Rat Model Of Thrombosis Research Articles

Abstract 9383: Selective Tissue Factor/Factor VIIa Inhibitor ER-410660 and its Prodrug E5539 Have an Anti-Venous Thrombotic Effect with Low Risk of Bleeding

The “extrinsic coagulation pathway” is activated by complex formation between tissue factor (TF) and factor VIIa (FVIIa). The onset of thrombosis is associated with the specific expression of TF. Compounds that inhibit the TF/FVIIa complex might therefore have therapeutic and/or prophylactic uses for diseases associated with thrombus formation. TF/FVIIa inhibitors might also reduce the risk of bleeding as they leave the intrinsic pathway intact and so maintain normal hemostasis. We assessed this hypothesis using a series of triazolone derivatives that acted as potent and selective TF/FVIIa inhibitors. Based on human intravenous pharmacokinetic data from a microdosing study, we identified ER-410660 as a candidate for prodrug formation to improve oral availability. ER-410660 showed potent and selective inhibition of the TF/FVIIa complex with an IC 50 of 5.7 nM. The compound selectively prolonged the prothrombin time (PT) in human, rhesus monkey, and rat plasma (a 50% increase of PT was obtained at 2.0, 1.7, and 19 μM, respectively). Intravenous (iv) administration of ER-410660 dose-dependently inhibited thrombin generation caused by TF-injection in the rhesus monkey with an ED 50 of 40 (95% CI, 25-55) μg/kg. ER-410660 (iv) reduced venous thrombus weights in a TF-administered stasis-induced venous thrombosis model in rats with an ED 50 of 0.18 (95% CI, 0.11-0.25) mg/kg. The anti-hemostatic effect of ER-410660 (iv) was evaluated in a rat tail-cut model. The total bleeding time was doubled at 7.1 (95% CI, 6.1-11) mg/kg, which was 39-fold higher than the ED 50 value in the rat venous thrombosis model. E5539, an orally available form of ER-410660, was discovered by prodrug formation. Oral E5539 at doses of 3, 10, and 30 mg/kg reduced plasma thrombin-antithrombin (TAT) complex levels elevated by TF-injection in a dose-dependent manner. The TAT level was reduced to 50% of the control at an ED 50 value of 12 (95% CI, 8.0-17) mg/kg. These proof-of-concept studies in animal models suggest that selective TF/FVIIa inhibitors such as ER-410660 and its prodrug E5539 could act as novel anticoagulants with a low risk of bleeding.

Biochemical and pharmacological profile of darexaban, an oral direct factor Xa inhibitor

Darexaban (YM150) is an oral factor Xa inhibitor developed for the prophylaxis of venous and arterial thromboembolic disease. This study was conducted to investigate the biochemical and pharmacological profiles of darexaban and its active metabolite darexaban glucuronide (YM-222714), which predominantly determines the antithrombotic effect after oral administration of darexaban. In vitro activity was evaluated by enzyme and coagulation assays, and a prothrombin activation assay using reconstituted prothrombinase or whole blood clot. In vivo effects were examined in venous thrombosis, arterio–venous (A–V) shunt thrombosis, and bleeding models in rats. Both darexaban and darexaban glucuronide competitively and selectively inhibited human factor Xa with Ki values of 0.031 and 0.020 μM, respectively. They showed anticoagulant activity in human plasma, with doubling concentrations of darexaban and darexaban glucuronide for prothrombin time of 1.2 and 0.95 μM, respectively. Anticoagulant activity was independent of antithrombin. Darexaban and darexaban glucuronide inhibited the prothrombin activation induced by prothrombinase complex or whole blood clot with similar potency to free factor Xa. In contrast, prothrombinase- and clot-induced prothrombin activation were resistant to inhibition by enoxaparin. In venous and A–V shunt thrombosis models in rats, darexaban strongly suppressed thrombus formation without affecting bleeding time, with ID 50 values of 0.97 and 16.7 mg/kg, respectively. Warfarin also suppressed thrombus formation in these models, but caused a marked prolongation of bleeding time at antithrombotic dose. In conclusion, darexaban is a selective and direct factor Xa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

An optimized method to assess in vivo efficacy of antithrombotic drugs using optical coherence tomography and a modified Doppler flow system

Introduction Animal models of venous and arterial thrombosis are extremely useful to study the efficacy of antithrombotic agents. Variability in efficacy data is often observed in those preclinical studies. The goal of this study was to optimize the methodology for assessing antithrombotic drug efficacy by the use of optical coherence tomography (OCT) and a modified Doppler flow system in rat models of thrombosis. Methods Thrombus formation was assessed in both the rat venous and arterial ferric chloride (FeCl 3) models of thrombosis. In the venous model, thrombus volume post-treatment was measured using OCT, and data were correlated against the thrombus weight. In the arterial model, the time to occlusion was measured using a Doppler flow probe connected to a perivascular flow module which allowed the reporting of dynamic blood flow data every 30 s. Heparin (130 or 165 U/kg), argatroban (4.5 mg/kg), bivalirudin (1.3 mg/kg) or saline were administered intravenously. Results In the venous model, for all treatment groups a strong linear correlation (R 2 = 0.998) was observed between thrombus volume measured by OCT and thrombus weight. In the arterial model, using a high sampling rate of a dynamic blood flow using a modified Doppler flow system provided data accuracy and precision of the time to occlusion measurement. Discussion This study demonstrates that OCT is a powerful tool for the assessment of antithrombotic drug efficacy. Furthermore, it shows that a high Doppler sampling rates of dynamic blood flow leads to data accuracy and precision.

Pyrolo[1,2:4,5]‐1,4‐dioxopyrazino[1,2:1,6]pyrido[3,4‐b]indoles: A Group of Urokinase Inhibitors, their Synthesis, and Stereochemistry‐Dependent Activity

Antifibrinolytic agents are required during complex surgeries to decrease bleeding; their pro-thrombotic potency and efficacy in causing hemostasis has attracted much attention. To discover new inhibitors of urokinase with high selectivity for antifibrinolytic effects over pro-thrombotic effects, the 12-position of (5aS,12S,14aS)- and (5aS,12R,14aS)-5,14-dioxo-1,2,3,5,5a,6,11, 12,14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indoles were modified with L-Ala, L-Asp, L-Phe, L-Trp, L-Lys, L-Ser, Gly, and L-Leu to provide 16 (5aS,12S,14aS) and (5aS,12R,14aS) derivatives. In a murine bleeding model, the (5aS,12S,14aS) derivatives containing L-Ala, L-Asp, L-Phe, and L-Trp induced blood coagulation for the treated mice; they also stimulated thrombus formation in a rat thrombosis model, but the other derivatives inhibited thrombosis. The most potent compound, the L-Asp derivative, showed a good therapeutic window: the minimum effective dose for coagulation was <1 nmol kg(-1), whereas at 10 nmol kg(-1), no pro-thrombotic effect was observed. This type of coagulation action was correlated with a mechanism of urokinase inhibition, and these results could lead to the discovery of novel urokinase inhibitors.

Heparin-triggered release of camouflaged tissue plasminogen activator for targeted thrombolysis

A targetable, heparin-triggered release system for tissue plasminogen activator (tPA) was designed to prevent the excessive ‘lytic’ state associated with the current tPA therapy for acute thrombotic conditions, such as myocardial infarction (MI). The strategy is, upon target accumulation, to trigger tPA release from a prodrug construct by a usual heparin dose. A relatively inactive form of tPA was constructed by conjugating tPA with low-molecular weight heparin followed by complexation with albumin-protamine conjugate, termed ‘camouflage’. The modified tPA was ~ 97% as active as native tPA. The prodrug construct of tPA significantly masked the enzymatic activity, which was fully recovered upon heparin addition. The camouflaged tPA was stable in human blood for at least 30 min and was able to trigger enzyme activation in vitro at heparin level of 0.4 U/mL. In vivo studies on jugular vein rat thrombosis model showed that the clot lysis of the heparin-triggered camouflaged tPA group was equivalent to the tPA + heparin group without prolongation of activated partial thromboplastin time (aPTT) before and after the treatment. This proof-of-principle study suggests that the activity of the tPA prodrug construct can be triggered at the thrombus site at therapeutic heparin concentration conjunctively used for MI with reduced bleeding risk.

New derivative of staphylokinase SAK-RGD-K2-Hirul exerts thrombolytic effects in the arterial thrombosis model in rats

SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul are recombinant proteins that are derivatives of r-SAK (recombinant staphylokinase). They are characterized by their fibrin-specific plasminogen activation properties and their antithrombin and antiplatelet activities. The difference between these proteins is the presence of the antithrombotic fragment (hirudin or hirulog) in the C-terminal portion of the r-SAK.The aim of the present study was to examine the thrombolytic potentials of SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul in an electrically induced carotid artery thrombosis model in rats and to compare the potentials to that of r-SAK.We determined that a bolus injection of SAK-RGD-K2-Hirul was more effective than one of r-SAK in the improvement and maintenance of carotid patency and in arterial thrombus weight reduction; however, it had the same potency as SAK-RGD-K2-Hir. The bleeding time, prothrombin time and activated partial thromboplastin time were significantly prolonged in the animals that were treated with either dose (1.5 or 3.0mg/kg) of SAK-RGD-K2-Hir or SAK-RGD-K2-Hirul, whereas no changes were observed in the plasma fibrinogen concentration or the α 2 plasmin inhibitor level. r-SAK alone did not change the bleeding time or coagulation parameters. In conclusion, our findings demonstrate the thrombolytic activity of intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hirul in rats. Although this protein compares favorably with r-SAK, we were unable to show the presence of any beneficial effects of SAK-RGD-K2-Hirul over those of SAK-RGD-K2-Hir. Furthermore, our results suggest that high doses of SAK-RGD-K2-Hirul bear the risk of bleeding.

Antithrombotic activity of a 2-kDa heparin fragment in an experimental model of carotid artery thrombosis in rats.

The antithrombotic activity of a 2-kDa heparin fragment was studied in a rat model of common carotid artery thrombosis that causes a completely occlusive thrombus with cessation of the blood flow within 10-15 min. The compound reduced thrombus formation in a dose-dependent manner, starting from an intravenous dose of 5 mg kg-1. A dose of 20 mg kg-1 completely prevented thrombus formation and apparently induced the almost complete lysis of the already formed occlusive thrombus. At none of the doses used did the compound cause increased bleeding or the formation of haematomas. The present results indicate that low molecular weight heparins, which have an established, highly beneficial effect in venous thromboembolism, are also highly effective in an animal model of arterial thrombosis.

An antithrombin-dependent sulfated polysaccharide isolated from the green alga Caulerpa cupressoides has in vivo anti- and prothrombotic effects

Red algae sulfated polysaccharides (SPs) have been widely described as anticoagulant and antithrombotic agents; however no description of antithrombotic activity regarding green algae SPs has been reported. Caulerpa cupressoides (Chlorophyta) has three different SPs fractions (SP1, SP2 and SP3). We investigated the effects of SP2 on thrombin activity by antithrombin and in an experimental model of venous thrombosis in rats. The inhibition of thrombin assay was evaluated using antithrombin (AT) in the presence of SP2 and the antithrombotic activity was investigated in rats with thromboplastin as the thrombogenic stimulus. The anticoagulant effects of SP2 are suggested be due to the potentiation of thrombin inhibition by antithrombin (IC50 ~ 10.0µg mL-1) and this mechanism of interaction is different when compared to other studied Caulerpa polysaccharides. SP2 exhibited antithrombotic effects at doses of 1.0 and 2.0mg kg-1 body weight, but at higher doses (&gt;2.0mg kg-1 body weight) this polysaccharide revert the antithrombotic property. No hemorrhagic effect (2.0mg kg-1) was observed. As occurs with red algae SPs, these results indicate that green algae SPs are also capable of exhibiting different in vivo properties.

Pharmacokinetic evaluation of an oral tablet form of low-molecular-weight heparin and deoxycholic acid conjugate as a novel oral anticoagulant

This study was designed to develop a solid oral dosage form of deoxycholic acid (DOCA)-conjugated low-molecular-weight heparin (LMWH) and to evaluate its oral absorption, distribution, and metabolic stability for the prospect of providing an orally bioavailable LMWH. The LMWH derivative (LHD) was synthesised and then formulated with solubilisers and other pharmaceutical excipients to form a solid tablet. Its absorption and distribution after oral administration were evaluated in mice, rats, and monkeys. The in vitro metabolic stability of LHD was examined by liver microsome assays. More than 80% of LHD was released from the tablet within 60 minutes, guaranteeing rapid tablet disintegration after oral administration. Oral bioavailability of LHD in mice, rats and monkeys were 16.1 ± 3.0, 15.6 ± 6.1, and 15.8 ± 2.5%, respectively. After the oral administration of 131I-tyramine-LHD, most of the absorbed drug remained in the blood circulation and was eliminated mainly through the kidneys. LHD was hardly metabolised by the liver microsomes and showed a stable metabolic pattern similar to that of LMWH. In a rat thrombosis model, 10 mg/kg of orally administered LHD reduced thrombus formation by 60.8%, which was comparable to the anti-thrombotic effect of the subcutaneously injected LMWH (100 IU/kg). Solid tablets of LHD exhibited high oral absorption and statistically significant therapeutic effects in preventing venous thromboembolism. Accordingly, LHD tablets are expected to satisfy the unmet medical need for an oral heparin-based anticoagulant as an alternative to injectable heparin and oral warfarin.

The Tick Protein Ir-Cpi Efficiently Delays Contact Pathway Induced Thrombin Generation and Displays In Vivo Antithrombotic Activity

AbstractAbstract 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 CaCl2 was used to activate the TF pathway whereas a lower TF concentration of 1 pM with 4 μM PL and 16.7 mM CaCl2 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 CaCl2 (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 FeCl3 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 ED50 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. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Anti-thrombosis effect of diosgenin extract from Dioscorea zingiberensis C.H. Wright in vitro and in vivo

Thrombus formation in blood vessel plays an important role in the pathogenesis and progression of atherosclerosis and cardiovascular diseases. Extract of Dioscorea zingiberensis C.H. Wright (D. zingiberensis) is demonstrated to posses activities for curing cardiovascular diseases such as coronary heart disease and angina pectoris. However, there were few studies on anti-thrombosis activity of it. We investigated the anti-thrombosis effect of diosgenin from D. zingiberensis (Dio) in vitro and in vivo on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model. We evaluated the protective effect of Dio by measuring the platelet aggregation, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and the venous thrombosis in rats and the bleeding time, clotting time and protection rate in mice. Results showed that Dio inhibited platelet aggregation, thrombosis and prolonged APTT, PT and TT in rats in a dose-dependent manner. They also prolonged the bleeding time, clotting time and increased protection rate in mice in a dose-dependent manner. Taken together, these findings suggested that Dio which contained 95% diosgenin had anti-thrombosis activity. Dio executives the anti-thrombosis activity through improving the anticoagulation function, inhibiting platelet aggregation and thrombosis.

Nitrophorin 2, a factor IX(a)-directed anticoagulant, inhibits arterial thrombosis without impairing haemostasis

Nitrophorin 2 (NP2) is a 20 kDa lipocalin identified in the salivary gland of the blood sucking insect, Rhodnius prolixus. It functions as a potent inhibitor of the intrinsic pathway of coagulation upon binding to factor IX (FIX) or FIXa. Herein we have investigated the in vivo antithrombotic properties of NP2. Surface plasmon resonance assays demonstrated that NP2 binds to rat FIX and FIXa with high affinities (KD = 43 and 47 nM, respectively), and prolongs the aPTT without affecting the PT. In order to evaluate NP2 antithrombotic effects in vivo two distinct models of thrombosis in rats were carried out. In the rose Bengal/laser induced injury model of arterial thrombosis, NP2 increased the carotid artery occlusion time by ≍35 and ≍155%, at doses of 8 and 80 μg/kg, respectively. NP2 also inhibited thrombus formation in an arterio-venous shunt model, showing ≍60% reduction at 400 μg/kg (i.v. administration). The antithrombotic effect lasted for up to 48 hours after a single i.v. dose. Notably, effective doses of NP2 did not increase the blood loss as evaluated by tail-transection model. In conclusion, NP2 is a potent and long-lasting inhibitor of arterial thrombosis with minor effects on haemostasis. It might be regarded as a potential agent for the treatment of human cardiovascular diseases.

Anti-thrombotic activity and chemical characterization of steroidal saponins from Dioscorea zingiberensis C.H. Wright

Steroidal saponins have long attracted scientific attention, due to their structural diversity and significant biological activities. Total steroidal saponins (TSS) extracted from the rhizomes of Dioscorea zingiberensis C.H. Wright (DZW) constitute an effective treatment for cardiovascular disease. However, the active constituents contained in DZW rhizomes and their pharmacological properties are not fully understood. The aim of this work is to determine and quantify the active constituents in DZW rhizomes using fingerprint technique, and evaluate its anti-thrombotic activity using inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model after being gavaged with TSS for 1 or 2 weeks. In the study, a chemical fingerprint method was firstly established and validated to quantify and standardize TSS from DZW rhizomes including parvifloside, protodeltonin, protodioscin, protogracillin, zingiberensis saponin, deltonin, dioscin and trillin. TSS extracted from DZW rhizomes were showed to have the inhibitions on platelet aggregation (PAG) and thrombosis, and prolong activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) in a dose-dependent manner in rats. TSS also prolonged the bleeding time and clotting time in a dose-dependent manner in mice. The results indicate that TSS could inhibit thrombosis by both improving the anticoagulation activity and inhibiting PAG action, suggesting that TSS from DZW rhizomes have the potential to reduce the risk of cardiovascular diseases by anti-thrombotic action.

MS381 THE EFFECT OF ROSUVASTATIN ON THE ARTERIAL PRESSURE OF HYPERLIPIDAEMIC AND HYPERTENSIVE PATIENTS

Vaccinium myrtillus L. (blueberry) leaf infusions are traditionally used as a folk medicine treatment of diabetes. To further define this therapeutical action, a dried hydroalcoholic extract of the leaf was administered orally to streptozotocin-diabetic rats for 4 days. Plasma glucose levels were consistently found to drop by about 26% at two different stages of diabetes. Unexpectedly, plasma triglyceride (TG) were also decreased by 39% following treatment. Subsequent to the latter observation, possible lipid-lowering properties of the extract were investigated on other models of hyperlipidaemia and ciprofibrate, a well-established hypolipidaemic drug, was used as a reference compound. Both drug reduced TG levels of rats on hyperlipidaemic diet in a dose-dependent fashion. When administered at single doses over the same experimental period, blueberry and ciprofibrate were effective in lowering TG concentrations in ethanol-treated normolipidaemic animals and in genetically hyperlipidaemic Yoshida rats. Unlike ciprofibrate, however, blueberry failed to prevent the rise in plasma TG elicited by fructose and did not affect free fatty acid levels in any of the above experimental conditions. In rats treated with Triton WR-1339, blueberry feeding induced an hypolipidaemic activity one hour after injection but proved to be ineffective at later time points, thus suggesting that its hypolipidaemic action may reflect improved TG-rich lipoprotein catabolism. In addition, ciprofibrate and the extract were tested for antithrombotic activity using a collagen-triggered model of venous thrombosis in diabetic and Yoshida rats. Only ciprofibrate, however, significantly reduced thrombus formation in diabetics, possibly because of its effects on free fatty acid metabolism, whereas no effect was observed in Yoshida rats. In conclusion, the present findings indicate that active consituent(s) of Vaccinium myrtillus L. leaves may prove potentially useful for treatment of dyslipidaemiae associated with impaired TG-rich lipoprotein clearance. Copyright © 1996 Elsevier Science Ltd

Effect of the Direct Factor Xa Inhibitor Apixaban in Rat Models of Thrombosis and Hemostasis

Apixaban is an oral, direct, and highly selective factor Xa inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. Apixaban was evaluated in rat thrombosis and hemostasis models. Thrombosis was produced in the carotid artery by FeCl2 application, in the vena cava by either FeCl2 application or tissue factor injection, and in an arterial-venous shunt. Hemostasis was assessed using cuticle, renal cortex, and mesenteric artery bleeding times. Intravenous apixaban infusions of 0.1, 0.3, 1, and 3 mg/kg per hour increased the ex vivo prothrombin time to 1.24, 1.93, 2.75, and 3.98 times control, respectively. The 0.3, 1, and 3-mg/kg per hour doses inhibited thrombosis in all models. Concentrations for 50% thrombus reduction ranged from 1.84 to 7.57 microM. The 3-mg/kg per hour dose increased cuticle, renal, and mesenteric bleeding times to 1.92, 2.13, and 2.98 times control, respectively. Lower doses had variable (1 mg/kg per hour) or no effect (0.1, 0.3 mg/kg per hour) on hemostasis. Heparin's prolongation of renal and cuticle bleeding time was twice that of apixaban when administered at a dose that approximated apixaban (3 mg/kg per hour) efficacy in arterial thrombosis. In summary, apixaban was effective in a broad range of thrombosis models at doses producing modest increases in multiple bleeding time models.

Anticoagulant Efficacy of Solid Oral Formulations Containing a New Heparin Derivative

The need for an efficacious and safe oral anticoagulant that does not require monitoring has been largely unmet. Many efforts have centered on preparing orally available heparin to improve patient compliance. In this study, novel orally active heparin derivatives (LHD), i.e. low molecular weight heparin (LMWH) conjugated with deoxycholic acid (DOCA), were evaluated in vitro and in vivo for their enhancement effect of oral heparin absorption. After oral administration of 10 mg/kg of water-soluble LHD, Ws-LHD1.5 showed optimum oral efficacy and its bioavailability was about 24% in rats. The oral absorption of LHD1.5 was also enhanced by several solubilizers, among which Poloxamer 407 provided the best results. When 5 mg/kg of LHD1.5 with Poloxamer 407 was orally administered to monkeys, the maximum anti-FXa activity in plasma was 0.26 +/- 0.04 IU/mL and its bioavailability was 17.4%. In a rat thrombosis model, 5 mg/kg of orally administered LHD1.5 formulated with Poloxamer reduced thrombus formation by 63.9 +/- 16.6%, which was higher than the efficacy of clinically used enoxaparin (49.4 +/- 17.8% at 100 IU/kg, sc). Considering the oral absorption efficacy and therapeutic effect, the conjugation ratio was optimized as about 1.5 molecules of DOCA per mole of heparin. Therefore, LHD1.5 with Poloxamer 407 can be further formulated as a solid oral anticoagulant drug.

Low-Level Mercury Can Enhance Procoagulant Activity of Erythrocytes: A New Contributing Factor for Mercury-Related Thrombotic Disease

BackgroundAssociations between cardiovascular diseases and mercury have been frequently described, but underlying mechanisms are poorly understood.ObjectivesWe investigate the procoagulant activation of erythrocytes, an important contributor to thrombosis, by low-level mercury to explore the roles of erythrocytes in mercury-related cardiovascular diseases.MethodsWe used freshly isolated human erythrocytes and ex vivo and in vivo thrombosis models in rats to investigate mercury-induced procoagulant activity.ResultsProlonged exposure to low-dose mercuric ion (Hg2+; 0.25–5 μM for 1–48 hr) induced erythrocyte shape changes from discocytes to echinocytes to spherocytes, accompanied by microvesicle (MV) generation. These MVs and remnant erythrocytes expressed phosphatidylserine (PS), an important mediator of procoagulant activation. Hg2+ inhibited flippase, an enzyme that recovers PS into the inner leaflet of the cell membrane, and activated scramblase, an enzyme that alters lipid asymmetry in the cell membrane. Consistent with these activity changes, Hg2+ increased intracellular calcium and depleted ATP and protein thiol. A thiol supplement reversed Hg2+-induced MV generation and PS exposure and inhibited the increase in calcium ion (Ca2+) and depletion of ATP, indicating that free-thiol depletion was critical to Hg2+-mediated procoagulant activity. The procoagulant activity of Hg2+-treated erythrocytes was demonstrated by increased thrombin generation and endothelial cell adhesion. We further confirmed Hg2+-mediated procoagulant activation of erythrocytes in ex vivo and in vivo rat thrombosis models, where Hg2+ treatment (0.5–2.5 mg/kg) increased PS exposure and thrombus formation significantly.ConclusionThis study demonstrated that mercury could provoke procoagulant activity in erythrocytes through protein-thiol depletion–mediated PS exposure and MV generation, ultimately leading to enhanced thrombosis.

Synthesis and evaluation of 1,4-diphenylbutadiene derivatives as inhibitors of plasminogen activator inhibitor-1 (PAI-1) production

Butadiene-imide 1 (T-686) derivatives were synthesized and evaluated for their inhibitory activity against PAI-1 production and their ADMET (DMPK and toxicology) profiles. Among these derivatives, compound 15k (T-2639) showed good antithrombotic activity in two rat thrombosis models without affecting bleeding time, indicating reduction of haemorrhagic risk. We also describe in this report a practical synthesis of 15k suitable for scale-up using Z, E-selective Stobbe condensation.

Nitrophorin 2, a factor IX(a)-directed anticoagulant, inhibits arterial thrombosis without impairing haemostasis

SummaryNitrophorin 2 (NP2) is a 20 kDa lipocalin identified in the salivary gland of the blood sucking insect, Rhodnius prolixus. It functions as a potent inhibitor of the intrinsic pathway of coagulation upon binding to factor IX (FIX) or FIXa. Herein we have investigated the in vivo antithrombotic properties of NP2. Surface plasmon resonance assays demonstrated that NP2 binds to rat FIX and FIXa with high affinities (KD = 43 and 47 nM, respectively), and prolongs the aPTT without affecting the PT. In order to evaluate NP2 antithrombotic effects in vivo two distinct models of thrombosis in rats were carried out. In the rose Bengal/laser induced injury model of arterial thrombosis, NP2 increased the carotid artery occlusion time by ≈35 and ≈155%, at doses of 8 and 80 μg/kg, respectively. NP2 also inhibited thrombus formation in an arterio-venous shunt model, showing ≈60% reduction at 400 μg/kg (i.v. administration). The antithrombotic effect lasted for up to 48 hours after a single i.v. dose. Notably, effective doses of NP2 did not increase the blood loss as evaluated by tail-transection model. In conclusion, NP2 is a potent and long-lasting inhibitor of arterial thrombosis with minor effects on haemostasis. It might be regarded as a potential agent for the treatment of human cardiovascular diseases.

Antithrombotic effects of bromophenol, an alga-derived thrombin inhibitor

Thrombin, the ultimate proteinase of the coagulation cascade, is an attractive target for the treatment of a variety of cardiovascular diseases. A bromophenol derivative named (+)-3-(2,3-dibromo-4, 5-dihydroxy-phenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroiso-benzofuran 1, isolated from the brown alga Leathesia nana exhibited significant thrombin inhibitory activity. In this study, we investigated the inhibition of human thrombin in vitro with this bromophenol derivative, and its antithrombotic efficacy in vivo using the arteriovenous shunt model and the ferric chloride-induced arterial thrombosis model in rats. The results show that the bromophenol derivative is a potential inhibitor of thrombin (IC(50) = 1.03 nmol/L). In antithrombotic experiments in vivo, the bromophenol derivative also shows good effect comparing with the control group. These data indicate that the bromophenol derivative is a potential drug for prophylaxis and the treatment of thrombotic diseases.