Multicenter evaluation of the hemostatic activity of emicizumab in patients with severe hemophilia A.

BACKGROUND: Emicizumab has been approved for the prophylaxis of patients with hemophilia A with or without inhibitors. However, spontaneous and trauma-induced breakthrough bleeds have been reported in patients on emicizumab prophylaxis, and no laboratory assay has been validated to evaluate the hemostatic activity of emicizumab. OBJECTIVES: The thrombin generation assay (TGA) could be a surrogate marker of the hemostatic efficacy of emicizumab. The correlation between TGA and the methods used to measure emicizumab blood concentration was evaluated in this study. METHODS: TGA was modified by the use of a trigger reagent combining a very low concentration of tissue factor and activated factor (F)XI. Emicizumab quantification was performed by 3 methods: the modified 1-step FVIII assay and 2 methods based on liquid chromatography and mass spectrometry (LC-MS). RESULTS: Using tissue factor/activated FXI-triggered TGA and platelet-poor plasma, a relationship was observed between the area under the thrombin generation curve (endogenous thrombin potential [ETP]) and the clinical response of patients to emicizumab. The ultrastructure of fibrin clots was consistent with ETP results and showed that emicizumab had a hemostatic activity equivalent to 20 to 30 IU/dL of FVIII. Finally, pharmacokinetic/pharmacodynamic analyses showed no correlation between ETP and LC-MS nor with modified 1-stage FVIII assay, but a statistically significant correlation between the LC-MS methods and the time-to-peak results of the TGA. CONCLUSION: Using a modified TGA, this study showed that patients who experienced breakthrough bleeds while on emicizumab had a lower thrombin-generating capacity compared with others with good clinical response to emicizumab.

The Ca2+- and phospholipid-binding protein Annexin A2 is able to increase and decrease plasma membrane order.

Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, HII). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.

Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths.

Biological membranes contain a large variety of lipids species compartmentalized in different domains heterogeneous in size, composition and dynamics. Cholesterol induces membrane ordered domains thanks to its affinity for saturated lipids. Membrane domains had been studied with fluorescent probes either linked to phospholipids and proteins or as individual fluorophore. However, no efficient formulation of a cholesterol probe has been available so far. Herein, we described a cholesterol-pyrene probe behaviour in heterogeneous membranes. We characterised the pyrene fluorescence spectra in liquid-ordered (Lo) and liquid-disordered (Ld) membranes. Using statistical multivariate analysis, we found out the most appropriate wavelengths for membrane domains studies. 373 nm and 379 nm were the most discriminant wavelengths to follow the liquid-ordered and the liquid-disordered environments. Cholesterol clustering behaviour was quantified by the modulation of the cholesterol-pyrene excimers peak (474 nm). In liquid-ordered membranes at low temperature, cholesterol-pyrene was found as multimers and as monomers. At high temperature, the liquid-ordered status of the membrane decreases and cholesterol-pyrene tends to cluster. In liquid-disordered membranes, cholesterol-pyrene was present mostly as monomers and the small quantity of excimers increased with temperature. Cholesterol-pyrene was used to test the ceramide effect on membranes, and presented a behaviour in agreement with the cholesterol behaviour reported in the literature. Overall, the presented data show that cholesterol-pyrene is an efficient sensor to study liquid ordered and liquid disordered organisation in membranes.