Objective:
Assaying thrombin generation (TG) in real time using fluorogenic substrates has been a popular approach for developing a true global hemostasis assay. Benefits over other assays include assessment of global hemostasis potential, not just the level of coagulation factor deficiencies. Recent experiments in our laboratory have suggested that adding factor XIa to the assay improves the sensitivity and robustness of this assay approach. Its effects on clot formation and lysis are also being assessed.
Methods:
To expand the utility of the TG test, we optimized the reaction mixture and protocol. We add FXIa to the substrate/calcium mixture as previous experiments in our laboratory have shown FXIa needs to be added during or after plasma recalcification in order to maintain activity. We are also observing, concurrently with TG by fluorescence, absorbance as a direct measurement of fibrin generation (FG). Congenitally FV, FVII, FVIII, and FIX-deficient plasma were supplemented with their respective purified factors to give known level of factor deficiency. Tissue plasminogen activator (tPA) and thrombomodulin (TM) are also added to our assay to induce and allow observation of clot lysis and thrombin-dependent lysis inhibition. We run equivalent samples on Thrombinoscope’s Calibrated Automated Thrombinography (CAT, Stago USA) platform to assess our variations from the current standard protocol.
Summary of results:
Adding FXIa improves the robustness and sensitive range of the TGT as applied to clotting factor deficiencies. For FVIII deficiency, adding FXIa results in thrombin peak heights begin rising at lower factor concentrationsand increases in thrombin peak heights. For FIX deficiency, the addition of FXIa gives a dose-dependent thrombin maximum response that would otherwise be absent or weak. However, FV deficiency showed dose- dependent TG trends with or without FXIa, while the addition of FXIa eliminates dose- dependent TG trends in FVII deficiencies. These trends are seen both using our in-house TGT and CAT. The addition of tPA and TM do not appear to produce additional factor- dependent TG or FG responses under conditions tested, while they diminished the factor- dependent time to peak thrombin trend for FIX-deficient plasma.
Conclusions:
The addition of FXIa to the TGT gives us a more sensitive assessment of global hemostasis in intrinsic pathway deficiencies and reveals patterns not seen using current standard protocols.
Disclaimer. The authors are employees of the US Food and Drug Administration (FDA). This presentation is an informal communication and represents authors’ own best judgment. These comments do not bind or obligate FDA.