The thrombograms were measured in a 96-well plate fluorometer (Ascent reader; Thermolabsystems OY, Helsinki Finland) equipped with a 390/460 filter set (excitation/emission) and a dispenser. Flat-bottomed 96-well plates (Thermo Scientific, Waltham, MA) were used. Each experiment needs two sets of readings, one from a well in which thrombin generation takes place (TG well) and a second one from a well to which the calibrator has been added (CL well). Experiments were performed in duplicate (i.e., a set of two TG wells was compared to a set of two CL wells). A dedicated software program (Thrombinoscope; Synapse BV, Maastricht, The Netherlands) enables the identification of the (sets of) wells and determines the duration of the experiment and the sampling rate (usually 4/min). To each well, 80 μL of plasma (PPP) was added. The TG wells received 20 μL of buffer, containing the trigger (including 5 μL of the ocular fluid ±5 μL of antibodies against TF) but no Ca2+, whereas the CL wells received 20 μL of the α2M-T solution. The trigger was the subretinal fluid or vitreous fluid obtained during ocular surgery, together with 24 μM PS/PC/PE vesicles in HEPES-buffered saline, in the absence of TF. The plate was placed in the fluorometer and allowed to warm to 37° C (minimum time, 5 minutes). The dispenser of the fluorometer was flushed with a warm 100-mM CaCl2 solution, emptied, and flushed with warm FluCa. At the start of the experiment, the instrument dispenses 20 μL of FluCa to all the wells to be measured, registers this as time 0, shakes them for 10 seconds, and starts reading. During the measurement, the program compares the readings from the TG and the CL wells, calculates thrombin concentration and displays the thrombin concentration in time.
A typical course of a thrombogram is illustrated in
Figure 1. The period in which no observable thrombin is formed reflects the lag time. After this phase, the concentration of thrombin increases rapidly until a peak is reached and then decreases again. The area under the curve is called the endogenous thrombin potential (ETP) and is obtained by adding an excess of thrombin substrate, so that the total amount of substrate converted is proportional to the area under the thrombogram. Other parameters of the thrombogram are the peak and the time it takes to reach the peak. Evident thrombin generation is defined as a peak of >50 nM.
In any experimental setting, the intrinsic pathway of the coagulation cascade is triggered through contact activation. To rule out the contribution of contact activation to the amount of thrombin generated, severely FXII-deficient plasma was used in addition to normal plasma in all the experiments. As a result, the use of severely FXII-deficient plasma prevented any thrombin from being generated in the absence of a trigger of the coagulation system.