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.

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.

scholarly journals Enhancement of Human Protein C Function by Site-directed Mutagenesis of the γ-Carboxyglutamic Acid Domain

1998 ◽  
Vol 273 (47) ◽  
pp. 31086-31091 ◽  
Author(s):  
Lei Shen ◽  
Amit M. Shah ◽  
Björn Dahlbäck ◽  
Gary L. Nelsestuen
Keyword(s):  
Protein C ◽  
Human Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carboxyglutamic Acid ◽  
Acid Domain

A SHARED EPITOPE ON HUMAN PROTEIN C, FACTOR X, FACTOR VII, AND PROTTOBIN DEFINED BY A MONOCLONAL ANTIBODY

1987 ◽  
Author(s):  
W Church ◽  
T Messier ◽  
P Howard ◽  
J Amiral ◽  
D Meyer ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Vitamin K ◽  
Light Chain ◽  
Calcium Ion ◽  
Protein C ◽  
Solid Phase ◽  
Antigenic Site ◽  
Factor Vii ◽  
Factor X ◽  
Prothrombin Fragment

A monoclonal antibody prepared against hunan protein C (HPC) was found to react with several other vitamin K-dependent blood proteins. Using a competitive inhibition solid-phase radioinminoassay with HPC, binding of 125I-HPC to the antibody was inhibited by purified prothrombin, Factor X, and Factor VII in addition to protein C. Other vitamin K-dependent proteins including Factor IX, protein S, and bone-GLA protein did not compete for binding of 125I-HPC to the antibody. The effect of calciun ion on the binding of antibody to 125I-HPC was examined in a solid-phase imnunoassay system with the antibody bound to rabbit anti-mouse inminoglobulin adsorbed to microtiter plates. In the presence of 5 mM calciun ion, radiolabeled protein C did not bind to the antibody; radiolabeled protein C did bind, however, in the presence of 5 nM EDTA suggesting that the epitope is expressed only after removal of calciun ion. The antibody bound to prothrombin and to decarboxylated prothrombin after adsorption of the antigens onto nitrocellulose indicating that the presence of GLA was not required for antibody binding. Iimunoblotting of proteins which were reduced, the peptides separated by SDS-PAGE, and transferred to nitrocellulose showed that the antibody reacts with a determinant found on the light chains of protein C and Factor X and with prothrombin Fragment 1. Comparison of the protein sequences of protein C light chain, Factor X light chain, Factor VII, and prothrombin Fragment 1 identified a segment of amino acid sequence that is highly conserved in all four proteins and might contain the antigenic site. The monoclonal antibody thus defines an antigenic determinant which is masked by calcium ion and is found on the surface of several related, yet different coagulation proteins. This antibody should prove useful in understanding the evolutionary relationships amongst the vitamin K-dependent proteins and also in understanding the effect of calcium ion on the structure of protein C, Factor X, prothrombin, Factor VII and possibly other related proteins. (Supported by NIH grant MHLBI HL35058)

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952 ◽  
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

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.

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