Localization of a Hydrophobic Binding Site for Anticoagulant Protein S on the β-Chain of Complement Regulator C4b-binding Protein

Webb, Joanna; Villoutreix, Bruno O.; Dahlbäck, Björn; Blom, Anna M.

doi:10.1074/jbc.m006541200

Cited by 22 publications

(36 citation statements)

References 41 publications

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“…The very high affinity of the interaction between protein S and C4BP, its hydrophobic nature, and the very slow rate of dissociation of the complex suggest that the two molecules circulate in blood as a stable complex (2,35,37). In protein S, both LG modules are involved in the binding of C4BP, suggesting that important residues for the interaction are distributed on both modules (37).…”

Section: Discussionmentioning

confidence: 99%

“…The ␣ and ␤ chains are composed of repeating domains of about 60 amino acids denoted complement control protein (CCP) domains. The binding site for protein S is contained in CCP1-CCP2 of the ␤ chain (33)(34)(35). Using a molecular model of the ␤ chain in combination with recombinant ␤ chain expression and site-directed mutagenesis, it has been shown that a solvent-exposed hydrophobic patch in CCP1 lined by a positively charged area on an otherwise negatively charged surface forms the key binding site for protein S (35).…”

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confidence: 99%

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Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein

Giri¹,

Linse²,

Frutos³

et al. 2002

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein

Giri¹,

Linse²,

Frutos³

et al. 2002

Journal of Biological Chemistry

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“…The PS binding is totally dependent on the presence of the ␤-chain, the PS binding site being located mainly in the N-terminal CCP (10 -12). The binding site has been further narrowed down by site-directed mutagenesis, highlighting the importance of four amino acids in the N-terminal CCP domain (13).…”

Section: Protein S (Ps)mentioning

confidence: 99%

Dependence on Vitamin K-dependent Protein S for Eukaryotic Cell Secretion of the β-Chain of C4b-binding Protein

Carlsson

Dahlbäck

2010

Journal of Biological Chemistry

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The anticoagulant vitamin K-dependent protein S (PS) circulates in plasma in two forms, 30% free and 70% being bound to the complement regulatory protein C4b-binding protein (C4BP). The major C4BP isoform consists of 7 ␣-chains and 1 ␤-chain (C4BP␤ ؉ ), the chains being linked by disulfide bridges.PS binds to the ␤-chain with high affinity. In plasma, PS is in molar excess over C4BP␤ ؉ and due to the high affinity, all C4BP␤ ؉ molecules contain a bound PS. Taken together with the observation that PS-deficient patients have decreased levels of C4BP␤ ؉ , this raises the question of whether PS is important for secretion of the ␤-chain from the cell. To test this hypothesis, HEK293 cells were stably and transiently transfected with ␤-chain cDNA in combinations with cDNAs for PS and/or the ␣-chain. The concentration of ␤-chains in the medium increased after co-transfection with PS cDNA, but not by ␣-chain cDNA, suggesting secretion of the ␤-chains from the cells to be dependent on concomitant synthesis of PS, but not of the ␣-chains. Thus, ␤-chains that were not disulfide-linked to the ␣-chains were secreted in complex with PS, either as monomers or dimers. Pulse-chase demonstrated that the complexes between PS and ␤-chain were formed intracellularly, in the endoplasmic reticulum. In conclusion, our results demonstrate that successful secretion of ␤-chains depends on intracellular complex formation with PS, but not on the ␣-chains. This provides an explanation for the decreased ␤-chain levels observed in PS-deficient patients. Protein S (PS)2 is an anticoagulant vitamin K-dependent protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa (1). Recent studies have also identified PS as a cofactor to tissue factor pathway inhibitor (2, 3). Tissue factor pathway inhibitor regulates the tissue factor-dependent initial step of coagulation by inactivating coagulation factors VIIa and Xa. In human plasma, 60 -70% of PS circulates bound to the complement regulator C4b-binding protein (C4BP) (4), and this fraction has impaired anticoagulant functions (5). PS is a 635-amino-acid-long single chain molecule with several distinct domains. The N-terminal Gla domain is followed by a thrombin-sensitive region, four epidermal growth factor (EGF)-like domains, and the large C-terminal sex hormone-binding globulin (SHBG)-like region, which comprises two laminin G-type domains. The binding site in PS for C4BP is located in the SHBG region of PS, and both laminin G domains are required (6). The binding site in PS has not been narrowed down further, even though there are several studies suggesting different regions of the SHBG domain to be of importance (7,8).C4BP acts as a cofactor for the down-regulation of the complement cascade and shares structural features with several other proteins participating in this system. It is composed of multiple disulfide-linked subunits (␣-and ␤-chains), each containing repeats of complement control protein (CCP) domains (9). There are several isoform...

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“…The ␤-chain CCP1 binds protein S, with a hydrophobic patch in CCP1 involving I16, V18, V31, and I33 being particularly important (Figure 4). 17,65 Recently it was found that protein S binds to the negatively charged phospholipid surface that is exposed on apoptotic cells and can mediate phagocytosis of the apoptotic cell. 66,67 In contrast, binding of the protein S-C4BP complex was found to inhibit the phagocytic process.…”

Section: Protein S Affects the Complement System And The Phagocytosismentioning

confidence: 99%

Regulation of Blood Coagulation by the Protein C Anticoagulant Pathway

Dahlbäck

Villoutreix

2005

ATVB

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Abstract-The protein C system provides important control of blood coagulation by regulating the activities of factor VIIIa (FVIIIa) and factor Va (FVa), cofactors in the activation of factor X and prothrombin, respectively. The system comprises membrane-bound and circulating proteins that assemble into multi-molecular complexes on cell surfaces. Vitamin K-dependent protein C, the key component of the system, circulates in blood as zymogen to an anticoagulant serine protease. It is efficiently activated on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The endothelial protein C receptor (EPCR) further stimulates the protein C activation. Activated protein C (APC) together with its cofactor protein S inhibits coagulation by degrading FVIIIa and FVa on the surface of negatively charged phospholipid membranes. Efficient FVIIIa degradation by APC requires not only protein S but also intact FV, which like thrombin is a Janus-faced protein with both procoagulant and anticoagulant potential. In addition to its anticoagulant properties, APC has antiinflammatory and antiapoptotic functions, which are exerted when APC binds to EPCR and proteolytic cleaves protease-activated receptor 1 (PAR-1). The protein C system is physiologically important, and genetic defects affecting the system are the most common risk factors of venous thrombosis. The proteins of the protein C system are composed of multiple domains and the 3-dimensional structures of several of the proteins are known. The molecular recognition of the protein C system is progressively being unraveled, giving us new insights into this fascinating and intricate molecular scenario at the atomic level. Key Words: factor V Ⅲ protein C Ⅲ protein S Ⅲ thrombomodulin P latelet-dependent primary hemostasis and blood coagulation have evolved as important defense mechanisms against bleeding. The initial occlusion of a vascular lesion is provided by the formation of the platelet plug. 1 This is temporally coordinated with the initiation of the coagulation system by the exposure of tissue factor (TF) to blood and the subsequent binding to TF of circulating factor VIIa (FVIIa). 2 The FVIIa-TF complex efficiently converts factor IX (FIX) and factor X (FX) to active enzymes FIXa and FXa, which together with factor VIIIa (FVIIIa) and factor Va (FVa), respectively, propagate the coagulation process (Figure 1). Factor VIII (FVIII) and factor V (FV) circulate in blood as high-molecular-weight inactive procofactors, which are converted into their active forms, FVIIIa and FVa, early during the coagulation process by thrombin or FXa. FVIIIa and FVa bind to negatively charged phospholipid membranes, eg, on activated platelets, and form complexes with FIXa and FXa, respectively. 3-5 The FIXa-FVIIIa complex (tenase) activates FX, whereas the FXa-FVa complex (prothrombinase) converts prothrombin to thrombin. In both the tenase and the prothrombinase complexes, the cofactors increase the catalytic efficiency of the respective enzyme by several orders ...

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Localization of a Hydrophobic Binding Site for Anticoagulant Protein S on the β-Chain of Complement Regulator C4b-binding Protein

Cited by 22 publications

References 41 publications

Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein

Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein

Dependence on Vitamin K-dependent Protein S for Eukaryotic Cell Secretion of the β-Chain of C4b-binding Protein

Regulation of Blood Coagulation by the Protein C Anticoagulant Pathway

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