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T. Abache et al.
was moderately affected (63±11.97% that of pdFX), acti- vation by FVIIa/TF was impaired, being 31±1.14%, and activation by the “tenase complex” was even more affect- ed since actiten had only 20±1.23 % of the capacity of pdFX.
The ability of actiten to be activated by thrombin was assessed in the absence of phospholipids (Figure 4). Phospholipids were omitted in this assay to eliminate the risk of auto-activation or degradation due to the appear- ance of FXa.20,21 Incubation of pdFX with or without thrombin or actiten alone for 48 h at 37°C did not generate any FXa. This result confirms that our preparation of actiten did not contain traces of FXa. In contrast, the incu- bation of actiten with thrombin led to the appearance of a regular amount of FXa, indicating that the modification of the activation peptide rendered actiten sensitive to throm- bin. The kcat/Km was calculated and was found to be 1.25±0.1 x 103 M-1s-1, a value close to the modified FX (FXfpa) generated by Louvain-Quintard et al.16
To complete this evaluation, the molecule was incubat- ed for 6 h in the presence of phospholipids and FXa, at 10% of the actiten concentration. There was no supple- mental generation of FXa other than the 10% added, sug- gesting that actiten resisted FXa cleavage (data not shown).21
The presence of FXa and the stability of actiten were
then evaluated in re-calcified FX- or FVIII-deficient plasma samples (Figure 5A, B). As a control, pdFXa shortened the spontaneous clotting time of these plasma samples to 11±8.5 min and 6.7±2.9 min, respectively. The presence of pdFX in FX-deficient plasma or rFVIII in FVIII-deficient plasma allowed the plasma to clot in 60±35 min and 25±8.7 min, respectively. The time to clot in the presence of actiten was much longer in both plasma samples (96±47 min and >120 min, respectively). This result fur- ther confirms the absence of contaminating FXa and did not reveal any instability of actiten in different plasma samples.
In vitro evaluation of actiten potency
Thrombin generation assays were performed to evalu-
ate the ability of actiten to correct coagulation factor defi- ciencies. In all experiments, the deficient plasma was eval- uated as a negative control and a pool of lyophilized nor- mal plasma as a positive control. The thrombin generation parameters of normal plasma were chosen as a reference and compared with the parameters from deficient plasmas spiked with 20 μg/mL actiten (Table 1).
In FVIII-deficient plasma, a dose-response relation was observed for actiten from 10 to 60 μg/mL before satura- tion at 120 μg/mL (Figure 6A). At 20 μg/mL of actiten, the
Figure 3. Binding of plasma-derived factor X and actiten to phospholipids. Plasma-derived factor X (pdFX) (●) and actiten (■) were incubated at increasing concentrations on coated phospholipids and detected by a polyclonal anti-FX coupled to horse radish peroxidase. (n=4). UDO: optical densi- ty unit.
Figure 4. Activation of plasma-derived factor X and actiten by thrombin. Plasma-derived factor X (pdFX) (100 nM) with 10 nM thrombin (■) or with- out thrombin (●), and actiten (100 nM) with 10 nM thrombin (▼) or without thrombin (▲) were incu- bated without phospholipid (PL) for increasing peri- ods of time. At different time points a sample was taken and the presence of activated factor X (FXa) was measured. The results are representative of three independent experiments. Signs for pdFX without thrombin (●) and actiten without thrombin (▲) are masked by (■)."
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haematologica | 2020; 105(9)