Possible mechanisms contributing to oxidative inactivation of activated protein C: Molecular dynamics study

2008 ◽  
Vol 100 (07) ◽  
pp. 18-25 ◽  
Author(s):  
Armen Nalian ◽  
Alexei V. Iakhiaev
Keyword(s):  
Molecular Dynamics ◽  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Md Simulations ◽  
Catalytic Triad ◽  
Methionine Oxidation ◽  
Oxidative Inactivation ◽  
Apc Resistance ◽  
Methionine Residues

SummaryActivated protein C (APC) is a serine protease, an effector enzyme of the natural anticoagulant pathway. APC is approved for treatment of severe sepsis characterized by the increased concentrations of H2O2 and hypochlorite. We found that treatment of APC with these oxidants markedly inhibits the cleavage of the APC-specific chromogenic substrate, suggesting that oxidants can induce changes in the structure of the active site of APC. Resistance of oxidant-treated APC to chemical digestion with cyanogen bromide (CNBr) implies that methionine oxidation can at least in part be responsible for inhibition of APC. Since methionine residues, the main targets of oxidants in APC, are not included in the active site, we hypothesize that oxidation induces allosteric changes in the architecture of the catalytic triad of APC. Using molecular dynamics (MD) simulations we found that methionine oxidation alters the distance between cSer 195Oγ and cHis57 Nε2 atoms placing them in positions unfavorable for the catalysis. At the same time, neither distances between Cα atoms of the catalytic triad cAsp102-cHis57-cSer195, nor the overall structure of APC changed significantly after oxidation of the methionine residues. Disruption of the H-bond between Nδ1 of cHis57 and carboxyl group of cAsp 102, which can take place during the hypochlorite-induced modification of cHis57,dramatically changed the architecture of the catalytic triad in oxidized APC. This mechanism could contribute to APC inactivation by hypochlorite concurrently with methionine oxidation. These are novel findings, which describe potentially pathophysiologically relevant changes in the functional stability of APC exposed to the oxidative stress.

“Pseudo Homozygous” Activated Protein C Resistance due to Double Heterozygous Factor V Defects (Factor V Leiden Mutation and Type I Quantitative Factor V Defect) Associated with Thrombosis: Report of Two Cases Belonging to Two Unrelated Kindreds

1996 ◽  
Vol 75 (03) ◽  
pp. 422-426 ◽  
Author(s):  
Paolo Simioni ◽  
Alberta Scudeller ◽  
Paolo Radossi ◽  
Sabrina Gavasso ◽  
Bruno Girolami ◽  
...  
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Type I ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
Thrombotic Risk ◽  
Apc Resistance ◽  
Quantitative Factor

SummaryTwo unrelated patients belonging to two Italian kindreds with a history of thrombotic manifestations were found to have a double heterozygous defect of factor V (F. V), namely type I quantitative F. V defect and F. V Leiden mutation. Although DNA analysis confirmed the presence of a heterozygous F. V Leiden mutation, the measurement of the responsiveness of patients plasma to addition of activated protein C (APC) gave results similar to those found in homozygous defects. It has been recently reported in a preliminary form that the coinheritance of heterozygous F. V Leiden mutation and type I quantitative F. V deficiency in three individuals belonging to the same family resulted in the so-called pseudo homozygous APC resistance with APC sensitivity ratio (APC-SR) typical of homozygous F. V Leiden mutation. In this study we report two new cases of pseudo homozygous APC resistance. Both patients experienced thrombotic manifestations. It is likely that the absence of normal F. V, instead of protecting from thrombotic risk due to heterozygous F. V Leiden mutation, increased the predisposition to thrombosis since the patients became, in fact, pseudo-homozygotes for APC resistance. DNA-analysis is the only way to genotype a patient and is strongly recommended to confirm a diagnosis of homozygous F. V Leiden mutation also in patients with the lowest values of APC-SR. It is to be hoped that no patient gets a diagnosis of homozygous F. V Leiden mutation based on the APC-resi-stance test, especially when the basal clotting tests, i.e., PT and aPTT; are borderline or slightly prolonged.

Molecular Characterization of a Type I Quantitative Factor V Deficiency in a Thrombosis Patient that Is “Pseudo Homozygous” for Activated Protein C Resistance

1997 ◽  
Vol 77 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Joan F Guasch ◽  
Ruud P M Lensen ◽  
Rogier M Bertina
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Type I ◽  
Factor V ◽  
Sequencing Analysis ◽  
Apc Resistance ◽  
Quantitative Factor ◽  
Factor V Deficiency ◽  
Fv Leiden

SummaryResistance to activated protein C (APC), which is associated with the FV Leiden mutation in the large majority of the cases, is the most common genetic risk factor for thrombosis. Several laboratory tests have been developed to detect the APC-resistance phenotype. The result of the APC-resistance test (APC-sensitivity ratio, APC-SR) usually correlates well with the FV Leiden genotype, but recently some discrepancies have been reported. Some thrombosis patients that are heterozygous for FV Leiden show an APC-SR usually found only in homozygotes for the defect. Some of those patients proved to be compound heterozygotes for the FV Leiden mutation and for a type I quantitative factor V deficiency. We have investigated a thrombosis patient characterized by an APC-SR that would predict homozygosity for FV Leiden. DNA analysis showed that he was heterozygous for the mutation. Sequencing analysis of genomic DNA revealed that the patient also is heterozygous for a G5509→A substitution in exon 16 of the factor V gene. This mutation interferes with the correct splicing of intron 16 and leads to the presence of a null allele, which corresponds to the “non-FV Leiden” allele. The conjunction of these two defects in the patient apparently leads to the same phenotype as observed in homozygotes for the FV Leiden mutation.

Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies

2013 ◽  
Vol 454 (3) ◽  
pp. 387-399 ◽  
Author(s):  
Patrick Masson ◽  
Sofya Lushchekina ◽  
Lawrence M. Schopfer ◽  
Oksana Lockridge
Keyword(s):  
Osmotic Stress ◽  
Osmotic Pressure ◽  
Active Site ◽  
Md Simulations ◽  
Catalytic Triad ◽  
Wild Type ◽  
Irreversible Inhibitor ◽  
Enzyme Active Site ◽  
Diffusion Controlled ◽  
Peripheral Site

CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E′. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon≈108 M−1·min−1). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp70 in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium E⇌E′, indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E′. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His438, leading to the less reactive form E′.

scholarly journals A Factor V Genetic Component Differing From Factor V R506Q Contributes to the Activated Protein C Resistance Phenotype

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1552-1557 ◽  
Author(s):  
F. Bernardi ◽  
E.M. Faioni ◽  
E. Castoldi ◽  
B. Lunghi ◽  
G. Castaman ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Protein C ◽  
Activated Protein C ◽  
Functional Assay ◽  
Factor V ◽  
Resistance Phenotype ◽  
Apc Resistance ◽  
Genetic Components ◽  
Factor V Gene ◽  
V Gene

AbstractFactor V gene polymorphisms were investigated to detect components that may contribute to the activated protein C (APC) resistance phenotype in patients with venous thromboembolism. A specific factor V gene haplotype (HR2) was defined by six polymorphisms and its frequency was found to be similar in normal subjects coming from Italy (0.08), India (0.1), and Somalia (0.08), indicating that it was originated by ancestral mutational events. The relationship between the distribution of normalized APC ratios obtained with the functional assay and haplotype frequency was analyzed in patients heterozygous for factor V R506Q (factor V Leiden). The HR2 haplotype was significantly more frequent in patients with ratios below the 15th percentile than in those with higher ratios or in normal controls. Moreover, the study of 10 patients with APC resistance in the absence of the factor V R506Q mutation showed a 50-fold higher frequency of HR2 homozygotes. The HR2 haplotype was associated with significantly lower APC ratios both in patients with venous thromboembolism and in age- and sex-matched controls. However, the two groups showed similar HR2 haplotype frequencies. Plasma mixing experiments showed that an artificially created double heterozygote for the factor V R506Q mutation and the HR2 haplotype had an APC ratio lower than that expected for a simple R506Q heterozygote. Time-course experiments evaluating the decay of factor V in plasma showed the normal stability of the molecule encoded by the factor V gene marked by the HR2 haplotype, which ruled out the presence of a pseudo-homozygous APC resistance mechanism. Our results provide new insights into the presence of factor V genetic components other than the factor V R506Q that are able to contribute to the APC resistance phenotype in patients with venous thromboembolism.

scholarly journals Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor

2003 ◽  
Vol 373 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Laurent O. MOSNIER ◽  
John H. GRIFFIN
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protease Activated Receptor 1 ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Dose Dependent

In a model of staurosporine-induced apoptosis using EAhy926 endothelial cells, inhibition of apoptosis by activated protein C was dose-dependent and required the enzyme's active site, implicating activated protein C-mediated proteolysis. Consistent with this implication, both protease-activated receptor-1 (PAR-1) and endothelial cell protein C receptor (EPCR) were required for the anti-apoptotic effects of activated protein C.

APC resistance during the normal menstrual cycle

2007 ◽  
Vol 98 (12) ◽  
pp. 1246-1251 ◽  
Author(s):  
Angela Silveira ◽  
Stella Thomassen ◽  
Jacob Odeberg ◽  
Anders Hamsten ◽  
Jan Rosing ◽  
...  
Keyword(s):  
Menstrual Cycle ◽  
Protein C ◽  
Activated Protein C ◽  
Serum Levels ◽  
Serum Estradiol ◽  
Menstrual Cycles ◽  
Apc Resistance ◽  
Exogenous Estrogen ◽  
Normal Menstrual Cycle ◽  
Estradiol Concentration

SummaryIncreased serum levels of endogenous as well as exogenous estrogen are regarded to be responsible for acquired activated protein C (APC) resistance. It was the objective of this study to evaluate whether the physiological increase in serum estradiol concentration during the normal menstrual cycle affects the individual’s sensitivity to APC. Seventy-two women with normal menstrual cycles were included in the study. Blood samples for analysis of estradiol (E2), progesterone (P4) and APC resistance were drawn at two time points of the menstrual cycle (day 3–5 and day 22–25). Two methods of measuring APC resistance were used: the activated partial thromboplastin time (aPTT)-based assay and the endogenous thrombin potential (ETP)-based APC resistance test. Independent of the method used, no changes in APC resistance were found, even though the E2 concentration increased significantly between the two menstrual phases. No correlations between E2 levels and APC resistance, P4 levels and APC resistance or changes in E2 concentrations and changes in APC resistance were detected. Ten women were carriers of the factor VLeiden mutation. Their baseline APC resistance was increased, but their response to elevated E2 during the menstrual cycle did not differ from that of non-carriers. In conclusion, our observations suggest that physiological differences in serum levels of estradiol and progesterone between the early follicular and the luteal phase in a normal menstrual cycle do not have any significant impact on the individual’s sensitivity to APC.

scholarly journals Impact of progestagens on activated protein C (APC) resistance among users of oral contraceptives

2004 ◽  
Vol 2 (9) ◽  
pp. 1594-1600 ◽  
Author(s):  
M. Alhenc-Gelas ◽  
G. Plu-Bureau ◽  
S. Guillonneau ◽  
J.-M. Kirzin ◽  
M. Aiach ◽  
...  
Keyword(s):  
Oral Contraceptives ◽  
Protein C ◽  
Activated Protein C ◽  
Apc Resistance

Differential impact of conventional and low-dose oral hormone therapy (HT), tibolone and raloxifene on functionality of the activated protein C system

2007 ◽  
Vol 97 (06) ◽  
pp. 938-943 ◽  
Author(s):  
Sigurd Liestøl ◽  
Marie-Christine Mowinckel ◽  
H. Coen Hemker ◽  
Per-Morten Sandset ◽  
Anette Eilertsen
Keyword(s):  
Hormone Therapy ◽  
Protein C ◽  
Low Dose ◽  
Acquired Resistance ◽  
Activated Protein C ◽  
System Sensitivity ◽  
Conventional Dose ◽  
Apc Resistance ◽  
The Difference ◽  
Progestin Therapy

SummaryRecent studies have shown that hormone therapy (HT) is associated with an acquired resistance to activated protein C (APC). The aims of the present study were to evaluate a possible dose-response relationship and differential effects of different HT regimens on functionality of the APC system. Two hundred two healthy women were randomly assigned to receive treatment for 12 weeks with tablets containing either low-dose HT containing 1 mg 17β-oestradiol + 0.5 mg norethisterone acetate (NETA) (n=50), conventional-dose HT containing 2 mg 17β-oestradiol and 1 mg NETA (n=50), 2.5 mg tibolone (n=51), or 60 mg raloxifene (n=51). Normalized APC system sensitivity ratios (nAPCsr) were determined in plasma collected at baseline and after 12 weeks using a thrombin generation-based APC resistance test probed with either recombinant APC (rAPC) or thrombomodulin (rTM). NAPCsr increased in both the conventional- and low-dose HT groups, consistent with reduced sensitivity to APC. The increase was slightly more pronounced in the conventional-dose group, but the difference between the two HT groups was not statistically significant. The sensitivity to APC was only marginally altered in those allocated to tibolone. Consequently, tibolone showed a different phenotype as compared with the low-dose HT group. A small increase in nAPCsr with both rAPC and rTM was seen in the raloxifene-group, but the increase was less than in the low-dose HT group. Our findings indicate that oestrogen-progestin therapy induces an APC resistant phenotype, which may be related to dose, whereas tibolone and raloxifene only marginally alter the sensitivity to APC.

scholarly journals Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 642-647 ◽  
Author(s):  
K Murakami ◽  
K Okajima ◽  
M Uchiba ◽  
M Johno ◽  
T Nakagaki ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Vascular Injury ◽  
Active Site ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Myeloperoxidase Activity ◽  
Granulocyte Elastase ◽  
Pulmonary Vascular Permeability ◽  
Elastase Inhibitor

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS- induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

Sign in / Sign up

Export Citation Format

Share Document