Determination of Coagulation Inhibitor Levels and Resistance to Activated Protein C in Patients Undergoing Gastric Surgery for Benign and Malignant Disorders

1997 ◽  
Vol 27 (4) ◽  
pp. 157-162
Author(s):  
B.S. Andersen ◽  
H.B. Rahr ◽  
J.V. Sørensen
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Gastric Surgery ◽  
Coagulation Inhibitor

Influence of blood handling on determination of plasma resistance against activated protein C

1996 ◽  
Vol 10 ◽  
pp. 153-154
Author(s):  
O.D. Pedersen ◽  
J. Sidelmann ◽  
J. Gram ◽  
J. Jespersen
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Plasma Resistance

Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4802-4807 ◽  
Author(s):  
Chandrashekhara Manithody ◽  
Philip J. Fay ◽  
Alireza R. Rezaie
Keyword(s):  
Protein C ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Factor Va ◽  
Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

Determination of Protein C Antigen Levels in Hospitalized Patients with Blood Coagulative Defects

2021 ◽  
Vol 2 (1) ◽  
pp. 1-04
Author(s):  
George Zhu
Keyword(s):  
Plasma Protein ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Promyelocytic Leukemia ◽  
Hospitalized Patients ◽  
Cancer Cell Proliferation ◽  
Healthy Individuals ◽  
Acute Leukemias

Protein C, a vitamin K-dependent anticoagulant serine protease, is involved in blood coagulation. Activated protein C inactivates Va and VIIIa in purified protein systems and stimulates fibrinolysis by indirectly increasing the level of circulating plasminogen activator. In this process, protein S serve as an important factor for activated protein C. In recent years, excess protein S drives cancer cell proliferation and cell survival through oncogenic receptor Axl (Anexelekto). We determined changes of plasma protein C antigen by using rocket immunoassay both in 50 healthy individuals and 103 distinct hospitalized patients. In healthy individuals protein C antigen(PC:Ag) ranges o.6439- 1.4752 µ/ml. The results showed that plasma protein C antigen was considerably high in 22 diabetes mellitus. In contrast, the PC:Ag was significantly decreased in 19 liver cirrhosis(p< 0.001) and in closely line with serum albumin levels(p< 0.05). In 31 acute leukemias, on the average, there was slightly lower values in PC:Ag, and accompanied with the distribution of significant decrease of PC:Ag values in 5 FAB M5 subtype and in 9 hyperleukocytic leukemias. However, the 3 acute promyelocytic leukemia (APL) with overt laboratory criteria of disseminated intravascular coagulation (DIC) had protein C concentration no lower than the remaining 2 patients with infectious DIC, which suggested the coagulopathy in APL might be due to mechanisms different from other forms of DIC.

AMIDOLYTIC DETERMINATION OF ANTI-ACTIVATED PROTEIN C ACTIVITY IN PLASMA

1987 ◽  
Author(s):  
F Nicham ◽  
J L Martinoli
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Type I ◽  
Peptide Substrate ◽  
Normal Plasma ◽  
Clinical Investigations ◽  
Normal Individuals ◽  
Significant Difference ◽  
Protein C Activity

Anti-activated protein C (anti-APC) potency of plasma was studied using purified bovine activated protein C (Bovine APC) and the chromogenic peptide substrate CBS 65.25. The choice of bovine instead of human APC was justified by a better sensitivity (Km = 0.14 and 0.42 mM respectively). Inhibition was shown to be dramatically enhanced by the presence of Heparin and calcium. No significant difference occurred for pH values up to 8.2 for both inhibition and hydrolysis reactions.In the final test, O.l ml of 1:5 diluted plasma (Tris buffer saline, pH 8.4, containing 5 U/ml of Heparin) were incubated at 37°C with 0.2 ml of Bovine APC (0.125 U/ml). After 10 minutes of inhibition, 0.2 ml of CBS 65.25 (1.5 mM/1) were added to the mixture and the change in absorbance was recorded at 405 nm for 2 minutes. In these conditions linearity of the dose-response curve was ensured from O up to 130 % of activity (normal plasma pool being assigned to 100 %) ; day to day precision was 1.9 %. When a normal plasma was overloaded with different purified inhibitors such as antithrombin III, cl-esterase inactivator, alpha 2 macroglobulin, the measured anti-APC activities were not affected at all. It could be concluded that this test measures protein C inhibitor described by Suzuki.Levels in 23 normal individuals averaged 97.7 %, giving a normal range of 77 - 118 %. Levels were below normal in 6 of 10 patients after surgery (54.1 +/- 4.8 %), in 18 of 19 patients with liver disease (49.5 +/- 9.6 %) and in 4 of 18 coumarin treated patients (54.9 +/- 6.5 %). In 9 of 10 patients previou sly characterized as type I protein C deficient, a statistically significant increase in anti-APC activity was observed (mean 110.7 +/- 7.7 %).The use of a chromogenic peptide substrate has led to a sensitive and fast assay for anti-APC activity in plasma. That could be of interest in clinical investigations and knowledge of regulatory mechanisms in thrombotic disorders.

NEW CHROMOGENIC SUBSTRATES FOR THE DETERMINATION OF COAGULATION AND FIBRINOLYSIS ENZYMES

1987 ◽  
Author(s):  
W Stüber ◽  
D Schiwek ◽  
U Becker ◽  
N Heimburger
Keyword(s):  
Serine Proteases ◽  
Spectral Properties ◽  
Protein C ◽  
Activated Protein C ◽  
Peptide Sequence ◽  
Blue Dye ◽  
Chromogenic Substrates ◽  
New Type ◽  
Derivatives Of

A new type of chromogenic substraces based on derivatives of phenoxazine is presented. Particularly interesting is the blue dye 7-amino-3-diethylamino-8-methylphenoxazine (ADMP) with a molar extinktion coefficient of about 80,000 at 624 nm. Peptides were linked to the aminogroup of the dye and red coloured substrates were obtained with a λmax value of about 540 nm. On account of the distinct difference of the λmax values and the negligable influence of the absorption peaks of the acylated and the free dye this chromophore is suitable for the synchesis of substrates. Besides the spectral properties of chese new chromogenic peptides we determined their characce-riscics using serine proteases involved in the process of coagu-lacion and fibrinolysis. In comparison co para-nitroaniline substrates the introduction of the relatively bulky ADMP into the peptide sequence led to products with superior properties in respect of sensitivity and specificity.It was found that the ADMP substrates are particularly favourable for the determination of thrombin, urokinase and activated protein C in the presence of other proteases.

Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia

2011 ◽  
Vol 106 (12) ◽  
pp. 1189-1196 ◽  
Author(s):  
Cornelis van ’t Veer ◽  
Joris Roelofs ◽  
Bruce Gerlitz ◽  
Brian Grinnell ◽  
Marcel Levi ◽  
...  
Keyword(s):  
Protein C ◽  
Pneumococcal Pneumonia ◽  
Therapeutic Response ◽  
Activated Protein C ◽  
Coagulation Activation ◽  
Cytokines And Chemokines ◽  
Anti Inflammatory ◽  
D Dimer ◽  
Bacterial Outgrowth

SummaryRecombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been associated with a better therapeutic response as compared to later treatment. In a mouse model it was recently confirmed that recombinant murine (rm-)APC decreases coagulation activation and improves survival in pneumococcal pneumonia; however, APC did not impact on the inflammatory response. The aim of this study was to determine the effect of APC treatment instigated early in infection on activation of coagulation and inflammation after induction of pneumococcal pneumonia. Mice were infected intranasally with viable S. pneumoniae. Mice were treated with rm-APC (125 μg) or vehicle intraperitoneally 12 hours after infection and were sacrificed after 20 hours, after which blood and organs were harvested for determination of bacterial outgrowth, coagulation activation and inflammatory markers. In this early treatment model, rm-APC treatment inhibited pulmonary and systemic activation of coagulation as reflected by lower levels of throm-bin-antithrombin complexes and D-dimer. Moreover, rm-APC reduced the levels of a large number of cytokines and chemokines in the lung. When administered early in pneumococcal pneumonia, rm-APC inhibits systemic and pulmonary activation of coagulation and moreover exerts various anti-inflammatory effects in the lung.

Multicenter Evaluation of a Kit for Activated Protein C Resistance on Various Coagulation Instruments Using Plasmas from Healthy Individuals

1994 ◽  
Vol 72 (02) ◽  
pp. 255-60 ◽  
Author(s):  
S Rosén ◽  
K Johansson ◽  
K Lindberg ◽  
B Dahlbäck ◽  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V ◽  
Clotting Time ◽  
Apc Resistance ◽  
Normal Plasma ◽  
The Individual ◽  
Anticoagulant Response ◽  
Hemostatic Disorder

SummaryRecently a new hemostatic disorder has been described which appears to be an important risk factor for familial thromboembolism. The disorder is characterized by a poor anticoagulant response to activated Protein C (APC) and has been shown to be due to lack of an APC cofactor activity which is a property of factor V.A kit for determining the response of plasma samples towards addition of APC in an APTT-based assay - COATEST APC Resistance -has been evaluated on 35 coagulation instruments in a multicenter study involving 32 laboratories. A lyophilized normal plasma and identical plasma aliquots from 20 individuals, one of whom had a borderline resistance to APC, were analysed in each laboratory and the sensitivity of each plasma to APC was determined as the ratio between the clotting times obtained in the presence and absence of APC (APC ratio).The plasma from the individual with a borderline resistance to APC activity was correctly classified as the lowest responder in each laboratory, with an APC ratio in the range 1.6-2.4. In comparison, plasmas from individuals with a pronounced response to APC activity resulted in APC ratios above 3.4 in most cases. Interestingly, although the actual APT time for a plasma from a given individual showed a more than 10 s difference due to the type of instrumentation used, the variation in the APC ratio was limited. A similar discrimination was also obtained from evaluation of the actual prolongation of the clotting time in the presence of APC.The intra-laboratory coefficient of variation for the clotting times were on average 2.0% and 3.9% in the absence and presence of APC, respectively, indicating that the precision for the prolonged clotting times obtained in the presence of APC is sufficient to allow a safe assignment of the APC response. The APC ratio for the lyophilized normal plasma was 2.7 ± 0.2 (2 S.D.) illustrating a narrow distribution between instruments which shows the feasibility of including such plasma for assay validation. Altogether, the results indicate that all the coagulation instruments included in the study can be used for detection of individuals with resistance to APC activity through determination of the APC ratio or the prolongation time.

Quantitative and functional assays compared for determination of zymogen and activated human protein C

1990 ◽  
Vol 36 (11) ◽  
pp. 1892-1896 ◽  
Author(s):  
S M Richards ◽  
T Olson ◽  
L D Keyes
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inhibition Assay ◽  
Functional Assays ◽  
Accuracy And Precision ◽  
Marked Variability ◽  
Normal Human ◽  
Nonlinear Curve

Abstract We evaluated quantitative and functional assays for protein C, using either purified protein C samples or pooled normal plasma as assay standards. The purified protein C samples were examined as the zymogen form and after activation by thrombin. Mass concentrations of protein C were determined by amino acid analysis and confirmed by enzyme-linked immunosorbent assay (ELISA). Functional activity was assessed in both standard clot inhibition and amidolytic assays. The accuracy and precision of the ELISA was acceptable, with all three preparations of protein C having similar linear curves. The clot inhibition assay demonstrated marked variability when used according to the manufacturer's instructions; however, modifications to the protocol significantly decreased the CV, to less than 10%. Both activated protein C and the zymogen gave linear standard curves. Pooled normal human plasma gave a nonlinear curve, which contributed to inaccurate sample recoveries. We obtained the most nearly accurate recoveries when we used activated protein C as the assay standard. Amidolytic assays provided no insights into the appropriateness of the preparations for that assay format. A uniform, consistent source of protein C, e.g., recombinant activated protein C, would be useful for standardizing all assays of protein C.

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