Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

Abstract The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

Blood ◽

10.1182/blood.v86.7.2653.bloodjournal8672653 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2653-2660

Author(s):

SJ Koppelman ◽

C van't Veer ◽

JJ Sixma ◽

BN Bouma

Keyword(s):

Monoclonal Antibodies ◽

Factor Viii ◽

Protein C ◽

Binding Protein ◽

Intrinsic Factor ◽

Protein S ◽

Inhibitory Effect ◽

Factor X ◽

Beta Chain ◽

Factor X Activation

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

Blood ◽

10.1182/blood.v86.3.1062.bloodjournal8631062 ◽

1995 ◽

Vol 86 (3) ◽

pp. 1062-1071 ◽

Cited By ~ 2

Author(s):

SJ Koppelman ◽

TM Hackeng ◽

JJ Sixma ◽

BN Bouma

Keyword(s):

Factor Viii ◽

Protein C ◽

Intrinsic Factor ◽

Activated Protein C ◽

Protein S ◽

Inhibitory Effect ◽

Factor X ◽

Ic50 Values ◽

Factor X Activation ◽

Factor X Activating Complex

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

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Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

Blood ◽

10.1182/blood.v86.3.1062.1062 ◽

1995 ◽

Vol 86 (3) ◽

pp. 1062-1071 ◽

Cited By ~ 45

Author(s):

SJ Koppelman ◽

TM Hackeng ◽

JJ Sixma ◽

BN Bouma

Keyword(s):

Factor Viii ◽

Protein C ◽

Intrinsic Factor ◽

Activated Protein C ◽

Protein S ◽

Inhibitory Effect ◽

Factor X ◽

Ic50 Values ◽

Factor X Activation ◽

Factor X Activating Complex

Abstract Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

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The Mechanism of Protein S as An Anticoagulant Independent of Activated Protein C.

Blood ◽

10.1182/blood.v114.22.3186.3186 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3186-3186

Author(s):

Rinku Majumder

Keyword(s):

Venous Thrombosis ◽

Protein C ◽

Activated Protein C ◽

Protein S ◽

Factor X ◽

Protein S Deficiency ◽

Factor Ixa ◽

S Deficiency ◽

Factor X Activation

Abstract 3186 Poster Board III-123 Thrombosis is a serious problem in the United States. The overall estimated incidence (annual occurrence) of deep venous thrombosis is 1 episode for every 1000 persons. Protein S, a vitamin K-dependent protein, is one of the natural anticoagulants found in the blood. Deficiency of protein S is most common protein deficiencies associated with familial venous thrombosis There are studies that suggest an association between arterial thrombosis (stroke, heart attack) in patients with protein S deficiency. At this time, the exact role of protein S deficiency and its relative importance in arterial disease is still being explored by physicians and scientists. Protein S is known as a non-enzymatic cofactor of activated Protein C in the inactivation of factors Va and VIIIa, as part of a negative feedback loop to regulate blood coagulation. Plasma coagulation assays in the absence of activated protein C suggest that Protein S may have other anticoagulant role(s). For example, it has been suggested that Protein S down-regulates thrombin generation by stimulating FXa inhibition by the tissue factor pathway inhibitor (Rosing, J., et al., Thromb Res, 2008. 122 Suppl 1: p. S60-3). It has also been proposed that protein S can directly inhibit the intrinsic Xase complex (Takeyama, M., et al.. Br J Haematol, 2008. 143(3): p. 409-20). But the exact mechanism of how Protein S exerts its anticoagulant effect on factor IXa/VIIIa complex is still unclear. In order to determine the role of Protein S as an anticoagulant in the intrinsic Xase Complex, we have used C6PS (a small six carbon chain synthetic Phosphatidylserine (PS) molecule) that does not occur in vivo, but has been used as a powerful tool in demonstrating the regulation of both factors Xa and Va by binding of molecular PS. Soluble lipid binding can offer invaluable insights into events that would be next to impossible to document on a membrane surface which is complicated as it has surface condensation effect and allosteric effects of different factors. We focus here on the conformation changes of the proteins by using C6PS as a tool. We have determined the binding of Protein S with C6PS by using tryptophan fluorescence and observed a stoichiometric Kd of ∼180 μM.We checked for micelles formation under each experimental condition. We have also determined the direct binding of factor IXa with Protein S by using DEGR-IXa ((5-(dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl chloromethyl ketone) in the presence and absence of C6PS. Our results show that the affinity of binding of DEGR-IXa to Protein S in the presence of C6PS is ∼22 fold tighter (Kd ∼15 nM compared to 324 nM) than without C6PS. We also measured the rate of factor X activation by factor IXa with the addition of increasing concentration of C6PS in the presence and absence of Protein S. We observed that Protein S decreased factor IXa mediated factor X activation by 14 fold. We had previously shown that apparent Kd of factor IXa binding to C6PS during factor X activation was ∼125 μM. But addition of Protein S had an effect on the apparent Kd as it increased to 700 μM indicating the affinity of factor IXa towards C6PS was decreased with the addition of Protein S during factor X activation. From these data we can speculate that Protein S induces a conformational change in factor IXa in the presence of C6PS which may affect the interaction of factor IXa with factor VIIIa, thus affecting the intrinsic Xase complex. Using this useful tool (C6PS), we will characterize the anticoagulant role of Protein S in the intrinsic Xase complex which in turn will give us some insights into this important protein which is a crucial target for therapeutic drugs for venous thrombosis. Disclosures No relevant conflicts of interest to declare.

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BINDING SITE OF VITAMIN K-DEPENDENT PROTEIN S ON C4b-BINDING PROTEIN

10.1055/s-0038-1644637 ◽

1987 ◽

Cited By ~ 1

Author(s):

K Suzuki ◽

J Nishioka ◽

H Kusumoto ◽

Y Deyashiki

Keyword(s):

Monoclonal Antibodies ◽

Binding Site ◽

Disulfide Bonds ◽

Protein C ◽

Binding Protein ◽

Gel Filtration ◽

Activated Protein C ◽

Protein S ◽

Cofactor Activity ◽

Carboxyl Terminal

Protein S, a cofactor for activated protein C, reversibly complexes with a regulatory complement component C4b-binding protein (C4bp) in plasma. In plasma of patients with congenital protein S deficiency, most protein S exists as a complex with C4bp, which has no cofactor activity. C4bp (Mw 550,000) is composed of approximately seven subunits with Mw 75,000 which are linked by disulfide bonds near the carboxy1-terminus. We report here about the complex formation between protein S and C4bp particularly on the binding site of protein S on C4bp molecule. Protein S and C4bp were purified from human plasma. Seventeen mouse monoclonal antibodies against C4bp were prepared. Chymotrypsin-digested C4bp was separated on gel filtration into a fragment with Mw 160,000 derived from the carboxyl-terminal core of the intact C4bp and fragments with Mw 48,000 from the amino-terminus. The carboxy1-terminal fragment with Mw 160,000 was found to be composed of approximately seven polypeptides with Mw 25,000, which were linked by disulfide bonds.The experiments using these fragments and the monoclonal antibodies showed that: (1) Protein S bound not only to the intact C4bp, but also to the fragment with Mw 160,000. (2) The fragment with Mw 160,000 inhibited the binding of protein S to C4bp, but the fragment with Mw 48,000 did not. (3) One of the seventeen monoclonal antibodies blocked the inhibition of C4bp on the cofactor activity of protein S. (4) This antibody inhibited C4bp binding to protein S. (5) The antibody bound to the fragment with Mw 160,000. Based on these results, protein S was suggested to lose its cofactor activity for activated protein C by binding to the carboxyl-terminal core of C4bp where seven subunits are linked by disulfide bonds.

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Functional Effects of Anticardiolipin Antibodies

Lupus ◽

10.1177/096120339600500507 ◽

1996 ◽

Vol 5 (5) ◽

pp. 372-377 ◽

Cited By ~ 10

Author(s):

EN Harris ◽

SS Pierangeli

Keyword(s):

Lupus Anticoagulant ◽

Protein C ◽

Inhibitory Effect ◽

Anticardiolipin Antibodies ◽

Factor X ◽

Free System ◽

Immunoglobulin Preparations ◽

Coagulation Protein ◽

Factor X Activation

The ‘lupus anticoagulant’ phenomenon is the best documented functional effect of antiphospholipid (aPL) antibodies, occurring either by inhibition of the prothrombinase and/or Factor X activation reactions. Understanding the mechanism by which aPL antibodies inhibit phospholipid dependent coagulation reactions may yield important clues about their ‘thrombogenic effects’ in vivo. We conducted a series of studies to determine the specificity, diversity, and mechanism by which aPL antibodies inhibit phospholipid dependent reactions. Results showed that purified immunoglobulins with lupus anticoagulant and anti-cardiolipin activities were absorbed by negatively charged phospholipids and both activities were recovered from the phospholipid-antibody precipitate. Purified aPL antibodies inhibited the prothrombinase reaction in a plasma free system in which β2-glycoprotein 1 (β2-GP1) was absent. Affinity purified aPL antibodies had 25–50 times the inhibitory activity of immunoglobulin preparations. The phospholipid binding proteins, β2-GP1 and placental anticoagulant protein I (PAP I), independently inhibited the prothrombinase reaction, and when these proteins were combined with aPL, inhibition of the prothrombinase reaction was additive. Antibodies of syphilis had no inhibitory effect, partially accounted for by lack of specificity for phosphotidylserine (PS). Although aPL antibodies inhibited the protein C activation reaction, there was no correlation of these activities with inhibition of the prothrombinase reaction. Together, these results show that aPL exert their effects by interaction with negatively charged phospholipids, in particular phosphotidylserine, but lack of correlation between inhibition of the prothrombinase and protein C activation reactions, suggests that the nature of the coagulation protein is also important.

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Regulation of factor VIIIa by human activated protein C and protein S: inactivation of cofactor in the intrinsic factor Xase

Blood ◽

10.1182/blood.v95.5.1714.005k40_1714_1720 ◽

2000 ◽

Vol 95 (5) ◽

pp. 1714-1720 ◽

Cited By ~ 59

Author(s):

Lynn M. O'Brien ◽

Maria Mastri ◽

Philip J. Fay

Keyword(s):

Protein C ◽

Intrinsic Factor ◽

Activated Protein C ◽

Protein S ◽

Similar Rate ◽

Factor X ◽

Preferential Cleavage ◽

Factor Ixa ◽

Factor Viiia ◽

Reduced Rate

Factor VIIIa is a trimer of A1, A2, and A3-C1-C2 subunits. Inactivation of the cofactor by human activated protein C (APC) results from preferential cleavage at Arg336 within the A1 subunit, followed by cleavage at Arg562 bisecting the A2 subunit. In the presence of human protein S, the rate of APC-dependent factor VIIIa inactivation increased several-fold and correlated with an increased rate of cleavage at Arg562. (Active site-modified) factor IXa, blocked cleavage at the A2 site. However, APC-catalyzed inactivation of factor VIIIa proceeded at a similar rate independent of factor IXa, consistent with the location of the preferential cleavage site within the A1 subunit. Addition of protein S failed to increase the rate of cleavage at the A2 site when factor IXa was present. In the presence of factor X, cofactor inactivation was inhibited, due to a reduced rate of cleavage at Arg336. However, inclusion of protein S restored near original rates of factor VIIIa inactivation and cleavage at the A1 site, thus overcoming the factor X-dependent protective effect. These results suggest that in the human system, protein S stimulates APC-catalyzed factor VIIIa inactivation by facilitating cleavage of A2 subunit (an effect retarded in the presence of factor IXa), as well as abrogating protective interactions of the cofactor with factor X.

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APC Resistance and other Haemostatic Variables during Pregnancy and Puerperium

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614518 ◽

1999 ◽

Vol 81 (04) ◽

pp. 527-531 ◽

Cited By ~ 114

Author(s):

U. Kjellberg ◽

N.-E. Andersson ◽

S. Rosén ◽

L. Tengborn ◽

M. Hellgren

Keyword(s):

Factor Viii ◽

Plasminogen Activator ◽

Protein C ◽

Activated Protein C ◽

Plasminogen Activator Inhibitor ◽

Protein S ◽

Reference Level ◽

Soluble Fibrin ◽

Prothrombin Fragment ◽

D Dimer

SummaryForty-eight healthy pregnant women were studied prospectively and longitudinally. Blood sampling was performed at 10-15, 23-25, 32-34 and 38-40 weeks of gestation, within one week and at eight weeks postpartum. Classic and modified activated protein C ratio decreased as pregnancy progressed. In the third trimester 92% of the ratios measured with the classic test were above the lower reference level whereas all modified test ratios were normal. Slight activation of blood coagulation was shown with increased levels of prothrombin fragment 1+2, soluble fibrin and D-dimer. Fibrinogen, factor VIII and plasminogen activator inhibitor type 1 and type 2 increased. Protein S and tissue plasminogen activator activity decreased. Protein C remained unchanged. No correlation was found between the decrease in classic APC ratio and changes in factor VIII, fibrinogen, protein S, prothrombin fragment 1+2 or soluble fibrin, nor between the increase in soluble fibrin and changes in prothrombin fragment 1+2, fibrinogen and D-dimer.

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Plasma Levels of Protein S, Protein C, and Factor X: Effects of Sex, Hormonal State and Age

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649925 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1271-1275 ◽

Cited By ~ 36

Author(s):

C M A Henkens ◽

V J J Bom ◽

W van der Schaaf ◽

P M Pelsma ◽

C Th Smit Sibinga ◽

...

Keyword(s):

Regression Analysis ◽

Postmenopausal Women ◽

Protein C ◽

Blood Donors ◽

Premenopausal Women ◽

Protein S ◽

The Other ◽

Factor X ◽

Normal Ranges

SummaryWe measured total and free protein S (PS), protein C (PC) and factor X (FX) in 393 healthy blood donors to assess differences in relation to sex, hormonal state and age. All measured proteins were lower in women as compared to men, as were levels in premenopausal women as compared to postmenopausal women. Multiple regression analysis showed that both age and subgroup (men, pre- and postmenopausal women) were of significance for the levels of total and free PS and PC, the subgroup effect being caused by the differences between the premenopausal women and the other groups. This indicates a role of sex-hormones, most likely estrogens, in the regulation of levels of pro- and anticoagulant factors under physiologic conditions. These differences should be taken into account in daily clinical practice and may necessitate different normal ranges for men, pre- and postmenopausal women.

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The Influence of Insulin, ß-Estradiol, Dexamethasone and Thyroid Hormone on the Secretion of Coagulant and Anticoagulant Proteins by HepG2 Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649798 ◽

1995 ◽

Vol 74 (02) ◽

pp. 686-692 ◽

Cited By ~ 11

Author(s):

René W L M Niessen ◽

Birgit A Pfaffendorf ◽

Augueste Sturk ◽

Roy J Lamping ◽

Marianne C L Schaap ◽

...

Keyword(s):

Thyroid Hormone ◽

Protein C ◽

Protein Production ◽

Culture Medium ◽

Protein S ◽

Cell Protein ◽

Hepatoma Cell Line ◽

Factor X ◽

Factor Ii ◽

At Iii

SummaryAs a basis for regulatory studies on the influence of hormones on (anti)coagulant protein production by hepatocytes, we examined the amounts of the plasma proteins antithrombin III (AT III), protein C, protein S, factor II, factor X, fibrinogen, and prealbumin produced by the hepatoma cell line HepG2, into the culture medium, in the absence and presence of insulin, β-estradiol, dexamethasone and thyroid hormone. Without hormones these cells produced large amounts of fibrinogen (2,452 ± 501 ng/mg cell protein), AT III (447 ± 16 ng/mg cell protein) and factor II (464 ± 31 ng/mg cell protein) and only small amounts of protein C (50 ± 7 ng/mg cell protein) and factor X (55 ± 5 ng/mg cell protein). Thyroid hormone had a slight but significant effect on the enrichment in the culture medium of the anticoagulant protein AT III (1.34-fold) but not on protein C (0.96-fold) and protein S (0.91-fold). This hormone also significantly increased the amounts of the coagulant proteins factor II (1.28-fold), factor X (1.45-fold) and fibrinogen (2.17-fold). Insulin had an overall stimulating effect on the amounts of all the proteins that were investigated. Neither dexamethasone nor ß-estradiol administration did substantially change the amounts of these proteins.We conclude that the HepG2 cell is a useful tool to study the hormonal regulation of the production of (anti)coagulant proteins. We studied the overall process of protein production, i.e., the amounts of proteins produced into the culture medium. Detailed studies have to be performed to establish the specific hormonal effects on the underlying processes, e.g., transcription, translation, cellular processing and transport, and secretion.

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