A small-molecule hemostatic agent for the reversal of direct oral anticoagulant-induced bleeding.

Background: The bleeding risk associated with direct oral anticoagulants (DOACs) remains a major concern, and rapid reversal of anticoagulant activity may be required. Although specific and nonspecific hemostatic biotherapies are available, there is a need for small-molecule DOAC reversal agents that are simple and cost-effective to produce, store, and administer. Objectives: To identify and characterize a small molecule with procoagulant activity as a DOAC reversal agent. Methods: We sought to identify a small procoagulant molecule by screening a chemical library with a plasma clotting assay. The selected molecule was assessed for its procoagulant properties and its ability to reverse the effects of the DOACs in a thrombin generation assay. Its activity as a DOAC reversal agent was also evaluated in a tail-clip bleeding assay in mice. Results: The hemostatic molecule (HeMo) dose-dependently promoted thrombin generation in plasma, with dose values effective in producing half-maximum response ranging between 3 and 5 µM, depending on the thrombin generation assay parameter considered. HeMo also restored impaired thrombin generation in DOAC-spiked plasma and reversed DOAC activity in the mouse bleeding model. HeMo significantly reduced apixaban-induced bleeding from 709 to 65 µL (vs 43 µL in controls; P < .01) and dabigatran-induced bleeding from 989 to 155 µL (vs 126 µL in controls; P < .01). Conclusion: HeMo is a small-molecule procoagulant that can counterbalance hemostatic disruption by a thrombin inhibitor (dabigatran) or factor Xa inhibitors (apixaban and rivaroxaban). The compound's effective clot formation and versatility make it a possible option for managing the inherent hemorrhagic risk during DOAC therapy.

Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia.

BACKGROUND: Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients. METHODS: In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice. RESULTS: In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation (P=0.00013, P<0.0001, and P<0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; P=0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; P<0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets. CONCLUSIONS: ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO.

Emicizumab does not interfere with the activated clotting time.

INTRODUCTION: The activated clotting time (ACT) is a useful marker of unfractionated heparin (UFH) activity during cardiopulmonary bypass (CPB) or cardiac catheterization. Emicizumab, recently approved for bleeding prevention in haemophilia A patients, acts like FVIII but does not need prior activation; it therefore shortens coagulation times in assays using intrinsic pathway activators and so is expected to shorten the ACT. AIM: To evaluated emicizumab's impact on heparin-induced ACT (Hemochron®) prolongation. METHODS: We measured the high-range (HR) ACT in citrated blood samples from healthy donors (HDs) (n = 9), CPB patients (n = 3) and emicizumab-treated patients (n = 5) after spiking with UFH and/or emicizumab. The low range (LR) ACT was also measured in spiked-samples from HDs and emicizumab-treated patients. RESULTS: In HDs, the median [interquartile range] baseline HR-ACTs were similar with and without emicizumab (129 [123-138] and 136 [115-141] s for 50 µg/ml, respectively); whatever the concentration of emicizumab (10 to 50 µg/ml), increasing the UFH concentration (1-5 UI/ml) prolonged the HR-ACT. In blood from patients undergoing CPB, the HR-ACT prolongation observed during this procedure was not masked by emicizumab at any concentration. Likewise, the addition of increasing concentrations of UFH to blood from emicizumab-treated patients induced a concentration-dependent prolongation of HR-ACT. Baseline LR-ACT were prolonged in emicizumab-treated patients but as for HR-ACT, emicizumab does not prevent heparin-induced prolongation of LR-ACT. CONCLUSION: Emicizumab does not interfere with UFH-induced ACT prolongation. The hemochron® ACT can be used to monitor UFH in patients receiving emicizumab during CPB or cardiac catheterization.

Endothelial Dysfunction as a Component of Severe Acute Respiratory Syndrome Coronavirus 2-Related Multisystem Inflammatory Syndrome in Children With Shock.

TRIAL REGISTRATION: NCT04420468. OBJECTIVES: Severe acute respiratory syndrome coronavirus 2-related multisystem inflammatory syndrome in children is frequently associated with shock; endothelial involvement may be one of the underlying mechanisms. We sought to describe endothelial dysfunction during multisystem inflammatory syndrome in children with shock and then assess the relationship between the degree of endothelial involvement and the severity of shock. DESIGN: Observational study. SETTING: A PICU in a tertiary hospital. PATIENTS: Patients aged under 18 (n = 28) with severe acute respiratory syndrome coronavirus 2-related multisystem inflammatory syndrome in children and shock, according to the Centers for Disease Control and Prevention criteria. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Correlations between endothelial marker levels and shock severity were assessed using Spearman coefficient. The median (interquartile range) age was 9 years (7.5-11.2 yr). Sixteen children presented with cardiogenic and distributive shock, 10 presented with cardiogenic shock only, and two presented with distributive shock only. The median left ventricular ejection fraction, troponin level, and lactate level were, respectively, 40% (35-45%), 261 ng/mL (131-390 ng/mL), and 3.2 mmol/L (2-4.2 mmol/L). Twenty-five children received inotropes and/or vasopressors; the median Vasoactive and Inotropic Score was 8 (5-28). Plasma levels of angiopoietin-2 (6,426 pg/mL [2,814-11,836 pg/mL]), sE-selectin (130,405 pg/mL [92,987-192,499 pg/mL]), von Willebrand factor antigen (344% [288-378%]), and the angiopoietin-2/angiopoietin-1 ratio (1.111 [0.472-1.524]) were elevated and significantly correlated with the Vasoactive and Inotropic Score (r = 0.45, p = 0.016; r = 0.53, p = 0.04; r = 0.46, p = 0.013; and r = 0.46, p = 0.012, respectively). CONCLUSIONS: Endothelial dysfunction is associated with severe acute respiratory syndrome coronavirus 2-related multisystem inflammatory syndrome in children with shock and may constitute one of the underlying mechanisms.