S62798, a potent TAFIa inhibitor, accelerates endogenous fibrinolysis in a murine model of pulmonary thromboembolism.

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.

GTP-cyclohydrolase deficiency induced peripheral and deep microcirculation dysfunction with age.

The present study assessed the impact of impaired tetrahydrobiopterin (BH4) production on vasoreactivity from conduit and small arteries along the vascular tree as seen during aging. For this purpose, the mutant hyperphenylalaninemic mouse (hph-1) was used. This model is reported to be deficient in GTP cyclohydrolase I, a rate limiting enzyme in BH4 biosynthesis. BH4 is a key regulator of vascular homeostasis by regulating the nitric oxide synthase 3 (NOS3) activity. In GTP-CH deficient mice, the aortic BH4 levels were decreased, by -77% in 12 week-middle-aged mice (young) and by -83% in 35-45 week-middle-aged mice (middle-aged). In young hph-1, the mesenteric artery ability to respond to flow was slightly reduced by 9%. Aging induced huge modification in many vascular functions. In middle-aged hph-1, we observed a decrease in aortic cGMP levels, biomarker of NO availability (-46%), in flow-mediated vasodilation of mesenteric artery (-31%), in coronary hyperemia response measured in isolated heart following transient ischemia (-27%) and in cutaneous microcirculation dilation in response to acetylcholine assessed in vivo by laser-doppler technic (-69%). In parallel, the endothelium-dependent relaxation in response to acetylcholine in conduit blood vessel, measured on isolated aorta rings, was unchanged in hph-1 mice whatever the age. Our findings demonstrate that in middle-aged GTP-CH depleted mice, the reduction of BH4 was characterized by an alteration of microcirculation dilatory properties observed in various parts of the vascular tree. Large conduit blood vessels vasoreactivity, ie aorta, was unaltered even in middle-aged mice emphasizing the main BH4-deletion impact on the microcirculation.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling.

OBJECTIVE: Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine. DESIGN AND METHODS: A monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice. RESULTS: The lower limit of detection was 0.5ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p<0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice. CONCLUSION: We have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis.