Inhibition of Thrombin by Peptides Containing Lysyl-α-Keto Carbonyl Derivatives

1995 ◽  
Vol 74 (04) ◽  
pp. 1107-1112 ◽  
Author(s):  
Sidney D Lewis ◽  
Assunta S Ng ◽  
Elizabeth A Lyle ◽  
Michael J Mellott ◽  
Sandra D Appleby ◽  
...  
Keyword(s):  
Factor Xa ◽  
Order Rate Constant ◽  
Cross Reactivity ◽  
Thrombin Inhibitors ◽  
Carbonyl Derivatives ◽  
Thrombosis Model ◽  
Diffusion Controlled ◽  
One Step ◽  
Orally Active ◽  
Inhibition Of Thrombin

SummarySeveral H-N-Me-D-Phe-Pro-Lysyl-α-keto carbonyl derivatives were shown to be potent thrombin inhibitors (Ki 0.2 to 27 nM). The inhibitory potencies of these compounds toward tissue plasminogen activator, plasmin and factor Xa were minimal; however, substantial cross-reactivity versus trypsin was observed (Ki values from 0.5 to 1500 nM). Inhibition of thrombin by α-keto carbonyl compounds appeared to occur via a one-step reversible reaction. The α-keto carbonyl inhibitors bound thrombin with a second order rate constant (k, 1–4 μM-1s-1) that was 10–100-fold slower than that expected for a diffusion-controlled reaction. Certain α-kelo earbonyl inhibitors were as potent (on a weight basis) as hirudin when evaluated in a rat arterial thrombosis model. The modest oral bioavailability (10–19%) in rats demonstrated for three of the α-keto carbonyl thrombin inhibitors suggests the possibility that α-keto amide containing thrombin inhibitors may have utility as orally-active antithrombotic agents.

Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

1990 ◽  
Vol 63 (02) ◽  
pp. 220-223 ◽  
Author(s):  
J Hauptmann ◽  
B Kaiser ◽  
G Nowak ◽  
J Stürzebecher ◽  
F Markwardt
Keyword(s):  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Factor Xa Inhibitors ◽  
Venous Stasis ◽  
Clotting Time ◽  
Thrombosis Model ◽  
Activity Factor ◽  
Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

1997 ◽  
Vol 78 (04) ◽  
pp. 1215-1220 ◽  
Author(s):  
D Prasa ◽  
L Svendsen ◽  
J Stürzebecher
Keyword(s):  
Thrombin Generation ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Thrombin Generation Assay ◽  
Ic50 Values ◽  
Fxa Inhibitor ◽  
20 Nm ◽  
Tick Anticoagulant Peptide ◽  
Inhibition Of Thrombin

SummaryA series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations.The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

Comparative Studies of an Orally-active Factor Xa Inhibitor, YM-60828, with other Antithrombotic Agents in a Rat Model of Arterial Thrombosis

1998 ◽  
Vol 79 (02) ◽  
pp. 410-416 ◽  
Author(s):  
Kazuo Sato ◽  
Yumiko Sakai ◽  
Fukushi Hirayama ◽  
Hiroyuki Koshio ◽  
Yuta Taniuchi ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Test Drug ◽  
Antithrombotic Agent ◽  
Antithrombotic Activity ◽  
Thrombosis Model ◽  
Treatment And Prevention ◽  
Orally Active

SummaryWe examined the antithrombotic activity of a novel synthetic inhibitor of factor Xa, YM-60828, in an electrically-induced carotid artery thrombosis model in rats. In the first experiment, the antithrombotic activity of YM-60828 after i.v. infusion was compared with those of heparin, darteparin and argatroban. Test drug was administered by i.v. infusion from 30 min before electrical stimulation to the end of the experiment. YM-60828 at 1 mg/kg/h significantly improved patency status, prolonged the time to occlusive thrombus formation and duration of patency. Heparin at 300 U/kg/h also improved these parameters, but were accompanied by a marked increase in systemic coagulation time. In the second experiment, the antithrombotic activity of YM-60828 after oral administration was compared with those of ticlopidine, cilostazol, aspirin, beraprost, ethyl icosapentate and warfarin. Test drug was orally administered to fasted rats 60 min before electrical stimulation. YM-60828 at 30 mg/kg p.o., but not ticlopidine, cilostazol, aspirin, beraprost, ethyl icosapentate or warfarin, significantly reduced the incidence of occlusion and improved carotid arterial patency. These results suggest that YM-60828 may be a promising antithrombotic agent for the treatment and prevention of arterial thrombosis which can be given by oral as well as intravenous administration.

Antithrombotic and Hemorrhagic Effects of DU-176b, a Novel, Potent and Orally Active Direct Factor Xa Inhibitor: A Wider Safety Margin Compared to Heparins and Warfarin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1851-1851 ◽  
Author(s):  
Taketoshi Furugohri ◽  
Yuko Honda ◽  
Chikako Matsumoto ◽  
Koji Isobe ◽  
Nobutoshi Sugiyama ◽  
...  
Keyword(s):  
Animal Studies ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Dose Range ◽  
Safety Margin ◽  
Vena Cava ◽  
Factor Xa ◽  
Selective Inhibition ◽  
Thrombosis Model ◽  
Orally Active

Abstract DU-176b is a novel potent, orally active and selective direct inhibitor of factor Xa (FXa). Direct FXa inhibitors have been reported to exert little effect on bleeding time at antithrombotic doses in animal studies. The aim of the present study was to compare the antithrombotic and hemorrhagic effects of DU-176b with unfractionated heparin (UFH), low molecular weight heparin (LMWH; dalteparin) and warfarin in rat models of thrombosis and hemorrhage. Rats were treated with DU-176b, UFH and LMWH by continuous intravenous infusion for 2 – 2.5 h, and with warfarin orally once daily for 4 days before thrombosis or hemorrhage. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava and left for 60 min. Tail template bleeding time was measured after an incision on the tail. DU-176b dose-dependently inhibited thrombus formation in the venous thrombosis model. The dose required for 50% inhibition (ED50) was 0.076 mg/kg/h. In contrast, the dose of DU-176b to double template bleeding time (BT2) was 0.75 mg/kg/h, indicating 10-fold dissociation of the doses of antithrombotic and hemorrhagic effects. UFH, LMWH and warfarin also prevented thrombus formation (ED50 = 56 U/kg/h, 66 U/kg/h and 0.16 mg/kg/day, respectively), but prolonged bleeding time at slightly higher doses (BT2 = 73 U/kg/h, 135 U/kg/h and 0.21 mg/kg/day, respectively) than the effective doses. The dissociation of the doses for these compounds was only 1.3, 2.0 and 1.3-fold, respectively. Moreover, the slope of dose-antithrombotic response curve of DU-176b was significantly slighter than those of UFH, LMWH and warfarin, indicating that the therapeutic dose range of DU-176b would be wider than those of the other anticoagulants. These results suggest that direct and selective inhibition of FXa by DU-176b is preferable for the treatment of thrombotic diseases in the aspect of lack of compromising primary hemostasis.

Design and characterization of dipetacompinR10H, a dipetalogastin II-derived, classical competitive thrombin inhibitor

2007 ◽  
Vol 97 (01) ◽  
pp. 139-145
Author(s):  
Mercedes López ◽  
Goetz Nowak ◽  
Thomas Bitter
Keyword(s):  
Serine Proteases ◽  
Anticoagulant Activity ◽  
Tight Binding ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Coagulation Assays ◽  
Competitive Kinetics ◽  
Inhibition Of Thrombin

SummaryThe design of small chimeric thrombin inhibitors based on the structure of dipetalogastin II has been previously described. These proteins are effective inhibitors of thrombin showing slow binding or slow, tight-binding kinetics. We report here about dipetacompinR10H, a new dipetalogastin II-derived chimeric thrombin inhibitor, which exhibits classical competitive kinetics. The dissociation constant Ki of dipetacompinR10H was determined to be 17.1 ± 0.8 pM. In various coagulation assays it showed a comparable anticoagulant activity like r-hirudin and r-dipetalogastin II. DipetacompinR10H’s inhibition of thrombin was specific, since no inhibition of other serine proteases like factor Xa, plasmin, trypsin or chymotrypsin has been observed.

Oral Anti-Factor Xa and Factor IIa Agent Mediated Inhibition Of Tissue-Factor Mediated Generation Of Thrombin In Prothrombin Complex Concentrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4810-4810
Author(s):  
Daneyal Syed ◽  
Debra Hoppensteadt ◽  
Daniel Kahn ◽  
Job Harenberg ◽  
Jawed Fareed
Keyword(s):  
Tissue Factor ◽  
Oral Anticoagulant ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Onset Time ◽  
Thrombin Inhibitors ◽  
Bleeding Complications ◽  
Prothrombin Complex Concentrates ◽  
Inhibition Of Thrombin

Introduction Several oral anti-factor IIa and factor Xa agents have recently been developed. These include the thrombin inhibitors Ximelagatran/Melagatran (M) and Dabigatran Etexilate/Dabigatran (D), which require endogenous conversion to the active agents D and M. The factor Xa inhibitors, Rivaroxaban (R) and Apixaban (A), are anti-Xa agents that do not require any endogenous activation. Ximelagatran was withdrawn from the market due to adverse reactions. Dabigatran, Rivaroxaban, and Apixaban are approved for various clinical indications. Antagonism of the anticoagulant effect may be required in bleeding complications. Contradictory results were reported for the efficacy of various prothrombin complex concentrates (PCCs) with these new oral anticoagulants (NOACs). The purpose of this study was to determine the differences in the thrombin generation inhibitory profiles of the newer oral anticoagulant agents. Methods Commercially available PCCs namely Octaplex and Beriplex, were used as a source of Factors II, VII, IX and X. To investigate the effect of each of these agents, a working solution of 1U/ml of both PCCs were supplemented in a graded concentration of 0-1250ng/ml with M, D, R and A. Thrombin generation studies were carried out using a thromboplastin activator (RC High, Technoclone Vienna, Austria). Total thrombin generated was measured in terms of nM’s. The IC-50 for each agent was calculated individually. The time course of thrombin generation was also measured following the kinetic profiles and AUC. Results Dabigatran and Melagatran produced relatively weaker inhibition of thrombin generation with the IC-50 values ranging from 410-110ng/ml in Beriplex and 350-1120ng/ml in Octaplex. Both Rivaroxaban and Apixaban produced strong inhibition of thrombin generation, with the IC-50 ranging from 58-62ng/ml in Octaplex; whereas, in Beriplex these values ranged from 48-50ng/ml. The onset time for thrombin generation and total thrombin formation was concentration dependent. The kinetics of thrombin generation with A and R were distinct from D and M. At concentrations below 310ng/ml the total amount of thrombin generated was comparable to the control; however, its formation was delayed. In both systems, D exhibited the weakest thrombin generation inhibitory potential. While the onset time of thrombin generation was delayed at concentrations below 310ng/ml the levels were comparable to or higher than the control. Discussion This data suggests that PCC’s such as Octaplex and Beriplex can be used to generate thrombin and it’s inhibition by new oral anticoagulant drugs. Octaplex generates much higher amount of thrombin than Beriplex at equivalent units. These results also show that in comparison to the oral anti-Xa agents, the oral anti-IIa agents are relatively weaker inhibitors of thrombin generation. These studies also suggest that the differential inhibition of the generation of thrombin through tissue factor by the anti-Xa and IIa agents may contribute to the potential neutralization profile of PCC’s for these drugs. Disclosures: No relevant conflicts of interest to declare.

The uncalibrated prothrombinase-induced clotting time test

2010 ◽  
Vol 30 (04) ◽  
pp. 212-216 ◽  
Author(s):  
R. Jovic ◽  
M. Hollenstein ◽  
P. Degiacomi ◽  
M. Gautschi ◽  
A. Ferrández ◽  
...  
Keyword(s):  
Factor Xa ◽  
Heparin Therapy ◽  
Thrombin Inhibitors ◽  
Coagulation Cascade ◽  
Diagnostic Tools ◽  
Direct Thrombin Inhibitors ◽  
Functional Assay ◽  
Clotting Time ◽  
Coagulation Time ◽  
Time Test

SummaryThe activated partial thromboplastin time test (aPTT) represents one of the most commonly used diagnostic tools in order to monitor patients undergoing heparin therapy. Expression of aPTT coagulation time in seconds represents common practice in order to evaluate the integrity of the coagulation cascade. The prolongation of the aPTT thus can indicate whether or not the heparin level is likely to be within therapeutic range. Unfortunately aPTT results are highly variable depending on patient properties, manufacturer, different reagents and instruments among others but most importantly aPTT’s dose response curve to heparin often lacks linearity. Furthermore, aPTT assays are insensitive to drugs such as, for example, low molecular weight heparin (LMWH) and direct factor Xa (FXa) inhibitors among others. On the other hand, the protrombinase-induced clotting time assay (PiCT®) has been show to be a reliable functional assay sensitive to all heparinoids as well as direct thrombin inhibitors (DTIs). So far, the commercially available PiCT assay (Pefakit®-PiCT®, DSM Nutritional Products Ltd. Branch Pentapharm, Basel, Switzerland) is designed to express results in terms of units with the help of specific calibrators, while aPTT results are most commonly expressed as coagulation time in seconds. In this report, we describe the results of a pilot study indicating that the Pefakit PiCT UC assay is superior to the aPTT for the efficient monitoring of patients undergoing UFH therapy; it is also suitable to determine and quantitate the effect of LMWH therapy. This indicates a distinct benefit when using this new approach over the use of aPPT for heparin monitoring.

Reactivity of a Hereditary Abnormal Antithrombin III Fraction in the Inhibition of Thrombin and Factor Xa

1980 ◽  
Vol 44 (02) ◽  
pp. 092-095 ◽  
Author(s):  
T H Tran ◽  
C Bondeli ◽  
G A Marbet ◽  
F Duckert
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Superficial Thrombophlebitis ◽  
Heparin Concentration ◽  
Heparin Binding Site ◽  
At Iii ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryTwo different AT-III fractions were purified from the plasma of a patient with recurrent superficial thrombophlebitis. The abnormal AT-III fraction (A-AT) was compared to the normal AT-III fraction (N-AT) in the inhibition of thrombin and factor Xa. Without heparin, both inactivate proteases in a similar manner and at the same rate. However, at low heparin concentration the thrombin inhibition proceeds more slowly with A-AT than with N-AT. At high heparin concentration the difference between A-AT and N-AT becomes very small. The inhibition of factor Xa follows a similar pattern. It is suggested that the heparin binding site of A-AT differs from that of N-AT resulting in a decreased heparin cofactor activity.

GAGs-Potentiated Inhibition of Thrombin, Factor Xa and Plasmin in Plasma and in a Purified System Containing Antithrombin III – Correlation with Total Charge Density

1981 ◽  
Vol 46 (04) ◽  
pp. 749-751 ◽  
Author(s):  
E Cofrancesco ◽  
A Vigo ◽  
E M Pogliani
Keyword(s):  
Charge Density ◽  
Chemical Structure ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Total Charge ◽  
Pure System ◽  
At Iii ◽  
Total Charge Density ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryThe ability of heparin and related glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, factor Xa and plasmin in plasma and in a purified system containing antithrombin III (At III) was studied using chromogenic peptide substrate assaysThere was a good correlation between the charge density of the mucopolysaccharides and the activities investigated. While the difference between potentiation of the antithrombin activity by GAGs in plasma and in the purified system was slight, the inhibition of factor Xa in plasma was more pronounced than in the presence of purified At III, indicating the mechanisms for GAGs-potentiated inhibition of thrombin and factor Xa are not identical.For the antiplasmin activity, there was a good correlation between the chemical structure and biological activity only in the pure system, confirming that the antithrombin-GAG complex plays a very limited role in the inactivation of plasmin in plasma.

Human Prothrombin Activation: Determination of Plasma and Serum Levels of Prothrombin Fragment 3 by Radioimmunoassay

1979 ◽  
Author(s):  
Daniel Walz ◽  
Thomas Brown
Keyword(s):  
Blood Clotting ◽  
Factor Xa ◽  
Heart Association ◽  
Serum Levels ◽  
Cross Reactivity ◽  
Assay Procedure ◽  
A Chain ◽  
Prothrombin Activation ◽  
Theoretical Amount

Human prothrombin activation is unique in that, in addition to the release of fragment 1.2 (FI.2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prethrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400 μL of plasma. Serum, obtained from whole blood clotting, contained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents only 5-10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).

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