5.5.3. Thrombin Inhibition by rDromaserpin in the Presence of Heparin

For assays in the absence or presence of heparin, the concentration of thrombin was held at 2U, and the concentration of rDromaserpin was maintained at 0.2 μM. The stock of unfractionated heparin concentration of 10 μg/mL used in this assay represents a concentration of ~400 μM (taking an average molecular mass of 25,000 g/mol). Heparin was added to the reaction in a range of 0.8–7.2 μM. Reactions were incubated for 15 min at 37 ◦C in thrombin assay buffer. The chromogenic substrate S-2238 was used at a final concentration of 0.2 mM, and residual activity was followed at OD405 nm.

#### 5.5.4. Evaluation of Binding to Heparin-Sepharose Resin

The ability of rDromaserpin, thrombin and the covalent complex to bind to heparin was evaluated using Heparin-Sepharose 6 Fast Flow affinity resin (Cytiva, Marlborough, MA, USA). First, 50 μL of the resin was equilibrated with 200 μL of buffer A (20 mM Tris pH 7.4, 150 mM NaCl, 0.1% BSA). In four different tubes, 32 μg rDromaserpin, 133 U thrombin, rDromaserpin-thrombin complex, or 50 μg antithrombin III were added to the resins in a final volume of 250 μL. The samples were incubated for 30 min with gentle shaking. The resins were washed three times with 200 μL of buffer A to remove unbound proteins. For elution, 1 M NaCl was added and incubated for 10 min. The resins were pelleted by centrifugation at 1000× *g* rpm for 5 min, and the supernatants were analyzed by 10% SDS-PAGE.

### 5.5.5. Effect of rDromaserpin on Platelet Aggregation

The effect of rDromaserpin on the function of thrombin in platelets was also investigated on washed platelets. To prepare washed platelets, approximately 20 mL of freshly human blood collected from healthy donors, in a tube containing 3.8% sodium citrate, was centrifuged at 900× *g* rpm for 20 min at room temperature. Platelet rich plasma (PRP) was recuperated and 2% EDTA was added at a ratio of 1:100, followed by 2000 rpm centrifugation for 15 min at room temperature. The pellet containing platelets was resuspended in 10 mL of wash buffer (140 mM NaCl, 10 mM NaHCO3, 2.5 mM KCl, 0.49 mM Na2HPO4, 1 mM MgCl2, 22 mM Sodium Citrate, 52.23 mM BSA-dissolved in 100 mL of water, pH adjusted to 6.5 with HCl), and centrifuged at 2000× *g* rpm for 15 min at room temperature. The wash step was repeated twice under the same conditions. The supernatant was discarded and the pellet resuspended in 2 mL of Tyrode buffer (1 mM CaCl2, 134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 10 mM HEPES-pH adjusted to 7.4 with HCl. Add glucose 1% at the time of use). The aggregometer (CHRONO-LOG® Model 700, Havertown, PA, USA), was calibrated with Tyrode buffer containing glucose. To determine the effect of rDromaserpin on platelet aggregation, 10 μM rDromaserpin or equal volume of 1 × PBS buffer pH 7.4, 1, 10% glycerol were pre-incubated for 10 min at 37 ◦C, with agonist 1 μg /mL NIH-U thrombin in a 50 μL reaction. Platelet aggregation was induced following the addition of pre-warmed washed platelets in a final volume of 500 μL. Platelet aggregation was monitored for 6 min and the percentage of platelet aggregation inhibition was deduced using The AGGRO/LINK®8 program. Data are presented as mean ± S.E.M. of triplicate platelet aggregation assays.
