scholarly journals α2-Macroglobulin Is a Significant In Vivo Inhibitor of Activated Protein C and Low APC:α2M Levels Are Associated with Venous Thromboembolism

2018 ◽  
Vol 47 (04) ◽  
pp. 630-638 ◽  
Author(s):  
Laura Martos ◽  
Luis Ramón ◽  
Julia Oto ◽  
Álvaro Fernández-Pardo ◽  
Santiago Bonanad ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Protein C ◽  
Activated Protein C ◽  
Detection Range ◽  
In Vivo Experiments ◽  
Α2 Macroglobulin ◽  
History Of ◽  
Apc Protein ◽  
Control Study

Background Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α2-macroglobulin (α2M) have been observed by immunoblotting. Here, we report an ELISA for APC:α2M complexes in plasma. Methods Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α2M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α2M, PCI and α1AT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls. Results In all the in vivo experiments, α2M was a significant APC inhibitor. The VTE case–control study showed that VTE patients had significantly lower APC:α2M and APC levels than the controls (p < 0.001). Individuals in the lowest quartile of APC:α2M or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:α2M or of APC. The risk increased for individuals with low levels of both parameters. Conclusion The APC:α2M assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:α2M level is associated with increased VTE risk.

Low Level of Circulating Activated Protein C Is a Risk Factor for Venous Thromboembolism

2001 ◽  
Vol 86 (12) ◽  
pp. 1368-1373 ◽  
Author(s):  
Amparo Vayá ◽  
Yolanda Mira ◽  
Pilar Medina ◽  
Amparo Estellés ◽  
Piedad Villa ◽  
...  
Keyword(s):  
Risk Factor ◽  
Venous Thromboembolism ◽  
Protein C ◽  
Independent Risk Factor ◽  
Activated Protein C ◽  
Control Group ◽  
Low Level ◽  
History Of ◽  
The Mean

SummaryThe levels of circulating activated protein C (APC) reflect in vivo protein C activation. The aim of this study was to determine whether a low APC level is an independent risk factor for venous thromboembolism (VTE). We measured APC in 160 patients with a history of VTE and without recognized thrombophilic defects, and in 199 healthy individuals. The mean (±SD) APC level was lower in patients (0.99 ± 0.44 ng/ml) than in controls (1.19 ± 0.41 ng/ml) (p <0.0001), and showed a different distribution in the two groups. Thirty-eight patients (23.7%) had APC levels below the 5th percentile of the control group (<0.69 ng/ml) and 57 patients (35.6%) had APC levels below the 10th percentile (<0.77 ng/ml). APC levels <0.69 ng/ml increased the risk of a single or recurrent episode of VTE 4.2-fold (95% confidence interval, 2.0-9.0) or 6.9-fold (2.6-17.9), respectively, and APC levels <0.77 ng/ml increased these risks 3.4-fold (1.9-6.2) or 5.1-fold (2.3-11.2), respectively, compared with controls. Familial studies revealed that in some cases the low APC phenotype seems to be hereditary. We conclude that a low level of circulating APC in individuals without any of the most recognized thrombophilic defects is a prevalent, independent risk factor for VTE, and that it predisposes to recurrent VTE.

Quantification of Circulating Activated Protein C in Human Plasma by Immunoassays - Enzyme Levels are Proportional to Total Protein C Levels

1996 ◽  
Vol 75 (01) ◽  
pp. 056-061 ◽  
Author(s):  
Francisco España ◽  
Isabel Zuazu ◽  
Vicente Vicente ◽  
Amparo Estellés ◽  
Pascual Marco ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Limiting Factor ◽  
Healthy Controls ◽  
Blood Samples ◽  
History Of ◽  
The Mean ◽  
The Difference ◽  
Circulating Levels

SummaryWe have developed a simple assay that measures the circulating activated protein C (APC) in plasma. The assay requires collection of duplicate blood samples, one in citrate plus heparin and the other in citrate plus inhibitors of the enzyme. In the heparin tube, APC reacts completely and irreversibly with its major plasma inhibitors, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and the complexes formed are measured by ELISAs. The amount of circulating APC is calculated from the difference between the total amount of complexed APC (sample in citrate plus heparin) and the amount of APC complexed in vivo (sample in citrate plus inhibitor). Over 95% of the APC added to blood collected with heparin was recovered in the assay. The assay can easily be performed in four hours, and had a detection limit of 0.1 ng/ml APC. The mean APC level in 18 protein C heterozygous members from seven kindreds was significantly lower (0.6 ± 0.3 ng/ml) than in 20 healthy controls (1.1 ± 0.3 ng/ml) (p <0.001), whereas the mean level in 10 non-affected members from the kindreds studied was 1.5 ± 0.3 ng/ml. In the group of 12 nonanticoagulated heterozygous protein C-deficient individuals, the three patients with a history of venous thrombosis had a mean APC level significantly lower than the nine asymptomatic members (p <0.01), both subgroups showing similar protein C levels. There was a significant correlation in all groups between the levels of APC and the levels of protein C antigen (r = 0.758, p <0.0001) and activity (r = 0.745, p <0.0001), which means that APC circulating levels are proportional to protein C levels and suggests that the protein C level is the limiting factor in the rate of protein C activation in vivo.

scholarly journals Cleaved Protein S (PS), Total PS, Free PS, and Activated Protein C Cofactor Activity as Risk Factors for Venous Thromboembolism

2003 ◽  
Vol 49 (4) ◽  
pp. 575-580 ◽  
Author(s):  
Delphine Borgel ◽  
Jean-Luc Reny ◽  
David Fischelis ◽  
Sophie Gandrille ◽  
Joseph Emmerich ◽  
...  
Keyword(s):  
Risk Factor ◽  
Venous Thromboembolism ◽  
Protein C ◽  
Activated Protein C ◽  
Hormonal Treatment ◽  
Protein S ◽  
Molecular Forms ◽  
Cofactor Activity ◽  
Control Study

Abstract Background: Although hereditary protein S (PS) deficiency is clearly associated with venous thromboembolism (VTE), the importance of low PS concentrations as a risk factor for VTE in other patients is still a matter of debate. To clarify this issue, we designed a case-control study to evaluate the role of different molecular forms of plasma PS. Methods: We quantified plasma cleaved, total, and free PS and activated protein C (APC) cofactor activity in 87 VTE patients and 174 controls matched for age, sex, and hormonal treatment. Free PS was measured by ELISA or by enzyme-linked ligand sorbent assay (ELSA). Cleaved and total PS were measured by ELISA. Results: In controls, the mean (SD) concentration of circulating cleaved PS was 39 (14) nmol/L, corresponding to 10% (3.5%) of total PS. Concentrations of cleaved PS and total PS were not significantly different in patients with VTE compared with controls. However, in our population, low free PS measured by ELISA or ELSA, as well as APC cofactor activity values were significantly associated with VTE with odds ratios (95% confidence intervals) of 2.9 (1.3–6.3), 2.5 (1.1–5.6), and 2.9 (1.3–6.4), respectively, in multivariate analyses. Conclusion: Phenotypic low PS detected by APC cofactor activity assay or by an assay specific for free PS should be considered a risk factor for VTE.

A Sensitive and Facile Assay for the Measurement of Activated Protein C Activity Levels in Vivo

1993 ◽  
Vol 69 (05) ◽  
pp. 441-447 ◽  
Author(s):  
Carolyn L Orthner ◽  
Billy Kolen ◽  
William N Drohan
Keyword(s):  
Protein C ◽  
Plasma Concentrations ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Microtiter Plate ◽  
Reversible Inhibitor ◽  
Activity Levels ◽  
Standard Curve ◽  
Agarose Beads

SummaryActivated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivity of the assay could be increased by measuring the amount of color produced after longer incubation times in the endpoint mode. The measured APC activity levels were little affected by varying protein C or prothrombin over the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard curve in protein C-deficient plasma, the concentration of APC activity in normal pooled plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C concentration. The assay was approximately 50-fold more sensitive than the identical assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the capture step.

Factor V Leiden Is Associated with Repeated and Recurrent Unexplained Fetal Losses

1997 ◽  
Vol 77 (05) ◽  
pp. 0822-0824 ◽  
Author(s):  
Elvira Grandone ◽  
Maurizio Margaglione ◽  
Donatella Colaizzo ◽  
Marina d'Addedda ◽  
Giuseppe Cappucci ◽  
...  
Keyword(s):  
Protein C ◽  
Strong Association ◽  
Activated Protein C ◽  
Fetal Loss ◽  
Factor V Leiden ◽  
Factor V ◽  
Significant Difference ◽  
History Of ◽  
Fv Leiden ◽  
Caucasian Women

SummaryActivated protein C resistance (APCR) is responsible for most cases of familial thrombosis. The factor V missense mutation Arg506>Gln (FV Leiden) has been recognized as the commonest cause of this condition. Recently, it has been suggested that APCR is associated with second trimester fetal loss. We investigated the distribution of FV Leiden in a sample (n = 43) of Caucasian women with a history of two or more unexplained fetal losses. A group (n = 118) of parous women with uneventful pregnancies from the same ethnical background served as control. We found the mutation in 7 cases (16.28%) and 5 controls (4.24%; p = 0.011). A statistically significant difference between women with only early fetal loss vs those with late events (p = 0.04) was observed. Our data demonstrate a strong association between FV Leiden and fetal loss. Furthermore, they indicate that late events are more common in these patients.

A reactive center loop–based prediction platform to enhance the design of therapeutic SERPINs

2021 ◽  
Vol 118 (45) ◽  
pp. e2108458118
Author(s):  
Wariya Sanrattana ◽  
Thibaud Sefiane ◽  
Simone Smits ◽  
Nadine D. van Kleef ◽  
Marcel H. Fens ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Serine Proteases ◽  
Protein C ◽  
Activated Protein C ◽  
Reactive Center Loop ◽  
Physiological Processes ◽  
Naturally Occurring ◽  
Reactive Center ◽  
Insight Into

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN–protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1’ residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence–function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4’ region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4’ region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4’ RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

Abstract 34: Endogenous Superoxide Dismutase Protects From Impaired Generation of Activated Protein C and Enhanced Susceptibility to Experimental Thrombosis in Mice

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Sanjana Dayal ◽  
Sean X Gu ◽  
Katinan M Wilson ◽  
Ryan Hutchins ◽  
Steven R Lentz
Keyword(s):  
Superoxide Dismutase ◽  
Protein C ◽  
Activated Protein C ◽  
Vena Cava ◽  
Oxidative Modification ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Experimental Thrombosis

In vitro studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo . We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that Sod1-/- mice formed stable occlusions significantly faster than Sod1+/+ mice (P<0.05). In an inferior vena cava (IVC) stasis model Sod1- /- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs. Sod1+/+ mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from Sod1-/- and Sod1+/+ mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in Sod1-/- and Sod1 +/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in Sod1 -/- mice (P<0.05 vs. Sod1 +/+ mice). Further, in vivo generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in Sod1-/- mice (P<0.05 vs. Sod1+/+ mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1 -/- mice vs. Sod1 +/+ mice, suggesting that altered generation of activated protein C in Sod1-/- mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.

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.

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