INHIBITION OF HUMAN PROTEIN C ACTIVATION BY VITAMIN K-DEPENDENT PROTEINS, INVOLVEMENT OF THE γ-CARBOXIGLUTAMIC ACID DOMAIN IN DISTINCT INTERACTIONS WITH THE HUMAN THROMBIN-THROMBOMODULIN COMPLEX AND PHOSPHOLIPIDS

1987 ◽  
Author(s):  
J M Freyssinet ◽  
A Beretz ◽  
C Klein-Soyer ◽  
J Gauchy ◽  
J Gauchy ◽  
...  
Keyword(s):  
Vitamin K ◽  
Protein C ◽  
Competitive Inhibition ◽  
Protein S ◽  
Factor X ◽  
Amidolytic Activity ◽  
Carboxyglutamic Acid ◽  
Human Thrombin ◽  
Gla Domain ◽  
Acid Domain

Protein C (PC) activation by thrombin (T) occurs at the endothelial cell (EC) surface in the presence of thrombomodulin (TM). Reconstitution of purified TM into phospholipid (PL) vesicles results in an increased activation of PC but not of Gla-domainless-PC (GD-PC), a chymotryptic derivative of PC lacking the γ-carboxyglutamic acid domain (Gla-domain). We show that several human vitamin K-dependent proteins can interfere in the activation of human PC by the human T-TM complex either in the presence or in the absence of PL. Prothrombin fragment 1 (F1), peptide 1 -4.1, the N-terminal chymotryptic Gla-domain of prothrombin (F II), FI I and factor X (FX) were able to inhibit PC activation. They had no effect on the amidolytic activity of activated PC. Non-competitive inhibition was observed in the presence of 10 yM F1 when PC, at various concentrations, was activated by the 8 μM T-TM complex, at 2 mM Ca2+, with or without PL-reconstituted TM. In any case the apparent Km remained unchanged at 2 μM. In the presence of optimal PL concentrations and in the absence of F1, the Vmax could be enhanced up to 9-fold. When F1 was present, the extents of inhibition with and without PL were comparable and resulted in a 3fold decrease of the Vmax. These effects were independent of Ca2+ between 1 and 5 mM and of T between 10 and 50 nM. At 0.6 μM PC, half maximal inhibition occurred at 8μM F1 and 1 μM peptide 1-41 in the presence or in the absence of PL. Protein S and factors VII and IX had only minimal effect. The inhibition due to F1 and FX was also noticed when PC was activated by T in the presence of cultured human vascular EC. A Ki of 4 μM could be determined for F1 with EC-bound TM. The non-competitive character was confirmed by the observation that F1 could also inhibit the activation of GD-PC by the T-TM complex. Incomplete heat-decarboxylation of F1 and FII, partially abolished their capacity to inhibit PC activation. These results suggest that the Gla domain of PC is involved in two distinct types of interactions. This vitamin K-dependent functional entity is necessary to allow the enhancement of PC activation by anionic PL and also interacts with the T-TM complex.

Unique secretion mode of human protein Z: its Gla domain is responsible for inefficient, vitamin K–dependent and warfarin-sensitive secretion

Blood ◽  
2009 ◽  
Vol 113 (16) ◽  
pp. 3857-3864 ◽  
Author(s):  
Masayoshi Souri ◽  
Hiroki Iwata ◽  
Wei Guang Zhang ◽  
Akitada Ichinose
Keyword(s):  
Vitamin K ◽  
Plasma Concentrations ◽  
Factor X ◽  
Protein Z ◽  
Highly Sensitive ◽  
Carboxyglutamic Acid ◽  
Secretion Mode ◽  
Gla Domain ◽  
The Difference ◽  
Acid Domain

Abstract Protein Z is a vitamin K–dependent plasma glycoprotein that is involved in the regulation of blood coagulation. Plasma concentrations of protein Z vary widely between subjects and are greatly reduced during warfarin therapy. We developed a sensitive and quantitative assay for protein secretion using a secretory luciferase to explore the mode of secretion of protein Z compared with that of factor X. Protein Z secretion was much less efficient than factor X and was totally dependent upon added vitamin K, while factor X secretion was not. Protein Z secretion was highly sensitive to warfarin treatment of the synthesizing cells. In contrast, although factor X secretion was not precluded by warfarin, its γ-carboxylation was completely blocked. An exchange of the propeptide and/or γ-carboxyglutamic acid domain between protein Z and factor X reproduced the inefficient and warfarin-sensitive secretion pattern of protein Z, and vice versa. Joining of the propeptide and γ-carboxyglutamic acid domain to luciferase also demonstrated that the γ-carboxyglutamic acid domain of protein Z was responsible for its warfarin-sensitive secretion. Thus, it was concluded that the difference observed in secretion patterns of protein Z and factor X was mainly based on the structure of their γ-carboxyglutamic acid domains.

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

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 122-130 ◽  
Author(s):  
François Saller ◽  
Bruno O. Villoutreix ◽  
Aymeric Amelot ◽  
Tahar Kaabache ◽  
Bernard F. Le Bonniec ◽  
...  
Keyword(s):  
Protein C ◽  
Functional Characterization ◽  
Activated Protein C ◽  
Protein S ◽  
Phospholipid Binding ◽  
Carboxyglutamic Acid ◽  
The Face ◽  
Gla Domain ◽  
Cofactor Activity ◽  
Acid Domain

Abstract We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin γ-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (GlaFII-PS) or in PS deleted of the thrombin-sensitive region (TSR) (GlaFII-ΔTSR-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 solvent-exposed 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 GlaFII-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.

scholarly journals Interference of blood-coagulation vitamin K-dependent proteins in the activation of human protein C. Involvement of the 4-carboxyglutamic acid domain in two distinct interactions with the thrombin-thrombomodulin complex and with phospholipids

1988 ◽  
Vol 256 (2) ◽  
pp. 501-507 ◽  
Author(s):  
J M Freyssinet ◽  
A Beretz ◽  
C Klein-Soyer ◽  
J Gauchy ◽  
S Schuhler ◽  
...  
Keyword(s):  
Vitamin K ◽  
Protein C ◽  
Serine Proteinase ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Human Protein ◽  
Factor X ◽  
Prothrombin Fragment ◽  
Carboxyglutamic Acid ◽  
Acid Domain

Human protein C is the precursor of a serine proteinase in plasma which contains nine 4-carboxyglutamic acid residues and functions as a potent anticoagulant. It is activated by thrombin in the presence of an essential endothelial-cell-membrane glycoprotein cofactor, thrombomodulin. In a purified human system, vitamin K-dependent proteins such as factor X, prothrombin and prothrombin fragment 1 were able to inhibit protein C activation by the thrombin-thrombomodulin complex, using either detergent-solubilized thrombomodulin or thrombomodulin reconstituted into vesicles consisting of phosphatidylcholine and phosphatidylserine (1:1, w/w). Factors VII and IX and protein S were much less efficient. Prothrombin fragment 1 behaved as a non-competitive inhibitor with apparent Ki values of 4 microM in the absence, and of 2-2.5 microM in the presence, of phospholipids. Heat decarboxylation of fragment 1 abolished its ability to interfere in protein C activation, and high phospholipid concentrations could attenuate its inhibitory effect and were responsible for a gradual loss of the non-competitive character. Fragment 1 also inhibited the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues, without any influence from phospholipids. At high thrombin concentrations, with respect to thrombomodulin, the inhibitory effect of fragment 1 was diminished. Fragment 1, at 3.8 microM, inhibited by 50% the activation of protein C (0.1 or 0.3 microM) by thrombin. These results suggest that the 4-carboxyglutamic acid domain of vitamin K-dependent proteins can act as a modulator of the protein C anticoagulant pathway through two distinct types of interaction. The functional 4-carboxyglutamic acid domain would be necessary to allow the enhancement of protein C activation in the presence of anionic phospholipids and it could recognize a phospholipid-independent binding site on the thrombin-thrombomodulin complex.

The Use of Ideal Amphipathic Helical Peptides To Reduce Hemorrhage In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3152-3152
Author(s):  
Sophie Charbonneau ◽  
Jorge G. Ganopolsky ◽  
Henry T. Pang ◽  
Pang N. Shek ◽  
Mark D. Blostein
Keyword(s):  
Factor Xa ◽  
Factor X ◽  
Amphipathic Peptide ◽  
In Vivo Experiments ◽  
Factor Ixa ◽  
Carboxyglutamic Acid ◽  
Direct Binding ◽  
Gla Domain ◽  
Acid Domain

Abstract We have previously demonstrated that a 22 amino acid ideal amphipathic peptide (IAP) of K7L15 composition dramatically accelerates both factor IXa and factor Xa activity. In the present work, we investigate the activity of IAP attached to a surface in view of designing a procoagulant surface to reduce hemorrhage. Our results show that IAP maintains its catalytic enhancing properties for factor IXa and factor Xa when attached to a surface. This enhancement is dependent on the presence of the gamma-carboxyglutamic acid domain of factor X, consistent with the hypothesis that IAP behaves as a phospholipid membrane, providing a surface for the assembly of procoagulant enzymes and substrates. To further confirm this hypothesis, we demonstrate direct binding between surface-bound IAP and the Gla domain of factor X using an ELISA-based binding assay. Based on the aforementioned evidence that immobilized IAP enhances procoagulant activity, we conducted in vivo experiments using an ear-bleeding model in rabbits. We incorporated IAP into DuraSeal, a commercially available sealing agent, and found that the addition of IAP decreases the bleeding time in rabbits by 25% (p=0.0065). In conclusion, the above data provide a rationale for designing procoagulant surfaces in vivo. Further evaluation in larger animal models is warranted.

DETECTION AND EFFECTS OF THROMBOMODULIN ACTIVITY IN CRUDE THROMBOPLASTIN PREPARATIONS FROM PLACENTA AND LUNG

1987 ◽  
Author(s):  
M L Wiesel ◽  
R Spaethe ◽  
J-M Freyssinet ◽  
T Tran ◽  
H-J Kolde ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vitamin K ◽  
Human Placenta ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Coagulation System ◽  
Factor X ◽  
Coagulant Activity ◽  
The Stability

The activation of protein C (PC) by thrombin requires the presence of an endothelial membrane cofactor, thrombomodulin (TM). Activated PC (APC) exerts its anticoagulant activity by degrading factors (F) Va and Villa in the presence of phospholipids and of a vitamin K-dependent cofactor, protein S. Tissue factor (TF) is the essential cofactor of factor Vll/VIIain the activation of factor X. TF is synthetized by several cell lines including endothelial cells. Using a specific TM assay, up to 0.85 units of TM activity could be detected in commercial thromboplastin (TP) preparations from human placenta or rabbit or porcine lung, when the amount of TP was adjusted to contain 1 unit of TF activity. Preparations from brain contained very low amounts, if any, of this activity (< 0.02 TM units). In order to evaluate the effects of the presence of TM activity in some TP preparations, the stability of F V and VIII activities was examined after activation of the coagulation system by these TP in various plasmas. PC deficient plasmas, plasmas lacking F V, VIII or IX and immunoadsorbed PC deficient plasma supplemented with purified human PC (5 Ug/ml) were used. After activation with placenta or lung TP, F V and VIII activities were markedly reduced ( ∼ 90 % reduction) in normal and hemophiliac plasmas, whereas they remained high after activation with brain TP. F V and VIII activities were preserved in protein C deficient plasma after activation by all TP preparations. The same decrease of F V and VIII activities was observed after activation of immunoadsorbed PC deficient plasma supplemented with purified PC with placenta or lung TP only. Preincubation of TP from human placenta with antibodies to human TM raised in laying hens abolished the capacity of this preparation to destroy F V activity of PC containing plasmas. These results establish the presence of TM activity in crude thromboplastin preparations from placenta or from lung. Surprisingly, this anti-coagulant activity seems to be absent from brain. TM from placenta or lung extracts is responsible for the degradation of F V and VIII.

scholarly journals The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex

1986 ◽  
Vol 238 (1) ◽  
pp. 151-157 ◽  
Author(s):  
J M Freyssinet ◽  
J Gauchy ◽  
J P Cazenave
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Amidolytic Activity ◽  
Anionic Phospholipids ◽  
Carboxyglutamic Acid ◽  
Human Thrombin ◽  
Triton X ◽  
Positive Effect ◽  
Rate Of Activation ◽  
Dependent Interaction

Human thrombomodulin, an endothelial-cell-membrane glycoprotein, has been purified from placenta by Triton X-100 extraction and by affinity chromatography on concanavalin A-Sepharose and thrombin-Sepharose. It has been characterized by its ability to promote the activation of human protein C by human alpha-thrombin in the presence of Ca2+ and fulfilled the requirements of a cofactor. Reconstitution of thrombomodulin into phospholipid vesicles containing anionic phospholipids resulted in an increased rate of activation of protein C. Cardiolipin and vesicles containing phosphatidylcholine/phosphatidylserine (1:1, w/w) were the most effective. The apparent Km of the thrombin-thrombomodulin complex for protein C was 2 microM. It was not changed in the presence of phospholipid, whereas the Vmax. could be apparently increased up to 3.2-fold depending on the phospholipid and on its concentration, the catalytic-centre activity reaching 15.7 mol of activated protein C formed/min per mol of thrombin. Above their optimal concentrations, phospholipids inhibited the amidolytic activity of activated protein C. Phospholipids had no effect on the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues. These results show that the positive effect of anionic phospholipids in the activation of protein C by the thrombin-thrombomodulin complex involves a Ca2+-dependent interaction between protein C and phospholipids. They suggest that the enhancement of thrombomodulin activity by such phospholipids may be of functional significance.

Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1416-1418 ◽  
Author(s):  
Blandine Mille-Baker ◽  
Suely M. Rezende ◽  
Rachel E. Simmonds ◽  
Philip J. Mason ◽  
David A. Lane ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Wild Type ◽  
Factor Va ◽  
Carboxyglutamic Acid ◽  
Epidermal Growth ◽  
Gla Domain ◽  
Cofactor Activity

Human protein S (PS), a cofactor of anticoagulant-activated protein C (APC), is a modular protein containing 4 epidermal growth factor (EGF)–like domains. EGF1 appears to mediate PS interaction with APC, but the roles of EGFs 2, 3, and 4 are less clear. We synthesized PS variants lacking single EGF domains (EGF2, 3, or 4) and assessed their APC cofactor activity in a factor Va inactivation assay. The variant lacking EGF2 (variant 134) showed the most dramatic loss of activity (∼10% of recombinant wild-type PS activity). Replacement of EGF2 by an additional EGF3 (variant 1334) resulted in a comparable loss of activity, suggesting that the loss of a specific rather than “spacer” function of EGF2 was responsible. We confirmed that the variant 134 had a functional γ-carboxyglutamic acid (Gla) domain and that EGF1 was correctly folded. This is the first clear evidence that EGF2 is required for the expression of PS activity.

Protein S and C4b-Binding Protein: Components Involved in the Regulation of the Protein C Anticoagulant System

1991 ◽  
Vol 66 (01) ◽  
pp. 049-061 ◽  
Author(s):  
Björn Dahlbäck
Keyword(s):  
Vitamin K ◽  
Complement System ◽  
Protein C ◽  
Protein S ◽  
High Affinity ◽  
Anticoagulant System ◽  
Dependent Protein ◽  
The Complement System ◽  
Β Chain ◽  
Dependent Domain

SummaryThe protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF)epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 γ-carboxy glutamic acid residues in the vitamin K-dependent domain, a β-hydroxylated aspartic acid in the first EGF-like domain and a β-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical α-chains and one β-chain. The α-and β-chains are linked by disulphide bridges. The cDNA cloning of the β-chain showed the α- and β-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the β-chain to contain the single protein S binding site on C4BP, whereas each of the α-chains contains a binding site for the complement protein, C4b. As C4BP lacking the β-chain is unable to bind protein S, the β-chain is required for protein S binding, but not for the assembly of the α-chains during biosynthesis. Protein S has a high affinity for negatively charged phospholipid membranes, and is instrumental in binding C4BP to negatively charged phospholipid. This constitutes a novel mechanism for control of the complement system on phospholipid surfaces. Recent findings have shown circulating C4BP to be involved in yet another calcium-dependent protein-protein interaction with a protein known as the serum amyloid P-component (SAP). The binding sites on C4BP for protein S and SAP are independent. SAP, which is a normal constituent in plasma and in tissue, is a so-called pentraxin being composed of 5 non-covalently bound 25 kDa subunits. It is homologous to C reactive protein (CRP) but its function is not yet known. The specific high affinity interactions between protein S, C4BP and SAP suggest the regulation of blood coagulation and that of the complement system to be closely linked.

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