From principle to practice: bridging the gap in patient profiling

Abstract

The standard clinical coagulation assays, activated partial thromboplastin time (aPTT) and prothrombin time (PT) cannot predict thrombotic or bleeding risk. Since thrombin generation is central to haemorrhage control and when unregulated, is the likely cause of thrombosis, thrombin generation assays (TGA) have gained acceptance as "global assays" of haemostasis. These assays generate an enormous amount of data including four key thrombin parameters (lag time, maximum rate, peak and total thrombin) that may change to varying degrees over time in longitudinal studies. Currently, each thrombin parameter is averaged and presented individually in a table, bar graph or box plot; no method exists to visualize comprehensive thrombin generation data over time. To address this need, we have created a method that visualizes all four thrombin parameters simultaneously and can be animated to evaluate how thrombin generation changes over time. This method uses all thrombin parameters to intrinsically rank individuals based on their haemostatic status. The thrombin generation parameters can be derived empirically using TGA or simulated using computational models (CM). To establish the utility and diverse applicability of our method we demonstrate how warfarin therapy (CM), factor VIII prophylaxis for haemophilia A (CM), and pregnancy (TGA) affects thrombin generation over time. The method is especially suited to evaluate an individual's thrombotic and bleeding risk during "normal" processes (e.g pregnancy or aging) or during therapeutic challenges to the haemostatic system. Ultimately, our method is designed to visualize individualized patient profiles which are becoming evermore important as personalized medicine strategies become routine clinical practice.

Figure 1. The kinetics of warfarin anticoagulation in patients with atrial fibrillation.

Thrombin generating capacity was simulated by inputting each subjects' factor composition into our mathematical model. Each point (circle) in the figure is representative of a single individual's thrombin generating capacity before and during warfarin therapy. A video showing the dynamic thrombin generating capacity over time can be viewed from Movie S1. All subjects, including the 3 highlighted (S1: subject 1, S2: subject 2 and S3: subject 3), show a time dependent reduction in thrombin generating capacity (marginally increased lag time, decreased maximal rate, decreased peak and total thrombin) in response to warfarin therapy. Note that the peak thrombin scale ranges from 0–500 nM.

Figure 2. The effect of the protein C pathway on the kinetics of Warfarin anticoagulation in patients with atrial fibrillation.

Thrombin generating capacity was simulated by inputting each subjects' factor composition into our mathematical model containing the protein C pathway. Each point (circle) in the figure is representative of a single individual's thrombin generating capacity before and during warfarin therapy. A video showing the dynamic thrombin generating capacity over time can be viewed from the Movie S2. All subjects show a time dependent reduction in thrombin generating capacity (increased lag time, increased maximal rate, decreased peak and total thrombin) in response to warfarin therapy. Most subjects, including the subjects highlighted (S1: subject 1, S2: subject 2 and S3: subject 3), have an increased maximal rate, peak and total thrombin and a marginally increased lag time 3 days after starting warfarin therapy. After day 3, every subjects' thrombin generating capacity decreases in a similar fashion to that shown using our “Base model” (figure 1). Note that the peak thrombin scale ranges from 0–200 nM.

Figure 3. Dynamic reduction of thrombin generation parameters over time in a severe haemophilia A population.

Thrombin generating capacity was simulated by inputting each subject's factor composition into our mathematical model. Each point (circle) in the figure is representative of a single individual's thrombin generating capacity. A video showing the effects of decaying fVIII on the dynamic thrombin generating capacity can be viewed from the Movie S3. Since each subject has clinically severe haemophilia A (fVIII <1%), the fVIII concentration was set at 100% at time zero (baseline). The thrombin generating capacity was followed over 7 half-lives of fVIII (t_1/2 = 12.2 hours) which represents the approximate time between prophylactic fVIII doses. All individuals, including subject H1, showed a decrease in thrombin generating capacity (decreased maximal rate and peak thrombin and marginally decreased total thrombin and marginally increased lag time) as fVIII decayed. Note that the peak thrombin scale ranges from 0–200 nM.

Figure 4. The effect of factor VIII product half-life on the dynamics of thrombin generation over time 32 hours post administration of fVIII.

Thrombin generating capacity was simulated by inputting subject H1's factor levels into our mathematical model. A video demonstrating the relative extension between doses when the half-life of fVIII is increased is provided in the Movie S4. The thrombin generation capacity is also shown at 32 hours for 4 hypothetical fVIII products with half-lives of 6, 12, 18 and 24 hours. The baseline (100% fVIII) thrombin generating capacity at time zero is shown as a reference. By 32 hours, the 6 hour product has decayed to ∼1% which coincides with the approximate timing between prophylactic doses of fVIII. Note that the peak thrombin scale ranges from 0–200 nM.

Figure 5. Dynamic thrombin generation during the course of pregnancy.

Thrombin generating capacity was determined empirically using a thrombin generation assay. Each point (circle) in the figure is representative of a single individual's thrombin generating capacity. A video showing changes in the dynamic thrombin generating capacity during pregnancy can be viewed from the Movie S5. All subjects, including the 3 highlighted (P1: pregnant subject 1, P2: pregnant subject 2 and P3: pregnant subject 3), have increased thrombin generating capacity (decreased lag time, increased maximal rate, increased peak and total thrombin) in early pregnancy. The thrombin generation capacity increases further in late pregnancy and post-pregnancy returns to near baseline levels for most individuals. Note that the peak thrombin scale ranges from 0–750 nM.