Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The development of a fibrin clot microassay to define both the kinetic behaviour and the anticoagulant activity of direct thrombin inhibitors targeting various domains of thrombin (catalytic site, anion binding exosite or both) is described. Since classical kinetics studies are difficult to perform in a fibrin-clot assay, methodological conditions were selected in order to obtain a linear relationship between fibrin formation and the thrombin concentration i.e. 0.67 nM thrombin, 6 microM fibrinogen, 5 minutes reaction. Under those conditions, the concentration of the complex thrombin-inhibitor can easily be calculated from a standard curve performed with increasing concentrations of thrombin and fitted versus the total inhibitor concentration using adapted equations. To detect the slow establishment of the thrombin inhibition, results obtained with a protocol in which the inhibitor is pre-incubated with thrombin before the addition of fibrinogen is compared to a protocol in which the inhibitor is pre-incubated with fibrinogen before thrombin is added. Our assay which is validated using different types of thrombin inhibitors (classical competitive: NAPAP and hirudin 55-65; tight binding: r-hirudin; slow tight binding: DUP-714), provides a rapid screening protocol allowing to evaluate the biochemical and anticoagulant properties of any direct thrombin inhibitor.
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PMID:A screening procedure to evaluate the anticoagulant activity and the kinetic behaviour of direct thrombin inhibitors. 763 2

Cardiopulmonary bypass causes hemorrhagic complications, and initiates a chemical and cellular inflammatory response. Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, complement, and contact systems. Despite the fact that cardiopulmonary bypass is carried out in the presence of high doses of heparin, there is significant activation of both platelets and neutrophils. Thrombin is protected on cell and fibrin surfaces from antithrombin, even in the presence of high doses of heparin (approximately 5 U/ml). We therefore studied the effect of a small (Mr = 497), highly effective (Ki = 41 pM), reversible tripeptide inhibitor of thrombin, DUP 714 (1 microM), in a well characterized model of simulated extracorporeal circulation. In the absence of DUP 714, platelet counts decreased by 75% 5 min after the start of extracorporeal bypass and increased to 48% at 120 min of recirculation. DUP 714 significantly preserved platelet counts, decreased plasma levels of platelet beta-thromboglobulin levels, but did not prevent a decrease in sensitivity of platelets to adenosine diphosphate. Kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively from 0.32 U/ml to 0.67 U/ml and from 4.45 U/ml to 7.25 U/ml, respectively, during 120 min of recirculation without DUP 714. Addition of DUP 714 significantly inhibited kallikrein-C1-inhibitor complex formation but did not affect C1-C1-inhibitor complexes. In the absence of DUP 714, human neutrophil elastase levels rose from a baseline of 0.01 +/- 0.00 microg/ml to 1.18 +/- 0.21 microg/ml during 120 min of recirculation. Human neutrophil elastase release at 120 min was significantly inhibited in the presence of DUP 714 to 37% of the value with heparin alone. These results indicated that addition of this novel thrombin (and kallikrein) inhibitor to heparin preserved platelet counts, decreased platelet secretion, and provided the additional benefit of partially blocking neutrophil activation during simulated extracorporeal circulation.
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PMID:Thrombin and human plasma kallikrein inhibition during simulated extracorporeal circulation block platelet and neutrophil activation. 979 91

The objective of this study was to determine if the thrombin inhibitors Argatroban and DUP 714 could anticoagulate whole blood without influencing the analyses of blood gases, electrolytes, ionized calcium or CO-oximetry. The anticoagulant potency of DUP 714 (0.5-68 micromol/l) and Argatroban (1.5-390 micromol/l) was evaluated using the activated partial thromboplastin time (APTT), prothrombin time (PT) and whole blood clot time (WBCT). APTT and the PT were measured using a Behring Fibrintimer. APTT was found to be more sensitive to prolongation by both of the thrombin inhibitors than were the PT or WBCT assays. DUP 714 was found to a more potent anticoagulant than Argatroban. DUP 714 anticoagulated specimens (>2.2 micromol/l) did not clot for at least 2 days, whereas Argatroban preserved specimens (390 micromol/l) clotted within 5.5 h of collection. No statistically significant changes in the measurement of pH, PCO2, PO2, Na, K, ionized calcium, oxyhaemoglobin, deoxyhaemoglobin, methaemoglobin or carboxyhaemoglobin (measured using a Corning 288 Blood Gas/Electrolyte Analyzer and a Coming 270 CO-oximeter) were detected in DUP 714 (34 micromol/l) or Argatroban (390 micromol/l) anticoagulated whole blood specimens. In conclusion, DUP 714 and Argatroban are suitable anticoagulants for preserving blood prior to blood gas and electrolyte analyses.
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PMID:Evaluation of argatroban and DUP 714 as anticoagulants for blood gas, electrolyte and ionized calcium analyses. 1075 50