BACKGROUND: Anticoagulants are expected to promote fibrinolysis by counteracting the antifibrinolytic effects of thrombin, which include thrombin-activatable fibrinolysis inhibitor (TAFI) activation and clot structure enhancement. However, the efficiency of anticoagulants may vary remarkably, and the ability of direct thrombin inhibitors to facilitate clot lysis remains controversial. OBJECTIVE: To evaluate the profibrinolytic effect of dabigatran, a new, direct thrombin inhibitor, using different in vitro models. METHODS AND RESULTS: The resistance of tissue factor-induced plasma clots to fibrinolysis by exogenous tissue-type plasminogen activator (t-PA) (turbidimetric method) was reduced by dabigatran in a concentration-dependent manner, with > or = 50% shortening of lysis time at clinically relevant concentrations (1-2 microm). A similar effect was observed in the presence of low (0.1 and 1 nm) but not high (10 nm) concentrations of thrombomodulin. Acceleration of clot lysis by dabigatran was associated with a reduction in TAFI activation and thrombin generation, and was largely, although not completely, negated by an inhibitor of activated TAFI, potato tuber carboxypeptidase inhibitor. The assessment of the viscoelastic properties of clots showed that those generated in the presence of dabigatran were more permeable, were less rigid, and consisted of thicker fibers. The impact of these physical changes on fibrinolysis was investigated using a model under flow conditions, which demonstrated that dabigatran made the clots markedly more susceptible to flowing t-PA, by a mechanism that was largely TAFI-independent. CONCLUSIONS: Dabigatran, at clinically relevant concentrations, enhances the susceptibility of plasma clots to t-PA-induced lysis by reducing TAFI activation and by altering the clot structure. These mechanisms might contribute to the antithrombotic activity of the drug.

Hemorrhagic transformation (HT) associated with recombinant tissue plasminogen activator (rt-PA) complicates and limits its use in stroke. Here, we provide a focused review on the involvement of matrix metalloproteinase 9 (MMP-9) in rt-PA-associated HT in cerebral ischemia, and we review emerging evidence that the selective inhibitor of the sulfonylurea receptor 1 (Sur1), glibenclamide (U.S. adopted name, glyburide), may provide protection against rt-PA-associated HT in cerebral ischemia. Glyburide inhibits activation of MMP-9, ameliorates edema formation, swelling, and symptomatic hemorrhagic transformation, and improves preclinical outcomes in several clinically relevant models of stroke, both without and with rt-PA treatment. A retrospective clinical study comparing outcomes in diabetic patients with stroke treated with rt-PA showed that those who were previously on and were maintained on a sulfonylurea fared significantly better than those whose diabetes was managed without sulfonylureas. Inhibition of Sur1 with injectable glyburide holds promise for ameliorating rt-PA-associated HT in stroke.

Paediatric obesity, like adulthood obesity, is associated with an increase of fibrinolysis inhibitors. No study, however, has evaluated the impact of these changes on plasma fibrinolytic capacity. We investigated plasma fibrinolysis and the role therein of the fibrinolytic changes associated with obesity in 59 obese children (body mass index > 95th percentile) and 40 matched controls. Fibrinolysis was investigated by measuring 1) the plasma levels of relevant fibrinolytic factors; 2) the in vitro fibrinolytic capacity under different conditions, using a microplate plasma clot lysis assay; 3) the circulating levels of markers of clotting and fibrinolysis activation. Plasminogen activator inhibitor 1 (PAI-1), total thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen levels were higher in obese children as compared to controls (p<0.01). Plasma clots from obese children lysed significantly slower than control clots when exposed to exogenous plasminogen activator, indicating a greater resistance to fibrinolysis. By the use of a selective inhibitor of activated TAFI and by regression analyses we found that fibrinolysis resistance in obese samples was attributable to PAI-1 increase and to enhanced TAFI activation. The ratio between the circulating levels of D-dimer and thrombin-antithrombin complex, a marker of in vivo fibrinolysis, was significantly lower in obese children, suggesting a reduced fibrinolytic efficiency. These data indicate that paediatric obesity is associated with a hypofibrinolytic state which might contribute to the increased thrombotic risk associated with this condition.

The β-D-glucose-containing compound 3, bearing 2-chlorothiophene and 1-isopropylpiperidine moieties as binders of the S1 and S4 pockets, respectively, proved to be potent competitive inhibitor of factor Xa (fXa, Ki = 0.090 nM) and thrombin (fIIa, Ki = 100 nM). The potency of 3 increases, over the parent compound 1, against fIIa (110-fold) much more than against fXa (7-fold). Experimental deconstruction of 3 into smaller fragments revealed a binding cooperativity of the P3/P4 and C3-alkyl-linked -D-glucose fragments, stronger in fIIa (15.5 kJ•mol-1) than in fXa (2.8 kJ•mol-1). The crystal structure of human fIIa in complex with 3 revealed a binding mode including a strong H-bond network between the glucose O1’, O3’ and O5’ and two critical residues, namely R221a and K224, belonging to the Na+-binding site which may allosterically perturb the specificity sites. The potential of 3 as antithrombotic agent was supported by its ability to inhibit thrombin generation and to stimulate fibrinolysis at submicromolar concentration.

Background: The resistance of platelet-rich thrombi to fibrinolysis is generally attributed to clot retraction and platelet PAI-1 release. The role of TAFI in platelet-mediated resistance to lysis is unclear. Objective: We investigated the contribution of TAFI to the antifibrinolytic effect of platelets in whole blood by thromboelastography. Methods: Platelet-poor (PP-WB, < 40 x 103 µL-1) and platelet-rich (PR-WB, > 400 x 103 µL-1) blood samples were obtained from normal human blood (N-WB, 150-220 x 103 µL-1). Clot lysis time was measured by thromboelastography in recalcified blood supplemented with t-PA (100 ng mL-1) and tissue factor (1:1000 Recombiplastin). Results: t-PA-induced lysis time increased in parallel to platelet concentration (up to 3-fold). Neutralization of TAFI, but not of PAI-1, shortened the lysis time by ~ 50% in PR-WB and by < 10% in PP-WB. Accordingly, prothrombin F1+2 and TAFIa accumulation was greater in PR-WB than in PP-WB. A similar TAFI-dependent inhibition of fibrinolysis was observed when clot retraction was prevented by cytochalasin D or abciximab, or when platelet membranes were tested. Moreover, in blood with an intact contact system, platelet-mediated fibrinolysis resistance was attenuated by an anti-FXI but not by an anti F-XII antibody. Finally, platelets made the clots resistant to the profibrinolytic effect of heparin concentrations displaying a strong anticoagulant activity. Conclusions: Our data indicate that TAFI activation is one major mechanism whereby platelets make clots resistant to fibrinolysis and underscore the importance of TAFI inhibitors as new antithrombotic agents.

TAFI (thrombin activatable fibrinolysis inhibitor) is the precursor of a basic carboxypeptidase (TAFIa) with strong antifibrinolytic and anti-inflammatory activity. Compelling evidence indicates that thrombin, either alone or in complex with thrombomodulin, is the main physiological activator of TAFI. For this reason derangements of thrombin formation, whatever the cause, may influence the fibrinolytic process too. Experimental models of thrombosis suggest that TAFI may participate in thrombus development and persistence under certain circumstances. In several models of pharmacological thrombolysis, the administration of TAFI inhibitors along with the fibrinolytic agent leads to a marked improvement of thrombus lysis, underscoring the potential of TAFI inhibitors as adjuvants for thrombolytic therapy. The role of TAFI in inflammatory diseases is more complex as it may serve as a defense mechanism, exacerbate the disease, or have no influence, depending on the nature of the model and the role played by the mediators controlled by TAFIa. Finally, the numerous clinical studies in patients with thrombotic disease support the idea that increased levels of TAFI and/or the enhancement of TAFI activation may represent a new risk factor for venous and arterial thrombosis.