Protein S Gla-domain mutations causing impaired Ca2+-induced phospholipid binding and severe functional protein S deficiency

Rezende, Suely Meireles; Lane, David A.; Mille-Baker, Blandine; Samama, M; Conard, J.; Simmonds, Rachel E.

doi:10.1182/blood-2002-03-0909

Cited by 24 publications

(37 citation statements)

References 44 publications

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“…Concentrated recombinant ProS from culture media was diluted in imidazole buffer with saline to provide a range of ProS concentrations (0, 10, 20, 40 U=dl). Then, a factor Va inactivation assay was performed by traditional methods, as previously described [41]. Briefly, recombinant ProS of each concentration was incubated with 0.1 nM APC, 3 nM factor Va, and 25 mM phospholipid vesicles (Boatman Biotech, Shanghai, China) for 5 min at 37 C in imidazole buffer.…”

Section: Factor Va Inactivation Assaymentioning

confidence: 99%

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Molecular basis of protein S deficiency in China

et al. 2013

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Protein S (ProS) is a physiological inhibitor of coagulation with an important function in the downregulation of thrombin generation. ProS deficiency is a major risk factor for venous thrombosis. This study enrolled 40 ProS-deficient probands to investigate the molecular basis of hereditary ProS deficiency in Chinese patients. A mutation analysis was performed by resequencing the PROS1 gene. Large deletions were identified by multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 20 different mutations, including 15 novel mutations, were identified in 21 of the 40 index probands. Small mutations were detected in 18 (45.0%) probands, and large deletions were found in 3 (7.5%) probands, leaving 19 (47.5%) patients without causative variants. To evaluate the functional consequences of 2 novel missense variants, ex vivo thrombin-generation assays, bioinformatics tools, and in vitro expression studies were employed. The p.Asn365Lys ProS variant was found to have moderately impaired secretion and reduced activated protein C cofactor activity. In contrast, the p.Pro410His mutant appeared to have severely impaired secretion but full anticoagulant activity. This study is the largest investigation of ProS deficiency in China and the first investigation of the influence of Type I ProS missense mutations on the global level of coagulation function. The p.K196E mutation, which is common in the neighboring Japanese population, was not found in our Chinese population, and null mutations were common in our Chinese population but not common in Japan. Further genetic analysis is warranted to understand the causes of ProS deficiency in patients without a genetic explanation. Am. J.

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Section: Factor Va Inactivation Assaymentioning

confidence: 99%

“…Culture medium was collected 48 hr after transfection. Stable transfection was performed in HEK-293 cells with 500 lg=ml G-418 (Sigma-Aldrich, St. Louis, MO), as described previously [41]. Neomycin-resistant cells were grown to confluence.…”

Section: Recombinant Pros Expression Experimentsmentioning

confidence: 99%

Molecular basis of protein S deficiency in China

et al. 2013

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“…The first cluster comprised residues 10, 33, 34, 35, 36, and 39 and is referred to here as face 1. The second cluster involved residues 21,23,24,28,33,34,35,41, and 45 and is referred to as face 2. When replaced by the corresponding prothrombin amino acids, the substitutions occurring on face 1 and face 2 of PS would be of sufficient nature to disrupt putative interactions of the PS Gla domain with APC.…”

Section: Structural Analysismentioning

confidence: 99%

“…Thus, we decided to replace the face 1 and face 2 residues of Gla FII -PS. In this mutant, designated F12 PS -Gla FII -PS, residues 21,23,24,28,33,34,35,36,39,41, and 45 were replaced by the corresponding residues of PS ( Figure 1). This mutant comprised an Ala residue of prothrombin origin at position 42, disrupting the integrity of the aromatic amino acid stack Pro35-Pro42 loop that is potentially important for PS functions.…”

Section: Recovery Of Apc Cofactor Activity Of Gla Fii -Ps By Mutationmentioning

confidence: 99%

“…27 In addition, naturally occurring mutations in the PS Gla domain are associated with qualitative (functional) defects in APC cofactor activity, probably because of impaired phospholipid binding. 28 We previously prepared a recombinant analog of PS lacking the TSR (TSR-less) to investigate the role of the TSR. 29 Because TSR-less failed to bind to negatively charged phospholipid vesicles or to activated platelets, 29 we attempted to restore the ability of TSR-less to bind to phospholipids by preparing a chimeric PS molecule in which the TSR was deleted and the Gla domain was replaced by the Gla domain of prothrombin (FII) (Gla FII -⌬TSR-PS).…”

Section: Introductionmentioning

confidence: 99%

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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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Molecular diversity and thrombotic risk in protein S deficiency: The PROSIT study

et al. 2005

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The Protein S Italian Team (PROSIT) enrolled 79 protein S (PS) deficient families and found 38 PROS1 variations (19 novel) in 53 probands. Of these, 23 variants were selected for expression in'vitro, to evaluate their role as possible causative variants. Transient expression showed high secretion levels (>75%) for three variants, which were considered neutral. Seven missense and five nonsense variants showed low (

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Protein S Gla-domain mutations causing impaired Ca2+-induced phospholipid binding and severe functional protein S deficiency

Cited by 24 publications

References 44 publications

Molecular basis of protein S deficiency in China

Molecular basis of protein S deficiency in China

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

Molecular diversity and thrombotic risk in protein S deficiency: The PROSIT study

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