Due 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 dabigatran 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 chromogenic 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 reproducibility. 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.

We validated a preclinical toxicological screening assay and provided guidelines to evaluate the potential impact of nanoparticles (NPs) on blood coagulation. Five NPs with various physicochemical properties were studied using several existing methods of clotting times and thrombin generation assays in human normal pool plasma. In both recalcification clotting time (RCT) and calibrated thrombin generation test (cTGT), the NPs exhibited procoagulant activity (SiO₂ ≥ SiC ≥ TiC > CuO > CB) but cTGT was more sensitive and relevant than RCT. Thus, the cTGT appears as a reference assay to investigate the nanoparticle (NP) procoagulant activity in human plasma. It should be used as the reference toxicity test for evaluating the effects of nanomaterials on coagulation cascade. In addition, we also showed that the use of the Pluronic F-108 dispersant and/or the sonication for the NP suspension preparation may mask their procoagulant activity and thus should be avoided.

Abstract 3336 Introduction: The search for new anticoagulants is a major challenge in medicine. Contact factors have never been considered as interesting targets for the development of new anticoagulant agents since they are not required for in vivo coagulation (i.e. deficiencies affecting these factors do not cause excessive bleeding). The discovery that FXI and FXII deficiency protects against thrombosis without causing spontaneous bleeding in mice makes FXII a unique and ideal target for drug design. We demonstrated in vitro that Ir-CPI, a 67 amino acids recombinant Kunitz-type protein from Ixodes ricinus , specifically interacted with activated human contact phase factors (FXIIa, FXIa, and kallikrein). Aims: The goal of this study was to investigate the potential anticoagulant and antithrombotic efficacy of Ir-CPI. Methods: The effects of Ir-CPI were investigated on the thrombin activity during the coagulation process in human plasma using the CAT method. Three different inducers were employed to specifically trigger the coagulation pathways and to generate thrombin. A standard concentration of 5 pM TF with 4 μM PL and 16.7 mM CaCl 2 was used to activate the TF pathway whereas a lower TF concentration of 1 pM with 4 μM PL and 16.7 mM CaCl 2 was selected for stimulating the TF pathway in combination with the contact phase pathway through a thrombin-mediated positive feedback. The contact phase pathway was also triggered alone by ellagic acid, PL and 16.7 mM CaCl 2 (25-fold diluted APTT reagent Actin FS). The effect of Ir-CPI on venous thrombus formation was assessed using a rat thrombosis model induced by complete venous stasis in the posterior vena cava and FeCl 3 topical application on the outer vessel wall. Results: When stimulating plasma coagulation trough the contact pathway, Ir-CPI caused a concentration-dependent prolongation of the lag time and the Tmax and a concentration-dependent decrease in the Cmax compared to the control curve (i.e. without inhibitor). When the coagulation cascade was triggered by the TF pathway (5 or 1 pM TF), only a slight concentration-dependent decrease of the Cmax and a concentration-dependent prolongation of the lag time and the Tmax were observed. For comparison purpose, the effects of the specific competitive FXIIa inhibitor corn trypsin inhibitor (CTI) were also investigated using the three same inducers than for Ir-CPI. For the contact pathway, a concentration-dependent decrease of the Cmax and a concentration-dependent prolongation of the lag time and the Tmax were found. When stimulating the TF pathway (5 or 1 pM TF), no modification of the thrombin generation curves was observed with the tested concentrations of CTI. When comparing the results of the two inhibitors acting trough the delay of the contact pathway, we found that Ir-CPI was about 30-fold more potent than CTI. We further evaluated the antithrombotic effect of Ir-CPI on a rat venous thrombosis model induced by endothelial damage and vessel ligation, close to the physiological venous thrombus formation in humans. We showed that Ir-CPI reduced thrombosis in a dose-dependent manner with an ED 50 close to 65 μg/kg. A maximum effect starting from 0.5 mg/kg was observed with a mean reduction in the clot weight/body weight of 75 ± 7%. This antithrombotic effect of Ir-CPI was exclusively mediated through the inhibition of thrombin generation since it did not interference with collagen-induced platelet aggregation. This is the first time that an inhibitor of the coagulation contact phase was shown to protect against the formation of venous thrombi. The antithrombotic effect of Ir-CPI was also confirmed using other venous and arterial thrombosis models. We also showed that the effective antithrombotic dose of Ir-CPI in these tests did not promote bleeding or impair blood coagulation parameters. Conclusions: The present study demonstrated that Ir-CPI, a recombinant protein from the tick Ixodes ricinus targeting the contact factors (FXII, FXI and kallikrein), displayed an anticoagulant activity mainly through the delay of the contact pathway induced thrombin generation. This drug also exhibited an antithrombotic activity in our venous and arterial thrombosis model. This drug may thus provide an interesting and innovative therapeutic tool for the prevention and the treatment of thromboembolic diseases with a minimal risk of therapy-associated bleeding. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3165 Introduction: Patients with cancer have a 7- to 10-fold overall increased risk of developing venous thromboembolism (VTE). Some tumors shed small membrane vesicles called microparticles (MPs) containing tissue factor (TF) and membrane phospholipids (PL) leading to procoagulant activity (PCA). TF, the most potent initiator of coagulation cascade, plays a critical role in hemostasis. The circulation of active TF associated with MPs has been considered as a risk factor for VTE in cancer. Aims: The primary objectives of this study were to characterize structure, size and PCA of tumor-cell derived MPs released by breast cancer cells MDA-MB231 (MDA) using thrombin generation (TGT), flow cytometry (FCM) and transmission electron microscopy (TEM). The secondary objectives were to determine the PCA of MDA and MPs derived from MDA in order to study the effect of stirring on MPs production and PCA. The study of the effects of the size of MP on PCA and the contribution of TF and PL to the PCA will also be performed. Methods: In vitro generation of MPs: Cultured MDA-MB-231 breast cancer cells were adjusted to the desired concentrations (600000 cells/ml) in PBS. Cells suspensions were incubated for 45 min at 37°C under stirring condition or without any stirring. Samples were then centrifuged at 4500g for 15 min and the cell supernatants (Sup) were used for EM, FCM and TGT. Alternatively, MP fractions were filtered through 0.1, 0.22, 0.45 or 0.65μm membranes (Ultrafree-MC) and subjected to activity assays. PCA: TGT was performed with Calibrated Automated Thrombogram (CAT). Cells suspensions or in vitro-generated MP fractions from 500000 cells were used as the source of TF and PL and added to normal pool plasma (NPP). Counting and expression of TF and MUC-1: Quantification of MPs and expression of TF (CD142) and MUC-1 (CD227) on MDA and MDA Sup were studied by flow FCM. The size of PMPs was defined using a blend of monodisperse fluorescent beads (Megamix). Tumor MPs sizing: A 5 μL sample of cells or MP fraction derived from 500000 cells diluted 100X was gently put onto a non-BSA precoated formvar grid and allowed to settle undisturbed for 48h before analysis under a TECNAI 10 TEM. Results: Effects of MDA-MB-231 and MDA-MB-231 Sup on PCA MDA and MDA Sup significantly increased the active thrombin in comparison to human NPP alone. Indeed, for example the lagtime was reduced by 11,7-fold and 7,2-fold, respectively. The difference between MDA and its Sup were highly significant (p<0,01) but this result is due a loss of MPs by centrifugation as shown by the differences in MPs concentration measured by FCM. Therefore, TF is mostly present in a non active encrypted configuration on the surface of MDA-MB-231. Effect of stirring on PCA of MDA-MB-231 Comparison of MPs concentration positive and negative for anti-MUC-1 and anti-TF in MDA before and after stirring showed that the stirring did not lead to a significantly increased number of MPs, as confirmed by TGT. Contribution of TF and PL to the PCA The relative importance of TF and PL in the PCA of MPs was assessed by comparing the TGT curves with or without addition of 4 μM PL. By comparing lagtime, TTP and Peak, we concluded that both PL and TF contributed to PCA of MPs, but at different stages of the coagulation cascade. The lagtime reduction with 0,1μm filtrated Sup MP showed that these particles provide FT to initiate the coagulation. This result supports that active TF mainly come from MPs. The difference in peak can be explained by the PL provided by tumor cells. Effects of MPs sizes on PCA The lagtimes of Sup and 0,65 μm filtered Sup (0,65μm Filt-Sup) were respectively reduced by 8,6-fold and 6,6-fold in comparison to NPP alone. The differences before and after 0,65 μm filtration of the MDA Sup were highly significant (p<0,01). The same PCA was found with cell Sup filtered at 0.45 and 0.65μm whereas it lowered progressively with filters from 0.45 to 0.1 μm. Indeed, in FCM and TEM, we found very few MPs between 0.45 and 0.65 μm. Conversely to FCM, EM showed that MPs derived from MDA-MB-231 are comprised between 30 nm and 200 nm and that the vast majority were under 100 nm. Such results were in agreement with FCM and TGT. Conclusions: TGT, FCM and TEM are very interesting methods that should be combined to adequately determine the phenotype of tumor-cell derived MPs whatever their size. MDA-MB-231 cells release spontaneously MPs of sizes comprised between 30 nm and 200 nm. These MPs have a strong PCA due to the expression of TF and PL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3175 Introduction: Patients with multiple myeloma (MM) have an increasing risk of developing venous thromboembolism (VTE). The use of thalidomide or lenalidomide, two antimyeloma agents has been associated with an increased risk of VTE especially when associated with dexamethasone or melphalan-prednisone. This increased risk in less pronounced with the proteasome inhibitor bortezomib. The pharmacological effects of thalidomide, lenalidomide and bortezomib have never been studied on platelet aggregation or coagulation. These preliminary data are necessary to understand the differential effects of these drugs on thrombotic events. Aims: The aim of our study is to investigate the pharmacological effects of these drugs on primary hemostasis by light transmission agregometry (LTA) and on secondary hemostasis using thrombin generation test (TGT). Methods: LTA was performed with a Chrono-Log aggregometer (Kordia). Platelet-rich plasma (PRP) of healthy blood donors (n=3 to 10) was adjusted to 300,000 platelets/μl. Platelet-poor plasma (PPP) and PRP were used respectively to adjust the photometric measurement to the minimum and maximum optical density. The platelet reactivity of the healthy donors was previously checked by LTA. Thalidomide (racemic, enantiomer (+) and enantiomer (-)), lenalidomide and bortezomib were spiked at final concentration of 200 μM in PRP except for studying the influence of thalidomide on ADP-induced platelet aggregation where the maximal concentration usable was 100 μM. LTA was performed without (spontaneous aggregation) and with addition of 5μM ADP, 190μg/ml collagen, 600μM arachidonic acid (AA) and 10 μM PAR1-AP (final concentrations). Negative controls (physiological saline and DMSO at concentrations used to dissolve the drugs) were also included. TGT was performed both on PPP (from normal pool plasma (NPP)) and on PRP. We first validated the method on NPP and PRP with two anticoagulants: a thrombin direct inhibitor (i.e. argatroban) and a FXa direct inhibitor (i.e. ZK-807834). Thalidomide ((+/−), (+) and (-)), lenalidomide and bortezomid were tested at the final concentrations of 5, 10, 50, 100 and 200 μM. For the experiments on NPP, PPP reagent (5 pM TF + 4 μM PL in the final assays), PPP reagent low (1 pM TF + 4 μM PL in the final assays) and MP reagent (4 μM PL in the final assays) were used whereas for the experiments on PRP, PRP reagent (1 pM TF in the final assays) was used. Results: LTA Both three drugs did not induce spontaneous aggregation until 200 μM. Lenalidomide and bortezomib showed no effect on induced platelet aggregation until 200 μM, whatever the agonist used. On the contrary, for racemic thalidomide, platelet aggregation was reduced at 50 and 100 μM with 5 μM ADP and at 150 and 200 μM with 600 μM AA. These effects were more pronounced with thalidomide (+) than with thalidomide (-). So, the half maximal inhibitory concentrations (IC50) for platelet aggregation induced by 600 μM AA were 127, 143 and 221 μM for racemic, enantiomer (+) and enantiomer (-), respectively. When 190μg/ml collagen or 10 μM PAR1-AP were used as agonists, no effect was observed on platelet aggregation until 200 μM thalidomide. TGT. With argatroban and ZK-807834, we observed a concentration-dependent decrease and delay of the thrombin activity profiles in PRP and NPP, whatever the inducer used. No significant effect on thrombin activity in NPP or PRP has been observed for antimyeloma drugs at all concentrations tested, whatever the inducer used. Conclusions: Lenalidomide, thalidomide and bortezomid do not induce spontaneous platelet aggregation and do not affect platelet aggregation induced by collagen or PAR1-AP. Conversely to lenalidomide and bortezomib, thalidomide, and especially its enantiomer (+), moderately inhibits platelet aggregation induced by ADP and AA. Moreover, these antimyeloma agents have no effect on thrombin generation. The increased risk of VTE by thalidomide or lenalidomide in patients with multiple myeloma is thus not mediated by a direct impact on primary or secondary hemostasis at therapeutic concentrations. Disclosures: No relevant conflicts of interest to declare.

Ethnopharmacological relevance: The leaf decoction of Croton zambesicus Muell. Arg. (Euphorbiaceae; syn. Croton amabilis Muell. Arg., Croton gratissimus Burch) is traditionally used in Benin to treat hypertension.Aim of the study: As hypertension and thromboembolism are often associated in several cardiovascular diseases, we studied the potential effects of leaf extracts from Croton zambesicus on hemostasis.Materials and methods: We prepared the dichloromethane and aqueous extracts from the air-dried leaves of Croton zambesicus and separated the aqueous extract in its aqueous and dichloromethane fractions. The potential effects of these four extracts/fractions were investigated on red blood cells integrity using spectrophotometric lysis assays, on primary hemostasis using platelet aggregation studies and on secondary hemostasis using calibrated automated thrombin generation assays and coagulation factors inhibition tests.Results: In the in vitro testing, we found that none of the tested extracts/fractions exhibit hemolytic or antiplatelet activity. However, they display a moderate but significant anticoagulant activity which would be mediated through the direct inhibition of thrombin, FXa and TF/FVIIa. The active anticoagulant compound(s) seem to be mainly in the aqueous extract and especially in its aqueous fraction.Conclusions: This experimental work reported for the first time the anticoagulant effect of leaf extracts from Croton zambesicus. These findings are of particular interest as the leaves from Croton zambesicus are commonly used in infusion by local population and may provide a new natural source for the development of original anticoagulant agents. Furthermore, this activity, associated with the vasorelaxant properties of some of its diterpenes may prove to be interesting for the prevention of cardiovascular diseases in traditional medicine. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Abstract Abstract 3335 Introduction: We previously reported that thrombin generation (TG) conducted in human PPP supplemented with synthetic phospholipids (PL) and triggered by 5 pM tissue factor (TF) was a very rapid, suitable and reliable pharmacological tool for screening thrombin and/or FXa inhibitors whatever their inhibition mode. However, a drawback of this work was that platelets, which are known to play a critical role in blood coagulation cascade by providing the catalytic surfaces for coagulation factors activation, were lacking in these experiments. Aims: The aim of this study was thus to compare the effects of various anticoagulant agents on TF-induced TG in platelet rich plasma (PRP)and platelet poor plasma (PPP) in order to define the most relevant and reliable assay to evaluate the potency and the behavior of drugs targeting thrombin and/or FXa. Methods: TG experiments were conducted with Calibrated Automated Thrombogram® (Thrombinoscope) in fresh PRP (150 000 platelets/μL) of 3 individual healthy donors with 1 pM TF + 16.7 mM CaCl2 and in normal pool frozen-thawed PPP of 32 healthy donors with 5 or 1 pM TF + 4 μM PL + 16.7 mM CaCl2. Five anticoagulants with various mode of action (argatroban, lepirudin, enoxaparin, fondaparinux and rivaroxaban) were spiked in the plasmas at increasing concentrations ranging from 10 nM to 5 μM. Results: In the absence of the drugs, the peak of active thrombin concentration was delayed (lag time increase), prolonged (Tmax increase) and reduced (Cmax decrease) when using PRP instead of PPP with 1 pM TF. These effects were still higher when comparing to PPP with 5 pM TF. However, the total amount of active thrombin (ETP) was similar in the three experimental designs. For all the parameters, the experimental CVs were <5% for PPP with 5 pM TF and < 15% for both PPP and PRP with 1 pM TF. In presence of the anticoagulant drugs, the peak of active thrombin concentration was concentration-dependently delayed, prolonged and reduced within all the assays. In PRP with 1 pM TF, all the drugs displayed close behaviors, excepting lepirudin. In PPP, and especially with 5 pM TF, more different profiles were found according to the mechanism of action of drugs. Regarding to the potency of the drugs, the inhibition parameters (i.e. 2x lag time, 2x Tmax, Cmax EC50 and Vmax EC50) were in the same range for argatroban and lepirudin whatever the chosen assay. For enoxaparin, rivaroxaban and fondaparinux, the drug effects varied among the assays but also following to the studied parameters. In all the assays, lepirudin was the most active drug to increase the lag time and the Tmax. The Cmax was mostly decreased by fondaparinux in PRP with 1 pM TF, by rivaroxaban in PPP with 5 pM TF and by both drugs in PPP with 1 pM TF. The ETP was mostly diminished by fondaparinux in PRP with 1 pM TF while the stronger effects were found with fondaparinux and enoxaparin using the two others inducers. Conclusions: The present study did not demonstrate a superiority of TF-induced thrombin generation assay conducted in PRP compared to PPP supplemented with synthetic PL. Although the potency of the drugs varied according to the studied parameter and the experimental design of the assay, experiments conducted in PL-supplemented PPP with 5 pM TF seemed to be the best suited to study the effects of anticoagulant drugs targeting thrombin and/or FXa due to their high reproducibility and their relevancy on drug mechanism of action. Moreover, the use of frozen-thawed normal pool plasma within these assays was more convenient regarding to the sample collection, storage and handling. It also excluded the inter-donor variability to be more relevant on the normal population hemostatic system. Disclosures: No relevant conflicts of interest to declare.

Contribution of platelet microparticles generation assay to the diagnosis of type II heparin-induced thrombocytopenia -

Abstract Abstract 3332 Introduction: Recently, 2 oral anticoagulants have been marketed: dabigatran, a direct thrombin inhibitor, and rivaroxaban, a direct FXa inhibitor. Thanks to their safety and efficacy profiles, routine coagulation monitoring is generally not required. However, the most commonly reported adverse reactions with these drugs are bleedings. A sensitive laboratory assay might be valuable to measure the pharmacodynamics of these 2 drugs in different situations including risks of drug interaction (i.e. strong P-gp inhibitors), overdose or to measure patient compliance. Due to their different mechanism of action, the measurement of their anticoagulant level in plasma may require different types of clotting assays. Aims: The aim of the present study was to determine which coagulation assay(s) could be used to measure the impact of rivaroxaban and dabigatran among a range of routine or more specific tests. Methods: Both drugs were spiked at increasing concentrations in pooled citrated normal human platelet-poor plasma (PPP). The final concentrations were ranging from 10 nM to 2 μM for dabigatran and from 25 nM to 5 μM for rivaroxaban. Such concentrations covered the plasma range in patients during orthopedic surgery. The following routine clotting assays were performed with the spiked plasma according to the manufacturer's protocols: Prothrombin Time (PT) with Neoplastin CI+ and Neoplastin R on STA-R (Roche Diagnostics), with Innovin on BCS (Siemens) and with Recombiplastin on ACL-TOP (Instrumentation Laboratories); activated Partial Thromboplastin Time (aPTT) with CK-Prest, PTT-A and Cephascreen on STA-R, with Actin FS on BCS and with Synthasil on ACL TOP and Thrombin Time (TT) on STA-R. More specific tests included Prothrombinase-induced Clotting Time (PiCT, Pefakit®, Pentapharm) and Ecarin Clotting Time (ECT) on STA-R; dilute PT (dPT) and activated Clotting time (ACT) on KC10. Thrombin Generation Tests (TGT) were also performed with Calibrated Automated Thrombogram (CAT) using PPP or PPP LOW reagents (Thrombinoscope). Sensitivity of a coagulation assay was defined as the concentration required to double clotting time (2XCT). Results: Dabigatran. A concentration-dependent prolongation of PT, dPT and aPTT was observed. However, at high concentrations, sensitivity of aPTT decreased whereas sensitivity of PT increased. The results varied depending on the clotting reagent used. TT was too sensitive leading to high variability for concentrations higher than 250nM. PiCT showed a linear concentration coagulation time (CT) relationship until a plateau at 750nM. ECT showed a high sensitivity (2X CT ≈ 93 nM), a linear relationship in the whole concentration range and a very low variability (CV≤ 1,8%). ACT gave a similar profile to PT (Innovin) with a slightly higher sensitivity (2X CT ≈ 716 nM vs 842nM). Dabigatran induced a concentration-dependent delay and inhibition of the tissue factor-induced thrombin generation with 5 pM TF or 1 pM TF with 4 μM PL and 16.7 mM CaCl2. The drug strongly increased the lag time and Tmax whereas it slightly decreased the Cmax and ETP. The lag time was the most sensitive CAT parameter with a high sensitivity (2x lag time ≈ 145 nM) and a low variability (CV≤6%). Rivaroxaban. A concentration-dependent prolongation of PT, dPT and aPTT was observed. However, at high concentrations, sensitivity of aPTT increased. Sensitivity of dPT (2X CT ≈ 109 to 550 nM) and PT (2X CT ≈ 138 to 764 nM) were similar and superior to aPTT (2X CT ≈ 897 to 2050 nM). The results varied depending on the clotting reagent used. TT was insensitive to rivaroxaban until 2500nM. Both PiCT and ECT showed a low sensitivity (2X CT ≈ 2536 nM and 5750 nM, respectively). A concentration-dependent prolongation of ACT was observed until 2500nM (2X CT ≈ 2275 nM). Rivaroxaban induced a concentration-dependent reduction and delay of the TF-induced thrombin generation. The Cmax was more strongly decreased than the ETP whereas the Tmax was more prolonged than the lag time, showing a major influence on the amplification phase. The Cmax was the most sensitive CAT parameter with a high sensitivity (Cmax EC50 ≈ 50 nM) and a low variability (CV<1%). Conclusions: ECT and PT are the most appropriate tests to study the pharmacodynamic effects of dabigatran and rivaroxaban respectively, thanks to high sensitivity, linear relationship in the whole concentration range and low variability. Disclosures: No relevant conflicts of interest to declare.

Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo.

Recently, FXIIa was highlighted as an original attractive target for the development of new anticoagulant drugs with low rates of therapy-related hemorrhages. In this work, we describe the development of a new series of 3-carboxamide-coumarins that are the first potent and selective nonpeptidic inhibitors of FXIIa.

The ex vivo testing emerges as an essential and critical step for the selection of the most promising prospective anticoagulant agents. The aim of the present study was to validate the thrombin generation assay as an ex vivo pharmacological screening test for measuring the anticoagulant behaviour and potency of molecules. The effects of six thrombin and/or factor Xa (FXa) inhibitors (argatroban, lepirudin, PPACK, enoxaparin, ZK-807834, fondaparinux) were investigated on the time course of thrombin catalytic activity triggered by the tissue factor pathway in platelet-poor plasma (PPP) of male healthy volunteers using the Calibrated Automated Thrombogram((R)) (CAT) method. In the presence of the anticoagulant drugs, the thrombin activity profiles were dose-dependently modified according to their specific enzyme inhibitory activity. ZK-807834 was the most potent drug for reducing the C(max) and the V(max) but also for prolonging the T(max). Lepirudin most efficiently delayed the lag time whereas enoxaparin was the most powerfully drug for diminishing the endogenous thrombin potential (ETP). In conclusion, the thrombin activity profile performed with the CAT method is a very rapid, suitable and reliable pharmacological tool for screening thrombin and/or FXa inhibitors whatever their inhibition mode. It consists of a powerful alternative for the classical PT clotting assay, especially regarding to the time course and the total amount of active thrombin generated. Last but not least, it provides insight into the mechanism of action of the compounds.

New 2-oxo-2H-1-benzopyran derivatives were prepared to optimize 2a,b, initially developed as mechanism-based alpha-chymotrypsin (alpha-CT) inhibitors, into potent and selective thrombin (THR) inhibitors. From this study, 22, characterized by a 2-(N-ethyl-2'-oxoacetamide)-5'-chlorophenyl ester side chain, was shown to be a good THR inhibitor (ki/KI = 3455 M(-1) x s(-1)), displaying an excellent selectivity profile against other serine proteases such as factor Xa, trypsin, and alpha-CT. Docking analysis of this compound into the different protein structures revealed the molecular basis responsible for its potency and selectivity.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.