Neutrophil extracellular traps block endogenous and intravenous thrombolysis-induced fibrinolysis in large vessel occlusion acute ischemic stroke

BackgroundIntravenous thrombolysis (IVT) failure in acute ischemic stroke (AIS) due to large vessel occlusion (LVO) is frequent but its causes remain elusive. Several non-exclusive mechanisms have been proposed to explain IVT failure, including failed delivery of tPA and inhibition of its activity. We investigated whether biologically relevant intrathrombus concentrations of t-PA were achieved in failed IVT in patients with LVO AIS, and whether neutrophil extracellular traps (NETs) contributed to IVT failure. MethodsIn this cohort study, a total of 205 thrombi from AIS patients with LVO were analyzed. 83 of these thrombi were compared for tPA content and 53 for their susceptibility to ex vivo thrombolysis according to IVT status. An additional subset of 69 AIS thrombi was used to decipher if and how NETs interfere with intrathrombus fibrinolysis. ResultsAIS thrombi from IVT patients contained more tPA than those from no-IVT patients (0.209 vs 0.093 {micro}g/mg of thrombus, p<0.0001). Plasminogen and tPA in AIS thrombi were found in association with fibrin and NETs. The ability of NETs to bind tPA and plasminogen, titrating them away from fibrin, was confirmed in a microfluidic model of thrombosis. While ex vivo addition of plasminogen did not cause lysis of either no-IVT or IVT thrombi, combining plasminogen with DNase 1 helped translate the increased tPA content of IVT thrombi into increased thrombolysis. We further show that DNase 1 enables tPA- and plasmin-mediated thrombolysis by eliminating fibrinolysis inhibitors from AIS thrombi. ConclusionsThese results indicate that intrathrombus tPA concentrations reached in failed IVT bear a therapeutic potential that is however impaired by NETs, which favor intrathrombus retention of fibrinolysis inhibitors and compete with fibrin for tPA and plasminogen binding. Our results stress the interest of DNase 1 to enhance the efficacy of current IVT tPA regimens. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIIntravenous thrombolysis increases thrombus tPA content even when it fails to cause arterial recanalization in acute ischemic stroke C_LIO_LIThe fibrinolytic activity of intravenously-administered tPA is blocked by neutrophil extracellular traps in acute ischemic stroke thrombi C_LIO_LINeutrophil extracellular traps participate in thrombolysis resistance by retaining fibrinolysis inhibitors and titrating tPA and plasminogen away from fibrin in acute ischemic stroke thrombi C_LIO_LIDNase 1 can convert increased tPA content into increased fibrinolysis by eliminating NETs-associated fibrinolysis inhibitors in acute ischemic stroke thrombi C_LI 2) What are the clinical implications?O_LIDespite therapeutic failure, biologically significant intrathrombus tPA concentrations are achieved following intravenous thrombolysis at current tPA regimens C_LIO_LISequential administration of DNase 1 prior to intravenous thrombolysis could clear the way for tPA and potentiate its fibrinolytic activity for improved arterial recanalization efficacy C_LI