Hemostase Inflammation Thrombosis

Unfractionated heparin (UFH) is the most widely used anticoagulant during extracorporeal membrane oxygenation (ECMO). Optimal dosing of UFH for children undergoing ECMO is unknown, leading to suboptimal exposure. We aimed to develop a population pharmacokinetic (PK) model for UFH in children undergoing venoarterial (VA) ECMO, using anti-Xa activity, to improve the initial dosing regimen. A retrospective observational study (March 2015 to May 2021) using nonlinear, mixed-effect modeling software (Monolix) and Monte Carlo simulations to optimize the dosing. Single-center study in a PICU in Paris, France. All children under 18 years old who needed VA-ECMO and received continuous UFH. None. We included 59 children and 1305 anti-Xa activity results, 13% of which were outside the limits of quantification. Median (range) initial UFH bolus and infusion rate were 50 international units/kg (20-100) and 20 international units/kg/hr (9-40), respectively. Among the 1305 anti-Xa activity results, 875 (67%) were in the therapeutic range (0.3-0.7 international units/mL), whereas 263 (20%) and 167 (13%) were below and above the PK target, respectively. A one-compartment model with first-order elimination and time-varying clearance best fitted the data. Body weight according to allometric scale on clearance and volume of distribution was the selected covariate. Initial clearance was low and increased until steady state was reached after 16 hours. Simulations showed that initial bolus of 40 international units/kg followed by continuous infusion of 25, 20, and 15 international units/kg/hr for patients under 10, between 10 and 15 and above 15 kg, respectively, improved exposure. In children undergoing VA-ECMO, body weight, and time-varying clearance explained the variability for UFH between subjects. This model can be used to optimize initial UFH dosing.

Extracorporeal membrane oxygenation (ECMO) is a critical rescue therapy for severe respiratory or cardiac failure. However, current blood pumps generate high shear stresses that can damage blood components, leading to hemolysis, loss of von Willebrand factor multimers, and increased risks of bleeding, thrombosis, and organ injury. We developed novel magnetostaltic pumps that use magnetic liquid interfaces instead of solid walls to transport blood, aiming to reduce mechanical stress on blood cells. Four magnetostaltic pump designs were tested in ex vivo ECMO circuits using human donor blood at clinically relevant flow rates and compared with standard centrifugal and peristaltic pumps. Across all flow rates, magnetostaltic pumps produced less hemolysis than conventional pumps. Under pediatric flow conditions (1 L/min for 48 h), the large-scale magnetostaltic pump (QR3) reduced hemolysis by approximately one-third compared with commercial centrifugal pumps and preserved high-molecular-weight von Willebrand factor multimers. Platelet function was unaffected. Small amounts of nanoparticle leakage from the magnetic fluid were detected but remained well below toxic thresholds. Magnetostaltic pumping offers a promising alternative to current ECMO pumps by reducing blood damage. These results support further testing in animal models to evaluate the potential for clinical translation.

The major anticoagulant Protein S (PROS1) also contributes to the phagocytosis of apoptotic cells by bridging exposed phosphatidylserine (PtdSer) to the MerTK receptor on macrophages (efferocytosis). Whether PROS1 is involved in the splenic clearance of PtdSer-positive senescent and altered erythrocytes such as erythrocyte ghosts (eryghosts) is unknown. Here, we investigate the contribution of PROS1 and MerTK to the phagocytosis of intact RBC and eryghosts in healthy subjects and patients with sickle cell disease (SCD). We show that PROS1 enhances the phagocytosis of ionomycin-treated PtdSer-positive erythrocytes and of eryghosts generated invitro. We confirm that eryghosts circulate in patients with SCD at higher levels than in healthy subjects and observe increased hemolysis and decreased levels of plasmatic PROS1 in patients with the highest concentration of eryghosts. The proportion of circulating eryghosts is correlated with the intensity of hyposplenism, and eryghosts are less frequently observed in sections of SCD compared to control spleens. We demonstrate that circulating eryghosts are procoagulant and adhere to endothelial cells. In SCD, PROS1 enhances their phagocytosis in a MerTK-dependent manner but has no such effect on intact erythrocytes. PROS1 is therefore involved in erythrophagocytosis, a physiological process insufficient in patients with SCD due to intense intravascular hemolysis and hyposplenism, leading to PROS1 consumption and the abnormal persistence of eryghosts in circulation. PROS1 deficiency may in turn initiate a pathogenic loop, enhancing unregulated activation of coagulation and defective clearance of procoagulant and adherent eryghosts. This deeper understanding of physiological and pathological erythrophagocytosis opens new therapeutic approaches targeting PROS1 in SCD.

Non-factor therapies are changing the treatment paradigm in hemophilia A, which was previously dominated by replacement-therapy using factor VIII (FVIII) concentrates. However, the FVIII equivalence of these new therapies has remained unclear, since in vitro assays generate variable responses. Here we used four different in vivo bleeding models to compare FVIII to emicizumab and to a sequence-identical analog of the tissue factor pathway inhibitor-targeting antibody marstacimab (SIA-marstacimab). The severity of these models was variable, each requiring different doses of FVIII to reduce blood loss to levels of wild-type mice. For example, a dose of 2.5 IU/kg FVIII was needed for full correction in the tail vein transection (TVT) model, whereas 25 IU/kg was needed in the saphenous vein puncture (SVP) model. Intermediate doses were required in the tail artery transection (TAT) model (5 IU/kg) and tail clip model (7.5 IU/kg). Importantly, FVIII treatment produced stable clots, without spontaneous rebleeds being observed. Both emicizumab and SIA-marstacimab (used at therapeutic doses of 55 μg/mL and 16 μg/mL, respectively) displayed a variable, model-dependent FVIII equivalence. For example, emicizumab proved equivalent to 5 IU/kg FVIII in the tail clip model, and to 10 IU/kg in the SVP model. Strikingly, both emicizumab and SIA-marstacimab treatment resulted in spontaneous rebleeds in the TVT, TAT and tail clip models, further distinguishing them from FVIII treatment. Our data suggest that there is unlikely to be a single FVIII equivalence for emicizumab, SIA-marstacimab, and similar molecules, because their activity is dependent on local conditions and severity of the injury.

Obesity is characterized by chronic low-grade inflammation and consequentially a hypercoagulable state, associating with an increased incidence of venous thromboembolism. Increased VWF (von Willebrand factor) plasma concentration and procoagulant function are independent risk factors for venous thromboembolism and are elevated in obese patients. Here, we explore the pathobiological role of VWF in obesity-associated venous thrombosis using murine models. Approach and Results: We first showed that diet-induced obese mice have increased VWF plasma levels and FVIII (factor VIII) activity compared with littermate controls. Elevated VWF levels appeared to be because of both increased synthesis and impaired clearance. Diet-induced obesity-associated venous thrombosis was assessed using the inferior vena cava-stenosis model of deep vein thrombosis. Diet-induced obese mice developed larger venous thrombi that were rich in VWF, erythrocytes, and leukocytes. Administering a polyclonal anti-VWF antibody or an anti-VWF A1 domain nanobody was protective against obesity-mediated thrombogenicity. Delayed administration (3 hours post-inferior vena cava stenosis) similarly reduced thrombus weight in diet-induced obese mice. This study demonstrates the critical role of VWF in the complex, thrombo-inflammatory state of obesity. It adds to the growing rationale for targeting VWF-specific interactions in thrombotic disease.

Replacement therapy with plasma-derived or recombinant FVIII (pdFVIII or rFVIII) concentrates is the standard of treatment in patients with hemophilia A. The reference method used for measuring factor VIII (FVIII:C) levels in patients treated by FVIII concentrates is the chromogenic substrate assay (CSA). However, the one-stage clotting assay (OSA) is predominantly used in current clinical practice, but this method depends on the activated partial thromboplastin time (APTT) reagent and the coagulation analyzer used, and wide variations in the measurements of FVIII recovery have been reported with some factor concentrates. The French study group on the biology of hemorrhagic diseases (a collaborative group of the GFHT and MHEMO network) presents a review of the literature and proposals for the monitoring of FVIII:C levels in treated hemophilia A patients. The use of CSA calibrated with a plasma reference tested against the current FVIII WHO (World Health Organization) International Standard is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. OSA are adequate for the monitoring of patients treated with pdFVIII or with most of rFVIII concentrates. However, preliminary comparison with CSA is mandatory before measuring FVIII:C by OSA in patients treated by Refacto AF®. For rFVIII-EHL, OSA are only acceptable for Elocta®. Great caution is therefore required when measuring FVIII:C levels by OSA in patients substituted by other EHL-rFVIII. Indeed, most of recent studies reported data obtained with spiked plasmas, which deserve to be confirmed on plasma samples collected in treated patients.