Thrombinoscope

Calibrated automated thrombin generation was performed as described by Hemker.^{34 (link)} Briefly, a 96-well microplate format and the calibrated automated thrombin generation assay (Thrombinoscope, Diagnostica Stago) were used to measure thrombin generation in plasma based on thrombin’s hydrolysis of a fluorogenic substrate (Z-GLY-GLY-Arg-AMC). All samples were run in duplicate and in parallel with calibrator wells.
The following parameters were measured: lag time (LT; min) = time needed for initial thrombin generation, (i.e., 10 nM); peak thrombin generation (nM), endogenous thrombin potential (ETP; nM* min) = amount of thrombin that can be generated after in vitro activation of coagulation; and time to thrombin peak (TTP, min).

Two sets of experiments were carried out to determine the biocompatibility of the polyP inhibitors and their polyP neutralization activities, respectively. In the first set of experiments, only polyP inhibitors were used (for determining biocompatibility) without adding polyP, and in the second set of experiments, both polyP inhibitors and different molecular weight polyPs (SC polyP and LC polyP) were used to evaluate neutralization activity of the inhibitors.
Thrombin generation assays were carried out at 37 °C by measuring the fluorescence intensity upon cleavage of a fluorogenic substrate, Z-Gly-Gly-Arg-AMC by the generated thrombin, using the supplied “FluCa” and “PPP-low” reagents on the Thrombinoscope (Diagnostica Stago) system. PNP (20 donors) from George Kind Bio-Medical was mixed 1:1 with HBS (20 mM HEPES with 100 mM NaCl at pH 7.4). Phosphatidylcholine/phosphatidylserine (80:20) (PCPS) liposomes were added to obtain a final concentration of 25 μM. Reaction wells contained 80 μL of the 50% plasma and 20 μL of the supplied substrate/calcium reagent. Serial dilutions of inhibitors were prepared in 50% plasma:HBS immediately before each experiment, and two technical replicates were carried out each time.
The fluorescence intensity was recorded at 37 °C every 30 s over a period of 1.5 h and converted to thrombin concentration using the supplied calibrator and software.