Standard measurement of clot-bound thrombin by using a chromogenic substrate for thrombin

Meddahi, Sadia; Bara, L.; Fessi, Hatem; Samama, M

doi:10.1016/j.thromres.2004.04.014

Cited by 9 publications

(9 citation statements)

References 8 publications

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“…The absorbance was zero in the second washing solution as described previously [13], probably because clots were blotted before washing under our experimental conditions. This indicated that two washings were sufficient to remove any measurable enzymatic activity.…”

Section: Determination Of Clot-bound Thrombin and Standardization Of mentioning

confidence: 92%

“…Kinetics of clot-associated thrombin activity We used an in-vitro model for the measurement of clot-bound thrombin as previously described [13]. Briefly, different plastic tubes containing human fresh PPP and calcium chloride solution were incubated in a water bath at 378C for 1 min.…”

Section: Determination Of Clot-bound Thrombin and Standardization Of mentioning

confidence: 99%

“…Determination of the enzymatic activity in washing buffer We also determined the number of washes which were necessary to remove any clot-bound enzymatic activity as described previously [13]. The blotted clots were washed twice in 1 ml of EDTA-tris buffer then 100 ml of each buffer solution was introduced into microtiter plate wells, and 100 ml of specific substrate for thrombin at 0.75 mmol/l was added to each solution.…”

Section: Determination Of Clot-bound Thrombin and Standardization Of mentioning

confidence: 99%

“…PPP clots were incubated at room temperature in 100 ml of EDTA-tris buffer for 15, 30, 60 or 120 min and in the microtiter plate wells. The optical density was measured in each well by using a substrate for thrombin, the specificity of which was tested previously [13].…”

Section: Determination Of Clot-bound Thrombin and Standardization Of mentioning

confidence: 99%

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Is the inhibition of both clot-associated thrombin and factor Xa more clinically relevant than either one alone?

Meddahi

Samama²

2009

Blood Coagulation &Amp; Fibrinolysis

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The procoagulant activity of a thrombus is essentially due to clot-associated factor IIa and factor Xa activities.The aim of this review is to underline that specific antithrombin and anti-Xa drugs, such as r-hirudin and DX 9065a, respectively, are complementary, and could be used in combination in clinical trials in patients with acute arterial thrombosis such as coronary syndromes. After standardization of the in-vitro techniques for clot-bound thrombin and clot-associated factor Xa, we have studied the anticoagulant effect of unfractionated heparin and a low-molecular heparin in an in-vitro model. We have confirmed the inability of heparins to inhibit clot-bound thrombin and clot-associated factor Xa. We have compared r-hirudin, a direct thrombin inhibitor, with DX9065a, a direct factor Xa inhibitor. We have observed that r-hirudin inhibited clot-bound thrombin but not clot-bound factor Xa. After r-hirudin treatment interruption, a hypercoagulation rebound has been reported and it could be in relation with the persistence of factor Xa activity in the clot. We have demonstrated that DX9065a inhibits clot-bound factor Xa but does not inhibit clot-bound factor IIa. The complementary effect of DX9065a and r-hirudin is demonstrated in this experimental model. It is likely that several other combinations of drugs may also exhibit an increase of the antithrombotic activity which could be of interest in clinical implication for the treatment of several groups of patients at high risk of arterial thrombosis.

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Section: Determination Of Clot-bound Thrombin and Standardization Of mentioning

confidence: 92%

Section: Determination Of Clot-bound Thrombin and Standardization Of mentioning

confidence: 99%

Section: Determination Of Clot-bound Thrombin and Standardization Of mentioning

confidence: 99%

Section: Determination Of Clot-bound Thrombin and Standardization Of mentioning

confidence: 99%

See 2 more Smart Citations

Is the inhibition of both clot-associated thrombin and factor Xa more clinically relevant than either one alone?

Meddahi

Samama²

2009

Blood Coagulation &Amp; Fibrinolysis

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“…Nowadays, the methods for thrombin determination include absorption spectrophotometry, fluorescence method, electrochemistrical assay, and piezoelectric sensor. The spectrophotometry [3,4] is the common method to detect thrombin with a detection limit of 0.0312 U/mL, but with some limitations such as high cost, complicated procedure, and low selectivity, so its application field was limited. Recently, the utilization of new technology and new material such as aptamer and nanomaterials is a new trend in biochemical analysis.…”

Section: Introductionmentioning

confidence: 99%

Highly selective resonance scattering detection of trace thrombin using aptamer-modified AuRe nanoprobe

Liang

Jiang

2010

Bioprocess Biosyst Eng

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The gold-rhenium (AuRe) composite nanoparticle was prepared by NaBH(4) reduction procedure, and was modified by the aptamer to obtain an AuRe nanoprobe (AuRessDNA) for thrombin. In pH 7.0 Tris-HCl buffer solution and in the presence of salt, the nanoprobe specifically combined with thrombin to form AuRe-aptamer-thrombin cluster that resulted in the resonance scattering intensity (I (560 nm)) increasing at 560 nm. The increased intensity ΔI (560 nm) was linear to the thrombin concentration in the range of 0.115-6.93 nmol/L, with a regression equation of ΔI (560 nm) = 53.0 C + 2.5, a correlation coefficient of 0.9989, and a detection limit of 13 pmol/L. This method was applied to detect thrombin in human plasma samples, with satisfactory results.

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Exploring the efficiency of thrombin inhibitors with a quantitative model of the coagulation cascade

2019

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A detailed mathematical description of the coagulation cascade is a challenging task due to a huge set of protein–protein interactions. Simplified models do not permit quantitative description of anticoagulants. The detailed mathematical model presented here was constructed with 98 reactions between 70 species. The model was verified using experimental data on thrombin generation. Four thrombin inhibitors, which have different inhibitory mechanisms, were incorporated into the model. All four thrombin inhibitors delayed prothrombin conversion into thrombin, but did not preclude it. At high inhibitor concentration, thrombin‐mediated positive feedback loops were strongly inhibited and the proportion of prothrombin, converted with factor Xa only, was considerably increased. The most potent inhibitor of prothrombin conversion was aptamer NU172, which also binds prothrombin and inhibits its conversion.

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Standard measurement of clot-bound thrombin by using a chromogenic substrate for thrombin

Cited by 9 publications

References 8 publications

Is the inhibition of both clot-associated thrombin and factor Xa more clinically relevant than either one alone?

Is the inhibition of both clot-associated thrombin and factor Xa more clinically relevant than either one alone?

Highly selective resonance scattering detection of trace thrombin using aptamer-modified AuRe nanoprobe

Exploring the efficiency of thrombin inhibitors with a quantitative model of the coagulation cascade

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