RESUMO
BACKGROUND: Bypassing agents are used for breakthrough bleedings in patients with hemophilia A with inhibitor (PwHAwI) receiving emicizumab prophylaxis. Previous study demonstrated a weak binding affinity between emicizumab and factor (F)X (Kd; 1.85 µM), and that this value was much greater than the plasma FX concentration (~130 nM). We speculated that increased FX levels could enhance coagulation potential in emicizumab-treated PwHA. AIMS: To investigate the relationship between FX concentrations and emicizumab-driven coagulation. METHODS: Plasma FX (up to 1,040 nM) and emicizumab (50 µg/mL) were added to FVIII-deficient plasmas, and plasma-derived FX (520 nM) or recombinant (r)FVIIa (2.2 µg/mL) was added to plasmas from three emicizumab-treated PwHAwI. The adjusted-maximum coagulation velocity (Ad|min1|) by clot waveform analysis and peak thrombin (PeakTh) by thrombin generation assay in them were evaluated. Emicizumab (3.0 mg/kg), human (h)FIX (100 IU/kg), and various doses of hFX (100-500 IU/kg) were intravenously administered to HA mice. Clotting time/clot formation time (CT/CFT) were assessed using rotational thromboelastometry, and blood loss was estimated by a tail-clip assay. RESULTS: The addition of FX to FVIII-deficient plasma with emicizumab increased Ad|min1| and PeakTh. The coagulation parameters in emicizumab-treated PwHAwI spiked with additional FX remained within the normal range as well as the additional rFVIIa. In animal models, hFX injection shortened the CT and CT+CFT. The shorter CT and CT+CFT, and the lower blood loss were evident after 200 or 500 IU/kg hFX administration, and those indices were comparable to those in wild-type mice. CONCLUSION: Supplementation with FX may improve emicizumab-driven hemostasis in PwHA.
RESUMO
Quantitative determination of the biological activity of botulinum toxin type A usually depends on the LD(50) method after intraperitoneal injection into mice. This method requires a large number of mice to determine the toxic activity at a high level of precision and 3-4 days to obtain the results. Techniques to replace the LD(50) method have been attempted at various institutes. As a substitute for this method, by directly measuring the inhibition of neuromuscular transmission after the administration of a toxin, a method to quantitatively assess the toxin's activity by determining the compound muscle action potential (CMAP) was examined. Toxin solutions were injected into the rat gastrocnemius muscle, and that of the CMAP amplitude was determined over time. The CMAP amplitude decreased over 4 days after the injection of the toxin, and then slowly recovered. A dose-response relationship was noted for each dose, and a linear relation was observed between 0.01 and 30 U on the 1st day. From these results, we propose the CMAP as a substitute for the LD(50) method to examine the activity of toxin products as it is simple and reliable, reduces the number of experimental animals required, and lowers pain levels.
