC results in increased levels of active APC on the membrane, which results in enhanced efficiency of FVa degradation.…”

Section: Discussionmentioning

confidence: 99%

“…Protein S serves as a cofactor to APC by increasing the affinity of APC for the membrane 13 and also by changing the distance of the active site of APC relative to the membrane. 14,15 In FVa degradation, APC-mediated cleavages at Arg506 and Arg306 result in inhibition of FVa activity, the Arg506 cleavage resulting in partial inhibition of FVa activity, 16,17 whereas the Arg306 cleavage completely inactivates FVa. [16][17][18] Protein S is an important cofactor for the Arg306 cleavage, whereas the Arg506 is less affected by protein S. 19 In the degradation of FVIIIa, the effect of APC is stimulated by a synergistic APC cofactor activity between protein S and the intact form of FV.…”

Section: Introductionmentioning

confidence: 99%

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Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Sun

Shen

Dahlbäck

2003

Blood

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Protein C is a member of the vitamin Kdependent protein family. Proteins in this family have similar ␥-carboxyglutamic acid (Gla)-rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 ( IntroductionNegatively charged phospholipid membranes play an important role in blood coagulation, providing a suitable surface for the assembly of enzyme-cofactor complexes, which efficiently convert circulating zymogens to active enzymes. Phosphatidylserine (PS) is a key component in the negatively charged membrane that supports the clotting reactions. [1][2][3] In most cells, PS is normally mainly present in the inner leaflet of the cell membrane, but certain cellular processes such as platelet activation result in the exposure of PS on the cell surface. The vitamin K-dependent coagulation proteins in plasma bind to the surface of negatively charged phospholipid with a common mechanism involving the ␥-carboxyglutamic acid (Gla)-rich domains of these proteins. 4 Vitamin K is required in the posttranslational carboxylation of glutamic acid residues to Gla. 5 The Gla domains comprise approximately 45 amino acid residues, and they are structurally very similar in the different vitamin K-dependent proteins. 6 Despite the extensive structural similarity between different Gla domains, they bind negatively charged phospholipid with very different affinity, with dissociation constants (K d 's) ranging more than 1000-fold. 6 This variation in phospholipid binding ability is related to amino acid sequence differences at relatively few positions. This has made it possible to modulate the phospholipid binding abilities of this group of proteins by site-directed mutagenesis. [7][8][9] The nonvitamin K-dependent cofactors of blood coagulation also interact with the phospholipid membranes, certain proteins being membrane spanning, whereas others, such as factor V (FV) and factor VIII (FVIII), contain binding sites for negatively charged phospholipid membranes. 2,10 FV circulates as a procofactor to activated FV (FVa), which functions as a cofactor to activated FX (FXa) in the activation of prothrombin to thrombin. Activated FVIII (FVIIIa) serves a similar function in the FIXa-mediated activation of FX. 2,3 The protein C anticoagulant system, which includes the vitamin K-dependent proteins protein C and protein S, regulates the activities of FVa and FVIIIa by limited proteolysis (reviewed by Dahlbäck et al 11 and Esmon et al 12 ). Protein C is activated on the endothelial cell surface by the thrombin-thrombomodulin complex, and the serine protease activated protein C (APC) down-regulates coagulation by cleaving a limited number of peptide bonds in each of FVa and FVIIIa. Protein S serves as a cofactor to APC by increasing the affinity of APC for the membrane 13 and also by changing the distance of the activ...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Sun

Shen

Dahlbäck

2003

Blood

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“…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. However, this hypothetical effect did not seem of particular importance in our experiments.…”

Section: Discussionmentioning

confidence: 99%

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

Saller

Villoutreix

Amelot

et al. 2005

Blood

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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...

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“…Binding of activated protein C to protein S has been shown to relocate the active site of this enzyme relative to the membrane surface (14). Fascinatingly, when the distance from the active site of activated protein C to the membrane surface was altered by the formation of a chimeric enzyme, it exhibited full proteolytic activity toward factor V even in the absence of protein S (15).…”

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confidence: 99%

Raising the Active Site of Factor VIIa above the Membrane Surface Reduces Its Procoagulant Activity but Not Factor VII Autoactivation

Waters

Yegneswaran

Morrissey

2006

Journal of Biological Chemistry

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Tissue factor, the physiologic trigger of blood clotting, is the membrane-anchored protein cofactor for the plasma serine protease, factor VIIa. Tissue factor is hypothesized to position and align the active site of factor VIIa relative to the membrane surface for optimum proteolytic attack on the scissile bonds of membrane-bound protein substrates such as factor X. We tested this hypothesis by raising the factor VIIa binding site above the membrane surface by creating chimeras containing the tissue factor ectodomain linked to varying portions of the membraneanchored protein, P-selectin. The tissue factor/P-selectin chimeras bound factor VIIa with high affinity and supported full allosteric activation of factor VIIa toward tripeptidyl-amide substrates. That the active site of factor VIIa was raised above the membrane surface when bound to tissue factor/P-selectin chimeras was confirmed using resonance energy transfer techniques in which appropriate fluorescent dyes were placed in the active site of factor VIIa and at the membrane surface. The chimeras were deficient in supporting factor X activation by factor VIIa due to decreased k cat . The chimeras were also markedly deficient in clotting plasma, although incubating factor VII or VIIa with the chimeras prior to the addition of plasma restored much of their procoagulant activity. Interestingly, all chimeras fully supported tissue factor-dependent factor VII autoactivation. These studies indicate that proper positioning of the factor VII/VIIa binding site on tissue factor above the membrane surface is important for efficient rates of activation of factor X by this membrane-bound enzyme/cofactor complex. Tissue factor (TF)3 is the cell surface, type I integral membrane protein that triggers blood clotting in normal hemostasis and many thrombotic diseases (1, 2). TF initiates blood coagulation by binding the plasma serine protease, factor VIIa (fVIIa). The membrane-bound complex of fVIIa and TF can therefore be considered a two-subunit enzyme, with fVIIa as the catalytic subunit and TF as the regulatory subunit. The major substrates for the fVIIa-TF complex are factors X (fX) and IX (fIX), which are converted to active serine proteases by limited proteolysis. Activation of fX and fIX leads to propagation of the clotting cascade and, ultimately, clotted fibrin and activated platelets. Binding of fVIIa to TF embedded in a suitable phospholipid membrane increases its proteolytic activity many thousandfold (3, 4). For optimal fVIIa-TF enzymatic activity, the phospholipid bilayer must contain negatively charged phospholipid, most especially phosphatidylserine (PS) (5, 6). How PS augments the enzymatic activity of fVIIa-TF is not completely understood, but one role is to promote interaction of vitamin K-dependent clotting factors such as fVII, fIX, and fX with phospholipid surfaces (7). These proteins all have an N-terminal domain containing multiple ␥-carboxyglutamate residues (Gla domain) that confers reversible binding to membranes containing PS. Incorporating PS in...

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Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

Cited by 52 publications

References 40 publications

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

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

Raising the Active Site of Factor VIIa above the Membrane Surface Reduces Its Procoagulant Activity but Not Factor VII Autoactivation

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