Reversal efficacy of novel single domain antibodies against rivaroxaban, edoxaban or apixaban in vitro and ex vivo: studying platelet-dependent clot formation under flow

Introduction Direct oral anticoagulants (DOACs) are standard care for stroke prevention in non-valvular atrial fibrillation and management of venous thromboembolism but can contribute to major bleeding in up to 5% of patients, necessitating reversal. Rapid reversal is also critical in trauma or emergency surgery. Andexanet alfa, now the only specific reversal option for FXa-dependent DOACs, has limitations including doubled thrombotic risk (compared to standard care, ANDEXA I trial) and high cost. This study aimed to evaluate the efficacy and specificity of novel single domain antibodies (sdAbs) targetting rivaroxaban, edoxaban or apixaban to reverse anticoagulation and restore clot formation under static and flow conditions in blood from healthy volunteers and DOAC-treated patients. Method The effects of rivaroxaban (0-800 nM), edoxaban (0-300 nM) and apixaban (0-800)±a dose range of specific sdAbs, or andexanet alfa, were assessed in (i) control and patient plasma samples by diluted prothrombin time (dPT, 10 pM tissue factor, TF) and (ii) control whole blood by coagulation-dependent platelet ATP release (1 pM TF). To assess the whole blood hemostatic potential we employed a novel microfluidic perfusion system, flowing (1000 s -1 ) over collagen/TF, at 37°C. The study included 16 patients (nursing home residents) treated with DOACs: rivaroxaban (4), edoxaban (4), apixaban (8). For patient samples peak DOAC levels (3 h after intake) were quantified by an anti-Xa chromogenic assay. Results In vitro DOAC addition(dose-dependently) prolonged dPT, inhibited coagulation-dependent platelet ATP release, and reduced fibrin formation under flow by ~50% (control: 67 vs. rivaroxaban: 37, edoxaban: 36, apixaban: 35% area covered by fibrin; all P≤0.009). DOAC-specific sdAbs reversed DOAC effects on dPT in a dose-dependent manner and restored coagulation-dependent platelet ATP release. Under flow, none of the DOACs affected platelet aggregation, but the DOAC-induced inhibitory effects on fibrin formation were reversed by the sdAbs for on average 85% for all DOACs (84-87%, all P>0.9999 compared to control); time to fibrin formation was normalized (92.5%-128.5%, P=0.05-0.001). Andexanet alfa is as effective as sdAbs in vitro. Patient peak DOAC levels were for rivaroxaban 340-1148 nM, edoxaban 181-565 nM, and apixaban 218-770 nM. Importantly, in these patients the sdAbs DOAC-dependently corrected the dPT (16/16, 43.8%-66.54%, P