Effect of prostaglandin E1 on the formation of experimental arterial and venous thrombosis

The effect of prostaglandin E1 on thrombus formation and haemostasis was investigated in a standardized experimental model in 20 rabbits. Arterial thrombus formation was reduced significantly by 70% and venous thrombosis by 68% in comparison with the controls. Collagen- and ADP-induced platelet aggregation was also inhibited significantly. Additionally, the time lag until onset of collagen-induced aggregation was significantly prolonged. Platelet count however, and plasma coagulation time showed no change. The data demonstrate a clear inhibition of arterial and venous thrombus formation, suggesting a powerful antithrombotic activity of prostaglandin E1. The effect may be explained by an influence on platelet function. The data further indicate that a full antithrombotic effect is achieved during prostaglandin E1 infusion.     

Antithrombotic properties of SSR182289A,a new,orally active thrombin inhibitor

N-[3-[[[(1S)-4-(5-Amino-2-pyridinyl)-1-[[4-difluoromethylene)-1-piperidinyl]carbonyl]butyl]amino]sulfonyl][1,1'-biphenyl]-2-yl]acetamide hydrochloride (SSR182289A) is a novel, potent, and selective thrombin inhibitor. We have examined the antithrombotic properties of SSR182289A administered by i.v. and p.o. routes in several different animal thrombosis models in comparison with reference antithrombotic agents. Oral administration of SSR182289A produced dose-related antithrombotic effects in the following models; rat venous thrombosis (ED(50) 0.9 mg/kg p.o.), rat silk thread arterio-venous (AV) shunt (ED(50) 3.8 mg/kg p.o.), rat thromboplastin-induced AV shunt (ED(50) 3.1 mg/kg p.o.), rat carotid artery thrombosis (ED(200) 5.9 mg/kg p.o.), and rabbit venous thrombosis (ED(50) 7.5 mg/kg p.o.). Administered as an i.v. bolus, SSR182289A showed antithrombotic activity in the above models with ED(50)/ED(200) values in the range of 0.2 to 1.9 mg/kg i.v. SSR182289A increased rat tail transection bleeding time at doses > or =10 mg/kg p.o. In the rat thromboplastin-induced AV shunt model, SSR182289A 10 mg/kg p.o. produced marked antithrombotic effects at 30, 60, 120, and 240 min after administration. Hence, SSR182289A demonstrates potent oral antithrombotic properties in animal venous, AV-shunt, and arterial thrombosis models.     

Activity of a synthetic hexadecasaccharide (SanOrg123781A) in a pig model of arterial thrombosis.

The activity of SanOrg123781A, a new synthetic antithrombotic drug inhibiting both factor Xa and thrombin through antithrombin (AT), was compared to that of unfractionated heparin (UFH) and of the synthetic pentasaccharide (fondaparinux, SP) in an ex vivo arterial thrombosis model in the pig. Six groups of four pigs were administered intravenously with SanOrg123781A (1, 3, 10 and 30 nmol kg(-1)), UFH (30 nmol kg(-1)) or SP (30 nmol kg(-1)). In this arterial model in which platelet thrombus was formed on a thrombogenic surface under a constant high shear rate, UFH and SP had moderate antithrombotic effects while SanOrg123781A exhibited a strong, dose-dependent inhibitory activity on platelet adhesion and platelet thrombus formation. In contrast to UFH, SanOrg123781A did not modify the activated partial thromboplastin time (aPTT) even at 30 nmol kg(-1), but strongly inhibited thrombin generation. At the same dose, despite a lower antithrombotic activity than SanOrg123781A, UFH significantly affected all the coagulation parameters. Taken together, these results show that SanOrg123781A, due to its potent and selective antifactor Xa and antifactor IIa activities is a promising new antithrombotic agent even in arterial setting.     

Murine model of ferric chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor

BACKGROUND/OBJECTIVE: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. In the present study, we describe the development of a murine model of vena cava thrombosis and its use to characterize the antithrombotic activity of potato carboxypeptidase inhibitor (PCI) of TAFIa (activated TAFI) in mice. METHODS/RESULTS: Vena cava thrombosis was induced by various concentrations of FeCl(3) in C57BL/6 mice. A relatively mild stimulus (3.5% FeCl(3)) induced thrombosis that was consistent and sensitive to reference antithrombotic agents such as clopidogrel and heparin. Dose-response studies identified a PCI dose (5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1), i.v.) that produced a maximum 45% decrease in vena cava thrombus mass as assessed by protein content (n = 8, P < 0.01 compared to vehicle) in the 3.5% FeCl(3)-induced model without exogenous tissue plasminogen activator administration. In contrast, PCI had no effect on 3.5% FeCl(3)-induced carotid artery thrombosis in mice. In a tail transection bleeding model, the 5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1) dose of PCI increased tail-bleeding time up to 3.5 times control (n = 8, P < 0.05). The ex vivo activity of antithrombotic doses of PCI was also demonstrated by the enhanced lysis of whole blood clots formed in a thrombelastograph with the addition of a sub-threshold concentration of tPA. CONCLUSION: These studies provide evidence for a role of TAFIa in venous thrombosis in mice, and describe an optimized vena cava injury model appropriate for the evaluation of antithrombotic drugs and the characterization of novel therapeutic targets.     

Production of thrombi on intact endothelium by use of antiheparin agents in vivo

It had been suggested that antithrombin activity on the surface of intact endothelial cells may play a role in inhibiting platelet adhesion and thrombus formation. The antithrombin activity may be due to thrombomodulin or to activation of antithrombin III by glycosaminoglycans or thrombomodulin, or possibly a combination of these. This inhibitory activity has been shown to be affected by such antiheparin agents as protamine, hexadimethrine bromide (Polybrene; Aldrich Chemical Co., Milwaukee, Wis.) and platelet factor 4, as well as by such enzymes as heparinase and heparitinase. We have used a hamster cheek pouch preparation to observe thrombus formation in vivo in a normal vascular flow, to determine whether the production of thrombi by thrombin can be enhanced by antiheparin agents. After intra-arterial injection or topical application of protamine or hexadimethrine bromide, platelet adhesion and thrombus formation on intact arteriolar endothelium was produced by a dose of thrombin, which when injected alone had no effect. No thrombi were found in venules or capillaries. Injection of heparin before or after the antiheparin agents necessitated a larger dose to enhance the action of thrombin. On electron microscopy the thrombi were found to consist primarily of platelets adherent to an intact endothelium. The possible clinical implications of these observations are discussed.     

DU-176b, a potent and orally active factor Xa inhibitor: in vitro and in vivo pharmacological profiles

Summary.  Background:  Factor Xa (FXa), a key serine protease that converts prothrombin to thrombin in the coagulation cascade, is a promising target enzyme for the prophylaxis and treatment of thromboembolic diseases. DU-176b is a novel antithrombotic agent that directly inhibits FXa activity. Objective:  To evaluate the in vitro pharmacological profiles and in vivo effects of DU-176b in animal models of thrombosis and bleeding. Methods: In vitro , FXa inhibition, specificity and anticoagulant activities were examined. Oral absorption was studied in rats and cynomolgus monkeys. In vivo effects were studied in rat and rabbit models of venous thrombosis and tail bleeding. Results: DU-176b inhibited FXa with Ki values of 0.561 n m for free FXa, 2.98 n m for prothrombinase, and exhibited >10 000-fold selectivity for FXa. In human plasma, DU-176b doubled prothrombin time and activated partial thromboplastin time at concentrations of 0.256 and 0.508 μ m , respectively. DU-176b did not impair platelet aggregation by ADP, collagen or U46619. DU-176b was highly absorbed in rats and monkeys, as demonstrated by more potent anti-Xa activity and higher drug concentration in plasma following oral administration than a prototype FXa inhibitor, DX-9065a. In vivo , DU-176b dose-dependently inhibited thrombus formation in rat and rabbit thrombosis models, although bleeding time in rats was not significantly prolonged at an antithrombotic dose. Conclusions:  DU-176b is a more potent and selective FXa inhibitor with high oral bioavailability compared with its prototype, DX-9065a. DU-176b represents a promising new anticoagulant for the prophylaxis and treatment of thromboembolic diseases.     

In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939--an oral, direct Factor Xa inhibitor.

BAY 59-7939 is an oral, direct Factor Xa (FXa) inhibitor in development for the prevention and treatment of arterial and venous thrombosis. BAY 59-7939 competitively inhibits human FXa (K(i) 0.4 nm) with > 10 000-fold greater selectivity than for other serine proteases; it also inhibited prothrombinase activity (IC(50) 2.1 nm). BAY 59-7939 inhibited endogenous FXa more potently in human and rabbit plasma (IC(50) 21 nm) than rat plasma (IC(50) 290 nm). It demonstrated anticoagulant effects in human plasma, doubling prothrombin time (PT) and activated partial thromboplastin time at 0.23 and 0.69 microm, respectively. In vivo, BAY 59-7939 reduced venous thrombosis (fibrin-rich, platelet-poor thrombi) dose dependently (ED(50) 0.1 mg kg(-1) i.v.) in a rat venous stasis model. BAY 59-7939 reduced arterial (fibrin- and platelet-rich) thrombus formation in an arteriovenous (AV) shunt in rats (ED(50) 5.0 mg kg(-1) p.o.) and rabbits (ED(50) 0.6 mg kg(-1) p.o.). Slight inhibition of FXa (32% at ED(50)) reduced thrombus formation in the venous model; to affect arterial thrombosis in the rat and rabbit, stronger inhibition of FXa (74%, 92% at ED(50)) was required. Calculated plasma levels in rabbits at the ED(50) were 14-fold lower than in the rat AV shunt model, correlating with the 14-fold lower IC(50) of FXa inhibition in rabbit compared with rat plasma; this may suggest a correlation between FXa inhibition and antithrombotic activity. Bleeding times in rats and rabbits were not significantly affected at antithrombotic doses (3 mg kg(-1) p.o., AV shunt). Based on these results, BAY 59-7939 was selected for clinical development.     

Biomarker optimization to track the antithrombotic and hemostatic effects of clopidogrel in rats

We determined the dose response of the ADP antagonist clopidogrel (0.3-50 mg/kg p.o.) in rat models of thrombosis and provoked bleeding and correlated these activities to ex vivo platelet activation. Carotid artery thrombosis was induced by FeCl(2). Bleeding time was measured by mesenteric vessel puncture and renal cortex or cuticle incision. Platelet biomarkers included standard ADP-induced aggregation, P2Y(12) receptor occupancy, and phosphorylation of vasodilator-stimulated phosphoprotein. Clopidogrel decreased thrombus weight up to 78%, caused maximal prolongation of cuticle and mesenteric bleeding, but had little effect on renal bleeds. Due to the steep mesenteric dose response, further comparisons concentrated on cuticle bleeding. The half-maximal inhibitory dose (ED(50)) for thrombus reduction was 2.4 +/- 0.4 mg/kg, with 10 mg/kg providing optimal blood flow preservation and thrombus reduction. The ED(50) for bleeding was 10.5 +/- 3.4 mg/kg. Increased bleeding was intermediate (3-fold) at 10 mg/kg and maximal (6-fold) at 30 mg/kg. All biomarkers were affected, but with differing sensitivity. ED(50)s for peak platelet aggregation to 10 microM ADP (11.9 +/- 0.4 mg/kg) and the vasodilator-stimulated phosphoprotein index (16.4 +/- 1.3 mg/kg) approximated the higher ED(50) for bleeding. ED(50)s for ligand binding (3.0 +/- 0.3 mg/kg) and late aggregation (5.1 +/- 0.4 mg/kg) better matched the lower ED(50) for antithrombotic activity. Aspirin exerted lesser effects on bleeding (42-70% increase in all models) and thrombosis (24% inhibition). In summary, antithrombotic doses of clopidogrel have limited effects on bleeding and standard measures of platelet aggregation. Other biomarkers may be more sensitive for tracking antithrombotic efficacy.     

Effect of protamine sulfate on antithrombin III activity

When protamine sulfate was added to heparinized plasma in vitro for neutralization of heparin, the activities on both thrombin and Xa known as heparin cofactor in antithrombin action were completely abolished. However, progressive activities on thrombin and Xa both recovered within 30 minutes after protamine sulfate addition. When equivalent heparin was again added, heparin cofactor activity was immediately restored. Based on the fact that protamine sulfate did not show any direct action on the antithrombin III molecule, the presence of AT III with progressive activity was considered to play an important role in the rebound phenomenon of heparin after heparin neutralization with protamine sulfate.     

Effects of factor XI deficiency on ferric chloride-induced vena cava thrombosis in mice

BACKGROUND: Increased plasma levels of coagulation factor (F) XI are a risk factor for venous thrombosis. OBJECTIVE: To further explore the relationship between FXI and venous thrombosis, we evaluated FXI-deficient and wild-type mice in a ferric chloride (FeCl(3))-induced vena cava thrombosis model. METHODS AND RESULTS: Thrombosis was induced by 3-min topical application of filter papers containing increasing concentrations of FeCl(3) and the thrombus was measured at 30 min. In contrast to wild-type mice, FXI-deficient mice failed to form a thrombus with 5% FeCl(3,) and were partially protected against 7.5% and 10% FeCl(3,) respectively. The protective effect was substantially stronger than a high dose of heparin (1,000 units kg(-1), i.v.), clopidogrel (30 mg kg(-1), p.o.) or argatroban (30 mg kg(-1), i.p.). These antithrombotic agents resulted in off-scale bleeding in a tail bleeding time assay, whereas the bleeding time of FXI-deficient mice was unchanged compared to wild-type mice. In addition to its known effect on the coagulation cascade, enhanced clot lysis was demonstrated in FXI-deficient mouse and human plasma compared to those supplemented with FXIa. CONCLUSION: Given the strong antithrombotic efficacy (possibly contributed by strong anticoagulant activity associated with increased fibrinolytic activity) and mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable therapeutic strategy to treat or prevent venous thrombosis.     

Fibrin moderates the catalytic action of heparin but not that of dermatan sulfate on thrombin inhibition in human plasma

Three recent studies have reported that fibrin in solution significantly inhibits the ability of heparin to catalyze the inhibition of thrombin by antithrombin III. In addition, heparin inhibits the release of fibrinopeptide A by clot-bound thrombin less effectively than it inhibits the release of fibrinopeptide A by thrombin in solution. We have also reported that dermatan sulfate, which catalyzes thrombin inhibition by heparin cofactor II, inhibits thrombus growth in rabbits more effectively than heparin. Because the results of these studies suggest that fibrin inhibits the reactivity of thrombin with antithrombin III-heparin but not with heparin cofactor II-dermatan sulfate, we compared the relative catalytic effects of heparin and dermatan sulfate on thrombin inhibition in plasma, both in the presence and absence of fibrin. We quantitated the rates of thrombin inhibition by antithrombin III and heparin cofactor II by specific enzyme-linked immunosorbent assays. When it was generated, fibrin was kept in solution by adding 2 mmol/L Gly-Pro-Arg-Pro to plasma. Fibrinogen-fibrin reduced the reactivity of thrombin with plasma antithrombin III, both in the presence of and in the absence of heparin. In contrast, the catalytic action of dermatan sulfate on thrombin inhibition by plasma heparin cofactor II was unimpaired by fibrinogen-fibrin. Based on the ability of dermatan sulfate to inhibit thrombus growth in rabbits, failure of fibrinogen-fibrin to moderate the catalytic action of dermatan sulfate may account for its greater antithrombotic effectiveness relative to that of heparin.     

S35972, a direct‐acting thrombin inhibitor with high oral bioavailability and antithrombotic efficacy

Summary. Objectives: Dabigatran etexilate is the first oral thrombin inhibitor to demonstrate superior efficacy to warfarin for stroke prevention in patients with atrial fibrillation. This study describes the in vitro, ex vivo anticoagulant and in vivo antithrombotic effects of an oral thrombin inhibitor, S35972, in comparison with dabigatran etexilate. Methods: Enzyme assays with thrombin and related serine proteases were performed. Clotting times, including activated partial thromboplastin time (APTT) and thrombin time (TT), were measured in vitro in different species and ex vivo in dogs and rats to determine pharmacologic bioavailabilities. The formation of occlusive venous and arterial thrombi in the rat vena cava and aorta was induced with stasis plus thromboplastin or ferrous chloride, respectively. Results: S35972 inhibited human thrombin with an IC₅₀ of 3.7 nm , and did not inhibit other serine proteases. The anticoagulant activities of S35972 in vitro were comparable in dog and human plasmas, and the sensitivity of the clotting times to S35972 was TT > APTT > prothrombin time. In the fasted dog, oral administration of 3 mg kg^?1 S35972 increased TT rapidly and for at least 8 h, and its pharmacologic bioavailability was 75.4% ± 0.1%. In the rat venous thrombosis model, 3 mg kg^?1 oral S35972 or dabigatran etexilate significantly decreased the thrombus weight. In the rat aortic thrombosis model, oral S35972 at 10 mg kg^?1 significantly decreased thrombus weight, by approximately 50%, whereas, at this dose, no effect was obtained with dabigatran etexilate. Conclusions: S35972 is a non‐prodrug thrombin inhibitor with high selectivity, oral bioavailability, and antithrombotic efficacy.     

Favorable therapeutic index of the direct factor Xa inhibitors, apixaban and rivaroxaban, compared with the thrombin inhibitor dabigatran in rabbits

Summary.  Background: Apixaban is an oral, direct factor Xa (FXa) inhibitor in late-stage clinical development. This study assessed effects of the direct FXa inhibitors, apixaban and rivaroxaban, vs. the direct thrombin inhibitor, dabigatran, on venous thrombosis (VT), bleeding time (BT) and clotting times in rabbits. Methods: We induced the formation of non-occlusive thrombus in VT models by placing threads in the vena cava, and induced bleeding by the incision of cuticles in anesthetized rabbits. Apixaban, rivaroxaban and dabigatran were infused IV to achieve a stable plasma level. Clotting times, including the activated partial thromboplastin time (aPTT), prothrombin time (PT), modified PT (mPT) and thrombin time (TT), were measured. Results: Apixaban, rivaroxaban and dabigatran exhibited dose-related efficacy in preventing VT with EC₅₀ of 65, 33 and 194 n m , respectively. At doses for 80% reduction of control thrombus, apixaban, rivaroxaban and dabigatran prolonged BT by 1.13 ± 0.02-, 1.9 ± 0.1-* and 4.4 ± 0.4-fold*, respectively (* P  <   0.05, vs. apixaban). In the treatment model, these inhibitors equally prevented growth of a preformed thrombus. Antithrombotic doses of apixaban and rivaroxaban prolonged aPTT and PT by <3-fold with no effect on TT. Dabigatran was ≥50-fold more potent in prolonging TT than aPTT and PT. Of the clotting assays studied, apixaban, rivaroxaban and dabigatran responded the best to mPT. Conclusion: Comparable antithrombotic efficacy was observed between apixaban, rivaroxaban and dabigatran in the prevention and treatment of VT in rabbits. Apixaban and rivaroxaban exhibited lower BT compared with dabigatran at equivalent antithrombotic doses. The clinical significance of these findings remains to be determined.     

Identification in vitro of an endothelial cell surface cofactor for antithrombin III. Parallel studies with isolated perfused rat hearts and microcarrier cultures of bovine endothelium.

Two in vitro systems were used to identify an antithrombin III cofactor activity on vascular endothelium. Langendorff rat heart preparations or columns packed with endothelium cultured on microcarrier beads were perfused with mixtures of purified thrombin and antithrombin III. With each preparation, accelerated inhibition of thrombin by antithrombin III occurred during passage over endothelium. Platelet factor 4, protamine sulfate and diisopropylphosphoryl thrombin, all antagonists of the antithrombin III cofactor activity of heparin, significantly reduced the capacity of the preparation to inhibit thrombin. It is concluded that a substance with the functional properties of a stationary phase cofactor for antithrombin III is present on the microvascular endothelium and there catalyzes the inactivation of circulating free thrombin.     

Repeated doses of oral and subcutaneous heparins have similar antithrombotic effects in a rat carotid arterial model of thrombosis

Although heparins are usually injected intravenously or subcutaneously, antithrombotic activity is observed in rat models following single oral heparin doses. Since repetitive dosing is usually needed for thromboprophylaxis, study objectives were to determine whether repetitive oral heparin prevented arterial thrombosis and to compare effectiveness to subcutaneous administration. Wistar rats were given subcutaneous or oral unfractionated heparin ([UFH] 1 mg/kg per 48 h), low-molecular-weight heparin ([LMWH] tinzaparin, 0.1 mg/kg per 12 h), or saline for 30 days. On the last day, thrombosis was initiated by placing 30% FeCl(3)-soaked filter paper on the distal carotid. Subsequent flow measurements, for a 60-minute period, included recorded time of initial thrombus formation (time till thrombus begins [TTB]), and time until carotid occlusion (time till occlusion [TTO]). The formed thrombus was dried and weighed. The activated partial thromboplastin time (aPTT), anti-factor Xa, and antithrombin activity were determined from the plasma. Both oral and subcutaneous heparins significantly increased TTB and TTO. Time of initial thrombus formations were 12.6 ± 1.1, 21.2 ± 2.2, 25.3 ± 3.9, 21.7 ± 3.1, and 21.3 ± 1.7 minutes and TTOs were 29.3 ± 3.6, 54.8 ± 4.0, 60.0 ± 0.3, 56.7 ± 3.3, and 58.3 ± 1.7 minutes (mean ± SEM) for control, subcutaneous UFH, oral UFH, subcutaneous LMWH, and oral LMWH, respectively. Thrombus weight was 2.52 ± 0.29 g in control and was reduced to 43%, 23%, 33%, and 28% of control weight for subcutaneous UFH, oral UFH, subcutaneous LMWH, and oral LMWH, respectively. Thrombus weight was significantly less for oral compared to subcutaneous UFH. The aPTT for oral UFH, and anti-factor Xa activity in the LMWH-treated groups were significantly greater than control (two-tailed t tests). These findings confirm that orally administered heparins are absorbed. Repeated treatment with oral heparin showed similar antithrombotic activity compared to subcutaneous heparin. Oral heparin use for arterial thromboprophylaxis should be further investigated.     

Antithrombotic and anticoagulant effects of the direct thrombin inhibitor dabigatran, and its oral prodrug, dabigatran etexilate, in a rabbit model of venous thrombosis

BACKGROUND: Oral anticoagulant therapies targeted at thrombin are being developed to overcome limitations associated with current standard therapies. OBJECTIVES: This study was undertaken to assess and compare the antithrombotic and anticoagulant effects of the novel, selective and reversible, direct thrombin inhibitor (DTI), dabigatran, and its oral prodrug dabigatran etexilate, to that of unfractionated heparin (UFH), hirudin and melagatran using a rabbit model of venous thrombosis. METHODS: A rabbit model of venous thrombosis consisting of endothelial damage with blood flow reduction was used with minor modifications. RESULTS: All compounds demonstrated a dose-dependent reduction in thrombus formation following i.v. administration with complete or almost complete inhibition at the highest doses. Dabigatran (in the dose range 0.03-0.5 mg kg(-1)) had a 50% effective dose of 0.066 mg kg(-1). By comparison, UFH (5-50 U kg(-1)), hirudin (0.01-0.05 mg kg(-1)) and melagatran (0.01-0.3 mg kg(-1)) had a 50% effective dose of 9.8 U kg(-1), 0.016 mg kg(-1) and 0.058 mg kg(-1), respectively. Similarly, oral dabigatran etexilate (1-20 mg kg(-1)) inhibited thrombus formation in a dose-dependent manner. Maximum inhibition was achieved within 1 h of administration, suggesting a rapid onset of action. For both routes of administration, inhibition of thrombus formation directly correlated with prolongation of the activated partial thromboplastin time. CONCLUSIONS: These findings demonstrate the potent anticoagulant and antithrombotic activity of dabigatran as a selective thrombin inhibitor in a rabbit model of venous thrombosis. Notably, dose-dependent and long-lasting antithrombotic efficacy was observed after application of its oral form dabigatran etexilate, which is currently undergoing phase III clinical development.     

Pharmacological interruption of acute thrombus formation with minimal hemorrhagic complications by a small molecule tissue factor/factor VIIa inhibitor: comparison to factor Xa and thrombin inhibition in a nonhuman primate thrombosis model

This study was designed to evaluate the antithrombotic efficacy and bleeding propensity of a selective, small-molecule inhibitor of tissue factor/factor VIIa (TF/VIIa) in comparison to small-molecule, selective inhibitors of factor Xa and thrombin in a nonhuman primate model of thrombosis. Acute, spontaneous thrombus formation was induced by electrolytic injury to the intimal surface of a femoral blood vessel, which results in thrombus propagation at the injured site. The TF/FVIIa inhibitor 3-amino-5-[1-[2-([4-[amino(imino)methyl]benzyl]amino)-2-oxoethyl]-3-chloro-5-(isopropylamino)-6-oxo-1,6-dihydropyrazin-2-yl]benzoic acid dihydrochloride (PHA-927F) was fully effective in prevention of thrombosis-induced vessel occlusion at a dose of 400 microg/kg/min, i.v., in the arterial vasculature (femoral artery). Neither the effective dose nor multiples up to 4.4-fold the effective arterial plasma concentration elicited any significant effect on bleeding time or blood loss from either the bleeding time site or the surgical (femoral isolation) site. Small-molecule inhibitors of factor Xa or thrombin were effective arterial antithrombotic agents; however, in contrast to the TF/FVIIa inhibitor, they both elicited substantial increases in bleeding propensity at the effective dose and at multiples of the effective plasma concentration. These data indicate that TF/VIIa inhibition effectively prevented arterial thrombosis with less impact on bleeding parameters than equivalent doses of factor Xa and thrombin inhibitors.     

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents

Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.     

Effect of tiplaxtinin (PAI-039), an orally bioavailable PAI-1 antagonist, in a rat model of thrombosis

Summary. Objective: To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI‐039), an orally bioavailable antagonist of PAI‐1, in rat models of thrombosis. Methods and results: Carotid artery and vena cava vascular injury was produced by application of FeCl₃ and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI‐039 was administered orally 90 min before injury (1–30 mg kg^?1). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI‐039 administration. PAI‐039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg^?1, respectively. Time to occlusive thrombosis was increased from 18.2 ± 4.6 min in controls to 32.5 ± 8.7 (P = ns), 46.1 ± 7.0 (P < 0.05), and 41.6 ± 11.3 min (P < 0.05) in the respective PAI‐039 treatment groups. In the vena cava protocol, PAI‐039 pretreatment significantly reduced thrombus weight at PAI‐039 doses of 3, 10 and 30 mg kg^?1. When PAI‐039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI‐039 doses of 3, 10 and 30 mg kg^?1. PAI‐039 (10, 30 and 100 mg kg^?1) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI‐039 had no effect on circulating, low‐levels of PAI‐1 activity. In contrast, circulating PAI‐1 activity increased 5‐fold following the induction of vascular injury, which was completely neutralized by PAI‐039. Conclusions: PAI‐039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.     

Antithrombotic therapy after left atrial appendage closure

It is important to prevent on-device thrombus formation without increasing the risk for bleeding complications after successful interventional left atrial appendage closure. Therefore, choosing the optimal antithrombotic therapy poses a challenging task. While major clinical studies investigated patients eligible for oral anticoagulation using vitamin K antagonists, the vast majority of implants in ‘real life’ are performed in patients with contraindications to oral anticoagulation after serious bleeding events. In this patient population, strategies using dual antiplatelet therapy were found to be a sound alternative; however, the optimal duration of antithrombotic therapy remains unclear. Future studies will have to investigate the role of direct anticoagulants for post-implant thrombus formation and address the question of whether left atrial appendage closure obviates the need for long-term aspirin therapy.