*2.4. Functional Characterization of the rDromaserpin*

2.4.1. Global Blood Coagulation Assays

The effect of rDromaserpin was investigated on blood clotting using assays that independently target the extrinsic, intrinsic and common coagulation pathways. Prothrombin Time (PT) assay, designed to monitor the extrinsic coagulation [37], detected no significant effect upon the addition of 1 μM of rDromaserpin, and only a very slight increase of clotting time with 5 μM of rDromaserpin (2 s) in the tested conditions (data not shown). Thus, the protein, under these conditions, has minimal or no effect on the extrinsic coagulation pathway. As well, the Thrombin Time (TT) assay conducted on PPP (Platelet-poor plasma) did not show any significant differences under these conditions (data not shown). However, when tested with prothrombin deficient plasma (FII-DP), 5 μM of rDromaserpin significantly increased TT by ~10.97 s, when 2 mU of FIIa was added without pre-incubation (Figure 8a). Similarly, clotting time was increased by ~9.83 s, when rDromaserpin was pre-incubated with FII-DP for 1 h (Figure 8a). The plasma became incoagulable when rDromaserpin was pre-complexed with thrombin, preventing thrombin from converting the fibrinogen to fibrin (Figure 8a).

On the other hand, by using the activated partial thromboplastin time (APTT) assay, a statistically significant delay was shown in blood clot formation when plasma was treated during 20 min with 0.5, 1 or 5 μM rDromaserpin, prolonging the intrinsic coagulation time by 7.4 s, 13.06 s and 42.56 s, respectively (Figure 8b). Our results therefore suggest that rDromaserpin is probably able to inhibit serine protease(s) involved in the intrinsic pathway of blood coagulation.

**Figure 8.** Effect of rDromaserpin on the intrinsic pathway of blood coagulation. (**a**) In vitro TT assay performed on FII-DP in the presence of 5 μM rDromaserpin and 2 mU FIIa. The assay was tested without pre-incubation or by pre-incubating samples in brackets for 1 h at room temperature. (**b**) In vitro APTT evaluation in isolated human plasma incubated for 20 min with 0.2, 0.5, 1, or 5 μM rDromaserpin. (-) refers to plasma used as control in the experiments containing the same volume of buffer. The results correspond to the mean ± SEM values acquired in three independent experiments; the asterisk indicates a significant difference between groups; \* (*p* < 0.05) \*\* (*p* < 0.005) \*\*\*\* (*p* < 0.0005).
