PB0190 - Platelet-dependent factor VIII activity is more susceptible to inhibitor antibodies than phospholipid vesicle-dependent factor VIII activity

Background: Inhibitor antibodies remain a major complication of hemophilia A. However, clinical assays do not adequately predict bleeding risk. We recently demonstrated that factor VIII activity (fVIII:C) supported by activated platelets is inhibited by anti-C2 mAbs in a manner that has no significant correlation with the degree of inhibition in a standard aPTT-based assay. These findings suggest that a platelet-based assay may better assess bleeding risk.

Aims: To measure residual fVIII:C in the presence of patient inhibitors utilizing the activated platelet time (aPT) adapted for the clinical laboratory.

Methods: Cryopreserved platelets were thawed at 37˚C and purified on a density gradient. The aPT was initiated by combining i) platelets reconstituted in fVIII-deficient plasma, ii) a fVIII sample in 10% plasma (with or without inhibitory antibodies and using either fVIII-deficient plasma or inhibitor patient plasma), and iii) an activation mix (factor XIa, PAR1/4 agonist peptides, Ca++). Time to fibrin polymerization was measured using viscosity-based clot detection (STA Compact Max, Diagnostica Stago).

Results: A 3rd-order relationship between time to fibrin formation and the log of fVIII concentration was reliable over a 3.5-log range (Fig 1A). The assay was robust over platelet concentrations of 300,000-10,000,000 platelets/mL and 2-60 pM fXIa. A panel of anti-fVIII-C2 antibodies caused increased inhibition of the aPT vs. the aPTT (Fig 1B) consistent with prior results (Fig 1C). Out of 6 patient inhibitor plasmas, 5 showed lower factor VIII residual activity when measured using the aPT assay (Fig 2). For three samples, the aPT measured fVIII:C ≤ 1% while the aPTT-based assay indicated fVIII:C ≥ 5% for all samples.

Conclusion(s): The aPT, refined for clinical use, measures inhibitor-suppressed fVIII:C low enough to rationalize a high bleeding risk for some patients. Further studies will be needed to indicate whether the aPT more accurately predicts bleeding risk than current clinical assays.