Sensitive Measurement of Clinically Relevant Factor VIII Levels in Thrombin Generation Assays Requires Presence of Factor XIa

Abstract

Background: Hemophilia A (HA) is characterized by decreased or absent FVIII. Current FVIII assays are based on clotting time and only provide information about the initiation of coagulation. In contrast, thrombin generation assays (TGA) can be used to measure the full coagulation spectrum of initiation, propagation and termination that provide information on the course of thrombin generation and inhibition. However, commercially available TG kits lack sensitivity for measurements of hemophilia plasma within lower FVIII ranges, which is essential for explaining differences in bleeding phenotypes in hemophiliacs at clinical low levels of FVIII. Aims: Optimization of thrombin generation for measurements of low FVIII levels in severe HA patients. Methods: TGA measurements were performed in severe HA pooled plasma (n=10). Investigation of several pre-analytical and analytical variables were performed in a stepwise process and adjusted based on sensitivity towards intrinsic coagulation activation. Results: TGA initiated by tissue factor (TF) alone at varying concentrations was unable to significantly differentiate between FVIII levels below 20%. In contrast, TGA activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. In addition, a representative TGA-curve at trough levels could only be produced using the dual TF/FXIa TGA. Conclusion: We propose a critical optimization for the setup of the thrombin generation assay for measurements in severe HA plasma. The dual TF/FXIa TGA shows increased sensitivity, especially in lower FVIII ranges, which allows for better individual characterization at baseline, predictions of interventions and follow-up.

Comparative Thrombin Generation in Animal Plasma: Sensitivity to Human Factor XIa and Tissue Factor

“…The assay is usually triggered via extrinsic pathway to mimic physiological coagulation activation by tissue factor (TF) that is exposed at the site of the injured endothelium [ 3 ]. Activated coagulation factor XI (FXIa), which is a product of the contact activation, can be used together with or instead of TF in order to stimulate the intrinsic pathway of the coagulation cascade, e.g., to measure the treatment of severe deficiencies of coagulation factors VIII and IX (hemophilia A and B) [ 4 , 5 , 6 ]. Compared to traditional functional hemostasis assays, such as Prothrombin Time (PT) and activated Partial Thromboplastic Time (aPTT), TG may show better sensitivity to both disease conditions and the effects of novel treatments.…”