Development of Prothrombinase Induced Clotting Time (Pefakit ®, PiCT) Assay and its Validation in the Monitoring of Anti-Xa and Anti-FIIa Drugs.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1858-1858
Author(s):  
Debra A. Hoppensteadt ◽  
Josephine Cunanan ◽  
Jeanine M. Walenga ◽  
Jawed Fareed
Keyword(s):  
Dose Range ◽  
Fibrin Clot ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Clotting Time ◽  
Prothrombinase Complex ◽  
Anticoagulant Drugs ◽  
Normal Human ◽  
Ecarin Clotting Time ◽  
Direct Acting

Abstract Unfractionated heparin (UFH), low molecular weight heparin (LMWH), fondaparinux, danaparoid, direct and indirect (via a cofactor) acting anti-FIIa and anti-Xa anticoagulant drugs are being used in patients for the prophylaxis and treatment of thrombosis. The aPTT does not reliably measure the effects of these drugs. Therefore, there is no simple/reliable method for monitoring patients administered these drugs, particularly those with renal insufficiency and obesity. The newly developed Prothrombinase Induced Clotting Time (PiCT) assay (Pefakit®; Pentapharm, Ltd.; Basel, Switzerland) was developed for monitoring heparins. The PiCT is based on the activation of plasma coagulation by a defined amount of activated FXa, phospholipid and Russell’s viper venom (RVV). The prothrombinase complex formed following recalcification is independent of endogenous thrombin mediated FV activation because the RVV directly activates FV. In this study, the PiCT was used to determine the relative anticoagulant actions of UFH, LMWH, anti-FIIa and anti-FXa drugs. Drugs were supplemented to normal human pooled plasma in a concentration range of 10- 0.08 μg/ml. Plasma samples were analyzed using the PiCT, aPTT, Heptest, thrombin time (TT), ecarin clotting time (ECT), chromogenic anti-FIIa, chromogenic anti-FXa and fibrinopeptide A (FPA) generation assays. In addition, plasma samples from patients treated with these drugs were also tested. The UFH, LMWH, anti-FIIa drugs and the indirect acting anti-FXa drugs produced a concentration-dependent prolongation of the PiCT. Different responses were obtained for each drug. The anti-FIIa drugs gave the strongest dose-response response followed by UFH, LMWH and fondaparinux, in that order. The direct acting anti-FXa agents showed a weak response in the PiCT. Compared to the other assays, the PiCT response showed better sensitivity, reproducibility and linearity over a broader dose range than the aPTT, Heptest, TT, ECT, anti-FIIa and anti-FXa assays. Samples from patients treated with enoxaparin (0.5 mg/kg IV) gave peak concentrations of 0.92± 0.11 U/ml by the PiCT, compared to 0.95± 0.24 U/ml by the anti-FXa assay. Samples from patients treated with bivalirudin, lepirudin or argatroban gave comparable values by the PiCT, aPTT and ECT. The relative prolongation of the PiCT was proportional to the inhibition of thrombin generation as measured by FPA. The PiCT is as simple to perform as the aPTT and can be adapted to any instrument capable of detecting a fibrin clot. Unlike other clot-based tests, the PiCT is sensitive to most old and new anticoagulant drugs and the results are linear through both the therapeutic and interventional dosing ranges. The PiCT may offer the first available single assay that can be universally used for monitoring most of the new anticoagulant drugs regardless of their mechanism of action.

Clinical and Experimental Evaluation of the Interaction of Anticoagulant Drugs with Radiologic Contrast Media

1979 ◽  
Author(s):  
R. Moncada ◽  
H. L. Messmore ◽  
J. Fareed ◽  
P. J. Scanlon ◽  
Z. Parvez
Keyword(s):  
Contrast Media ◽  
Media Studies ◽  
Oral Anticoagulants ◽  
Thrombin Time ◽  
Clotting Time ◽  
Activated Clotting Time ◽  
Anticoagulant Action ◽  
Human Volunteers ◽  
Anticoagulant Drugs ◽  
Vascular Angiography

Although clinical incompatibilities of antihistamines and protamine with radiologic contrast media are well recognized, no report is available on the interaction of heparin, Coumadin, dextrans and other anticoagulants with these agents. We have employed the automated activated clotting time (ACT), prothrombin time (FT), partial thromboplastin time (PTT) and the thrombin time (TT) methods to monitor the anticoagulant actions of contrast media and its interaction with various anticoagulant drugs in patients undergoing angiography, A strong synergism of the anticoagulant action of heparin was observed in patients given heparin along with contrast media. Studies conducted in human volunteers revealed that contrast media at a 1-5 mg/ml level (clinical, 0.5-0.6 mg/ml) produce a strong synergistic effect on the anticoagulant action of heparin, oral anticoagulants, dextrans, and antiplatelet drugs. When blood obtained from patients undergoing angiography was supplemented with 0.25 u/ml heparin, the ACT, PTT and TT were equal to 1.5-2.0 units of heparin. Conventional amounts of protamine are incapable of neutralizing this synergistic interaction. These studies show that contrast media temporarily augments the degree of anticoagulation in patients undergoing angiography, which should be taken into consideration in patients undergoing vascular angiography.

Impact of dabigatran on a large panel of routine or specific coagulation assays

2012 ◽  
Vol 107 (05) ◽  
pp. 985-997 ◽  
Author(s):  
Séverine Robert ◽  
Christian Chatelain ◽  
Jonathan Douxfils ◽  
François Mullier ◽  
Bernard Chatelain ◽  
...  
Keyword(s):  
Linear Correlation ◽  
Individual Variability ◽  
Thrombin Inhibitor ◽  
Thrombin Time ◽  
Clotting Time ◽  
Thrombin Generation Assay ◽  
Coagulation Assay ◽  
Ecarin Clotting Time ◽  
The Impact ◽  
Higher Sensitivity

SummaryDue to low bioavailability and high inter-individual variability, monitoring of dabigatran may be required in specific situations to prevent the risk of bleedings or thrombosis. The aim of the study was to determine which coagulation assay(s) could be used to assess the impact of dabig-atran on secondary haemostasis. Dabigatran was spiked at concentrations ranging from 4.7 ng/ml to 943.0 ng/ml in pooled citrated human platelet-poor plasma. The following clotting assays were performed: prothrombin time (PT); activated partial thromboplastin time (aPTT); thrombin time (TT); ecarin clotting time (ECT); ecarin chromo-genic assay (ECA); prothrombinase-induced clotting time (PiCT); activated clotting time (ACT); Hemoclot Thrombin Inhibitor (HTI) and thrombin generation assay (TGA). A concentration-dependent prolongation of PT, dPT, and aPTT was observed with aPTT being the more sensitive test. The results varied mostly due to the clotting reagent. HTI, ECT and TGA were the most sensitive tests but are not available 24 hours a day. In addition, HTI showed a linear correlation with a good reproduci-bility. Dabigatran induced a concentration-dependent delay and inhibition of tissue factor-induced TGA. Cut-offs related with higher risk of bleedings or thrombosis were defined for each reagent of aPTT and HTI. In conclusion, aPTT could be used for the monitoring of dabigatran and as screening test for the risk of overdose. However, because of its higher sensitivity, good reproducibility, excellent linear correlation at all doses, its simplicity of use, and possibilities of automation, HTI should be considered as the gold-standard.

In Vitro Effects of Urokinase — Prevention by Different Inhibitors

1990 ◽  
Vol 64 (03) ◽  
pp. 402-406 ◽  
Author(s):  
M D Oethinger ◽  
E Seifried
Keyword(s):  
In Vitro Study ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Temperature Dependent ◽  
Chloromethyl Ketone ◽  
Control Value ◽  
Dose Time ◽  
In Vitro Effects ◽  
Full Protection

SummaryThe present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activato(u-PA, urokinase) on normal citrated plasma. When 10 μg/ml u-PA wereadded to pooled normal plasma and incubated for 30 min at an ambient temperature (25° C), α2-antiplas-min decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control,whereas α2-antiplasmin was fully consumed at 37° C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25° C. Thrombin time prolonged to 190% of control.Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KlU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase-induced plasmin generation without interfering with haemostatic assays. The anti-u-PA-antibody afforded full protection ofα2-antiplasmin at therapeutic levels of u-PA.It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody.

Monitoring of r-Hirudin Anticoagulation during Cardiopulmonary Bypass – Assessment of the Whole Blood Ecarin Clotting Time

1997 ◽  
Vol 77 (05) ◽  
pp. 0920-0925 ◽  
Author(s):  
Bernd Pötzsch ◽  
Katharina Madlener ◽  
Christoph Seelig ◽  
Christian F Riess ◽  
Andreas Greinacher ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Whole Blood ◽  
Heart Surgery ◽  
Ex Vivo ◽  
Open Heart Surgery ◽  
Clotting Time ◽  
Blood Samples ◽  
Alternative Approach ◽  
Ecarin Clotting Time

SummaryThe use of recombinant ® hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor Ila assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin- spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 µg/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin- spiked blood samples obtained from 50 healthy blood donors. CV- values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 µg/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

scholarly journals Ecological Effect of Ceftaroline-Avibactam on the Normal Human Intestinal Microbiota

2015 ◽  
Vol 59 (8) ◽  
pp. 4504-4509 ◽  
Author(s):  
Mamun-Ur Rashid ◽  
Staffan Rosenborg ◽  
Georgios Panagiotidis ◽  
Karin Söderberg-Löfdal ◽  
Andrej Weintraub ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Intestinal Microbiota ◽  
Ecological Effect ◽  
New Combination ◽  
Plasma Samples ◽  
Ceftaroline Fosamil ◽  
Content Type ◽  
Human Intestinal Microbiota ◽  
Normal Human ◽  
Lactamase Inhibitor

ABSTRACTCeftaroline-avibactam is a new combination of the antibiotic ceftaroline with a novel non-β-lactam β-lactamase inhibitor, avibactam. The purpose of the present study was to investigate the effect of ceftaroline-avibactam on the human intestinal microbiota. Fourteen healthy volunteers received ceftaroline-avibactam (600 mg ceftaroline fosamil and 600 mg avibactam) intravenously over 2 h every 8 h on days 1 to 6 and as a single dose on day 7. Fecal samples were collected on day −1 (within 24 h of the first infusion on day 1) and on days 2, 5, 7, 9, 14, and 21.Escherichia colinumbers decreased during the study and normalized on day 21. An increased number ofKlebsiellabacteria appeared on day 14 and normalized on day 21. The number of other enterobacteria decreased during the study, and the number of enterococci decreased from days 2 to 7 and normalized on day 9.Candidanumbers increased from days 5 to 9 and normalized after day 14. The number of lactobacilli decreased during the study and recovered on day 14. The number of bifidobacteria decreased on day 2 and normalized on day 21. The number ofBacteroidesbacteria was unchanged.Clostridium difficilenumbers decreased on days 7 and 9 and increased on days 14 and 21. A toxigenicC. difficilestrain was detected in one volunteer on day 21 with no reported adverse events. Plasma samples were collected on days −1, 2, 5, and 7. Ceftaroline and avibactam concentrations were 0 to 34.5 mg/liter and 0 to 61.6 mg/liter, respectively, in plasma and 0 to 35.4 mg/kg and 0 to 98.5 mg/kg, respectively, in feces. (This study is registered in the European Clinical Trials Database [https://eudract.ema.europa.eu/] under number EudraCT 2012 004921-25.)

scholarly journals Targeting exosites on blood coagulation proteases

2005 ◽  
Vol 77 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Robson Q. Monteiro
Keyword(s):  
Blood Coagulation ◽  
High Specificity ◽  
Heparin Binding ◽  
Zymogen Activation ◽  
Prothrombinase Complex ◽  
Blood Sucking ◽  
Anticoagulant Drugs ◽  
Coagulation Inhibitors ◽  
Tissue Factor Pathway ◽  
Surface Domains

The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed in this short revision. Bothrojaracin is a snake venom-derived protein that binds to thrombin exosites 1 and 2. Complex formation impairs several exosite-dependent activities of thrombin including fibrinogen cleavage and platelet activation. Bothrojaracin also interacts with proexosite 1 on prothrombin thus decreasing the zymogen activation by the prothrombinase complex (FXa/FVa). Ixolaris is a two Kunitz tick salivary gland inhibitor, that is homologous to tissue factor pathway inhibitor. Recently it was demonstrated that ixolaris binds to heparin-binding exosite of FXa, thus impairing the recognition of prothrombin by the enzyme. In addition, ixolaris interacts with FX possibly through the heparin-binding proexosite. Differently from FX, the ixolaris-FX complex is not recognized as substrate by the intrinsic tenase complex (FIXa/FVIIIa). We conclude that these inhibitors may serve as tools for the study of coagulation exosites as well as prototypes for new anticoagulant drugs.

scholarly journals A novel missense mutation in the FGB g. 3354 T>A (p. Y41N), Fibrinogen Caracas VIII

2011 ◽  
Vol 105 (04) ◽  
pp. 627-634 ◽  
Author(s):  
Héctor Rojas ◽  
Michael Meyer ◽  
Oscar Castillo ◽  
Arlette De Sáez Ruiz ◽  
John Weisel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fibrin Clot ◽  
Polymerization Process ◽  
Confocal Laser ◽  
Fibrinogen Concentration ◽  
Thrombin Time ◽  
Normal Range ◽  
Amino Terminal ◽  
Fibrin Network ◽  
Β Chain

SummaryA novel dysfibrinogenaemia with a replacement of Tyr by Asn at Bβ41 has been discovered (fibrinogen Caracas VIII). An asymptomatic 39-year-old male was diagnosed as having dysfibrinogenaemia due to a mildly prolonged thrombin time (+ 5.8 seconds); his fibrinogen concentration was in the low normal range, both by Clauss and gravimetric determination, 1.9 g/l and 2.1 g/l, respectively. The plasma polymerization process was slightly impaired, characterised by a mildly prolonged lag time and a slightly increased final turbidity. Permeation through the patients´ clots was dramatically increased, with the Darcy constant around four times greater than that of the control (22 ± 2 x10–9 cm2 compared to 6 ± 0.5 x10–9 cm2 in controls). The plasma fibrin structure of the patient, by scanning electron microscopy, featured a mesh composed of thick fibres (148 ± 50 nm vs. 120 ± 31 nm in controls, p<0.05) and larger pores than those of the control fibrin clot. The viscoelastic properties of the clot from the patient were also altered, as the storage modulus (G‘, 310 ± 30) was much lower than in the control (831 ± 111) (p ≤0.005). The interaction of the fibrin clot with a monolayer of human microvascular endothelial cells, by confocal laser microscopy, revealed that the patients´ fibrin network had less interaction with the cells. These results demonstrate the significance of the amino terminal end of the β chain of fibrin in the polymerisation process and its consequences on the clot organisation on the surface of endothelial cells.

scholarly journals Evaluation of anticoagulant activity of aqueous extract of Cestrum nocturnum

2017 ◽  
Vol 8 (4) ◽  
pp. 525
Author(s):  
Chandra Kishore Tyagi ◽  
Deenanath Jhade ◽  
Sunil Kumar Shah
Keyword(s):  
Aqueous Extract ◽  
Anticoagulant Activity ◽  
Ethanolic Extract ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Clotting Time ◽  
Standard Procedures ◽  
Prolonged Aptt ◽  
Pharmacologically Active

<p>The study evaluated anticoagulant properties of the aqueous extract of <em>Cestrum nocturnum</em> using aPTT-Activated Partial Thromboplastin Time, PT- Prothrombin Time &amp; TT-Thrombin Time as standard procedures.</p><p>For <em>in vitro</em> coagulation assays, aqueous extract of plant prolonged APTT, TT, and PT clotting times in a dose-dependent manner (Table 7). It prolonged APTT clotting time from 45 ± 2 (2mg/mL) to 82.2 ± 2.63s (10mg/mL), PT clotting time from 20.4 ± 1.49 (2mg/mL) to 31.4 ± 2.15s (10mg/mL), and TT clotting time from 9.2 ± 1.16 (2mg/mL) to 17.4 ± 1.01s (10mg/mL) at the concentration of 2 to 10mg/mL. Heparin prolonged APTT and PT clotting times more than 111.8s and 40.8s, respectively, at a concentration of 1 IU/mL. Heparin prolonged TT clotting times more than 20.6s at a concentration of 1 IU/mL.</p><p>The phytochemical screening of the plant confirm the presence of saponin in the water and ethanolic extract, Alkaloid in all the extract except hexane extract, tannin in water, ethanol and methanol extract, amino acid in water and ethanolic extract, carbohydrate in water and methanolic extract and triterpenoids and glycoside were absent in all the extracts. The results demonstrated that the aqueous extract of <em>Cestrum nocturnum</em> possesses pharmacologically active anticoagulant principles that could be isolated and evaluated for clinical or physiological purposes.</p>

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