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Enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Venous thrombosis and thromboembolism risks are associated with increased plasma levels of TAFI (Thrombin Activatable Fibrinolysis Inhibitor) as well as its active form TAFIa. A new TAFIa inhibitor, namely S62798 has been identified. Its ability to enhance fibrinolysis was investigated both in vitro and in vivo in a mouse model of pulmonary thromboembolism, as well as its effect on bleeding. S62798 is a highly selective human, mouse and rat TAFIa inhibitor (IC50 = 11; 270; 178 nmol/L, respectively). It accelerates lysis of a human clot in vitro, evaluated by thromboelastometry (EC50 = 27 nmol/L). In a rat tail bleeding model, no effect of S62798 treatment was observed up to 20 mg/kg. Enhancement of endogenous fibrinolysis by S62798 was investigated in a mouse model of Tissue Factor-induced pulmonary thromboembolism. Intravenous administration of S62798 decreased pulmonary fibrin clots with a minimal effective dose of 0.03 mg/kg. Finally, effect of S62798 in combination with heparin was evaluated. When treatment of heparin was done in a curative setting, no effect was observed whereas a significantly decreased pulmonary fibrin deposition was observed in response to S62798 alone or in combination with heparin. This study demonstrates that S62798 is a potent TAFIa inhibitor with minimal risk of bleeding. In vivo, curative S62798 intravenous treatment, alone or associated with heparin, accelerated clot lysis by potentiating endogenous fibrinolysis and thus decreased pulmonary fibrin clots. S62798 is expected to be a therapeutic option for pulmonary embolism patients on top of anticoagulants.

Selective and potent inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) have the potential to increase endogenous and therapeutic fibrinolysis and to behave like profibrinolytic agents without the risk of major hemorrhage, since they do not interfere either with platelet activation or with coagulation during blood hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients for the treatment, prevention, and secondary prevention of stroke, venous thrombosis, and pulmonary embolisms. In this paper, we describe the design, the structure-activity relationship (SAR), and the synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.

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.

SummaryTo investigate the role of active plasminogen activator inhibitor 1 (PAI-1) in the evolution of a microthrombus generated in the arteriolar microcirculation, the monoclonal antibody, 33H1F7, which transforms active PAI-1 to a tissue type plasminogen activator (t-PA) substrate, was evaluated in an arteriolar thrombosis model in the rat mesentery. Arterioles (200-300 μm) were stimulated electrically to create an endothelial lesion; ADP was then perfused for 2 min to induce the formation of a platelet-rich thrombus which lysed spontaneously in 140 ± 24 s. Two successive ADP superfusions produced comparable thrombi which lysed in comparable times. Different doses of 33H1F7 were infused to rats for 30 min and the dose which inactivates rapidly and totally active rat PAI-1 (300 μg/kg/min) was selected to be tested on the thrombosis model. Infusion of 33H1F7 beginning 10 min before the ADP application significantly reduced the lysis time in comparison to the control (123 ± 30 s versus 169 ± 33 s, P < 0.05, paired Student’s t-test) and the cumulative thrombus area during the lysis period was decreased by 56 ± 7%. These results demonstrate that inactivation of PAI-1 is able to accelerate lysis of a platelet-rich clot in a mesenteric arteriole of the rat. Thus active PAI-1 most likely participates to the resistance to thrombolysis in the arteriolar microcirculation and its inactivation may shorten ischemic periods after microvascular obstruction such as e.g. during cerebral stroke.

1 The present study was designed to investigate a possible role of thromboxane A2 (TXA2) on arteriolar vasomotion (spontaneous rhythmic variations of the vessel diameter). Therefore the microcirculatory effects of the thromboxane-receptor (TP-receptor) agonist, U 46619, as well as the effects of the TP-receptor antagonists S 17733 and Bay U3405 were evaluated in the hamster cheek pouch microcirculation. For comparison some effects of angiotensin II were also investigated. 2 For microcirculatory measurements, the cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. The TV monitor display was used to obtain arteriolar internal diameter measurements by means of an image shearing device. 3 Superfusion (0.1 nM to 1 μM) or bolus application (1 pmol to 10 nmol) of U 46619 concentration- or dose-dependently decreased the arteriolar diameter and induced vasomotion in arterioles with a mean initial diameter of 24±2 μm. Both the vasoconstriction and the vasomotion induced by U 46619 were inhibited by the TP-receptor antagonists S 17733 (100 mg kg−1, i.v.) and Bay U3405 (10 mg kg−1, i.v.). 4 Bolus applications of angiotensin II (0.1 pmol to 1 nmol) induced transient vasoconstriction followed by vasodilatation in the cheek pouch arterioles. The dilatation but not the constriction, was sensitive to treatment with the NO-synthase inhibitor Nω-nitro-L-arginine (L-NOARG; 100 μM). Angiotensin II did not induce vasomotion in control conditions or in the presence of L-NOARG. 5 Bolus application of phenylephrine (10 pmol) induced vasoconstriction but no vasomotion in previously quiescent hamster cheek pouch arterioles. 6 These results indicate that activation of TP-receptors causes vasomotion in the hamster cheek pouch arterioles. These spontaneous rhythmic variations in arteriolar diameter are not observed with equipotent doses of angiotensin II and phenylephrine. Thus, the vasoconstriction by itself cannot explain the occurrence of vasomotion observed with the TP-receptor agonist. British Journal of Pharmacology (1997) 122, 859–866; doi:10.1038/sj.bjp.0701464

The role of von Willebrand factor (VWF) in thrombosis involves its binding to a number of ligands. To investigate the relative importance of these particular interactions in the thrombosis process, we have introduced mutations into murine VWF (mVWF) cDNA inhibiting VWF binding to glycoprotein (Gp) Ib, GPIIbIIIa, or to fibrillar collagen. These VWF mutants were expressed in VWF-deficient mice (VWF−/−) by using an hydrodynamic injection approach, and the mice were studied in the ferric chloride–induced injury model. Expression of the collagen and the GPIIbIIIa VWF-binding mutants in VWF−/− mice resulted in delayed thrombus growth and significantly increased vessel occlusion times compared with mice expressing wild-type (WT) mVWF (30 ± 3 minutes and 38 ± 4 minutes for the collagen and GPIIbIIIa mutants, respectively, vs 19 ± 3 minutes for WT mVWF). Interestingly, these mutants were able to correct bleeding time as efficiently as WT mVWF. In contrast, VWF−/− mice expressing the GPIb binding mutant failed to restore thrombus formation and were bleeding for as long as they were observed, confirming the critical importance of the VWF-GPIb interaction. Our observations suggest that targeting the VWF-collagen or VWF-GPIIbIIIa interactions could be an interesting alternative for new antithrombotic strategies.

The synthesis and activity of novel benzothiophene derivatives are described. In the t-Pa-induced fibrin clot lysis assay, several compounds inhibit the formation of the tPa–PAI-1 complex with submicromolar IC 50 . This class of compounds potentially represents a new generation of antithrombotic-fibrinolytic agents.

Thromboxane (TXA2) causes platelet aggregation, vasoconstriction and cell proliferation through the activation of specific cell membrane TP-receptors1, 2. TXA2 is implicated in thrombotic disorders and TP-receptor antagonists have been described as an anti-thrombotic approach3. S 18204 (Fig. 1) is a recently developed orally active and long acting TP-receptor antagonist4. Since S 18204 is a racemic compound, the aim of this study was to select the active isomer (S 18885 or S 18886) of S 18204 on platelet and contractile responses induced by the TP-receptor agonist U 46619 under in vitro, in vivo and ex vivo conditions.