Innovative thrombolytic strategy using a heterodimer diabody against TAFI and PAI-1 in mouse models of thrombosis and stroke

Abstract

• Early thrombolytic treatment with a bispecific inhibitor against TAFI and PAI-1 is effective without exogenous tPA.• Even at the highest dose tested, the bispecific inhibitor against TAFI and PAI-1 does not prolong bleeding time.Circulating thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) are causal factors for thrombolytic failure. Therefore, we evaluated an antibody-engineered bispecific inhibitor against TAFI and PAI-1 (heterodimer diabody, Db-TCK26D6x33H1F7) in several mouse models of thrombosis and stroke. Prophylactic administration of the diabody (0.8 mg/kg) in a thromboplastin-induced model of thromboembolism led to decreased lung fibrin deposition. In a model of cerebral ischemia and reperfusion, diabody administration (0.8 mg/kg, 1 hour postocclusion) led to a mitigated cerebral injury with a 2.3-fold reduced lesion and improved functional outcomes. In a mouse model of thrombin-induced middle cerebral artery occlusion, the efficacy of the diabody was compared to the standard thrombolytic treatment with recombinant tissuetype plasminogen activator (tPA). Early administration of diabody (0.8 mg/kg) caused a twofold decrease in brain lesion size, whereas that of tPA (10 mg/kg) had a much smaller effect. Delayed administration of diabody or tPA had no effect on lesion size, whereas the combined administration of diabody with tPA caused a 1.7-fold decrease in lesion size. In contrast to tPA, the diabody did not increase accumulative bleeding. In conclusion, administration of a bispecific inhibitor against TAFI and PAI-1 results in a prominent profibrinolytic effect in mice without increased bleeding. (Blood. 2015;125(8):1325-1332 IntroductionPlasminogen activators are the only thrombolytic agents approved to rapidly revascularize a thrombosed vessel. Reperfusion of the ischemiaaffected organ leads to an improved outcome in patients when applied within the first hours after ischemic onset.1 Despite this evidence-based beneficial effect, current thrombolytic agents remain widely underutilized due to life-threatening side effects such as cerebral hemorrhages and possible neurotoxicity.2,3 For acute ischemic stroke, in particular, the only licensed treatment option consists of a high systemic dose of recombinant tissue-type plasminogen activator (tPA), which is actually given to ,10% of the patients. Therefore, there is an unmet clinical need to explore novel therapeutic avenues to enhance fibrinolysis without plasminogen activator-associated adverse effects. Endogenous intravascular fibrinolysis is driven on the release of tPA from the endothelium, which in turn enzymatically activates plasminogen into the fibrin-degrading enzyme, plasmin.4 This activation step is attenuated by circulating thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1). Activated TAFI (TAFIa; encoded by the CPB2 gene) eliminates C-terminal Lys exposed on partially degraded fibrin, which ultimately leads to a diminished efficiency and localizati...

Changes in Activated Thrombin-Activatable Fibrinolysis Inhibitor Levels Following Thrombolytic Therapy in Ischemic Stroke Patients Correlate with Clinical Outcome

“…In a rabbit model of arterial thrombosis, it was shown that several parameters associated with the efficiency of t-PA induced thrombolysis were enhanced and the time to reperfusion was reduced in the presence of the TAFIa inhibitor [56] . Likewise, it had been demonstrated that the combined administration of a heterodimer diabody against TAFI and PAI-1 in mice further decreases the brain lesion volume following rt-PA administration [57] .…”

Hypofibrinolysis in diabetes: a therapeutic target for the reduction of cardiovascular risk

“…Recent work in mouse models of thrombotic stroke have shown that simultaneous inhibition of PAI-1 and TAFI with a bispecific antibody results in a significant enhancement of fibrinolysis in mice, without increased bleeding [157]. To dissect out the contribution of PAI-1 inhibition and TAFI inhibition during ischaemic stroke, Denorme et al used monoclonal antibodies to identify the effect of inhibiting each of these antifibrinolytic factors.…”

Inhibition of Thrombin-Activatable Fibrinolysis Inhibitor and Plasminogen Activator Inhibitor-1 Reduces Ischemic Brain Damage in Mice

“…These results are strongly suggestive for a co-operative effect of PAI-1 and TAFI in ischemic stroke and further support the use of a bispecific diabody targeted against both TAFI and PAI-1. 9 Massberg et al 3 showed that as early as 5 minutes after initiation of reperfusion, fibrin(ogen) is accumulating in the postischemic vasculature, actively recruiting platelets and inflammatory cells. Our findings further support the idea that early fibrin(ogen) generation hampers efficient reperfusion of the affected brain tissue.…”

“…7,8 We recently showed a profibrinolytic effect of a novel bispecific inhibitor against TAFI and PAI-1. 9 However, the precise contributions of inhibiting PAI-1 and TAFI during ischemic stroke remain largely unknown. In this study, we therefore used well-characterized monoclonal antibodies (MAs) that inhibit either TAFI or PAI-1 to dissect their role in cerebral ischemia/reperfusion injury.…”