FIXa-triggered thrombin generation correlates with FVIII levels less than 1% in reconstituted plasma mimicking samples obtained from patients with severe hemophilia A

INTRODUCTION: More than 10% of severe hemophilia A patients are paradoxically mild bleeders despite factor VIII (FVIII) levels being <1 IU/dl. Quantitation of FVIII levels <1 IU/dl is a challenge due to the detection limits of traditional one-stage activated partial thromboplastin time, two-stage chromogenic assay, and tissue factor-initiated thrombin generation assay (TF-TGA), impeding a precise characterization of the bleeding phenotype in severe hemophilia patients. OBJECTIVE: The study aimed to enhance the sensitivity of TGA by modifying the trigger reagent for FVIII measurement. METHODOLOGY: We optimized the fluorometric quantitation of thrombin generation triggered by factor IXa (FIXa) in artificial reconstituted plasma by blending varying proportions of normal pooled plasma (NPP) and FVIII-immunodepleted plasma containing a normal level of von Willebrand factor. RESULTS: FIXa-initiated thrombin generation depended on FVIII level, while TF could bypass FVIII deficiency to activate thrombin, making TF-TGA incapable of quantifying low levels of FVIII. When triggering with 0.6 nM FIXa, in the presence of 4 μM phospholipids and 40 μg/ml corn trypsin inhibitor, thrombin generation was highly dependent on FVIII levels even below 1 IU/dl. Among the five routinely used TGA parameters, peak thrombin, endogenous thrombin potential, and velocity index demonstrated the strongest linear correlation with FVIII levels down to 0.1 IU/dl. CONCLUSION: The study evaluated the performance of a modified TGA activated with FIXa. In addition to traditional TF-TGA, FIXa-TGA may provide additional information when assessing the bleeding tendency of patients with severe hemophilia A, facilitating personalized factor replacement therapy.