OC 59.5 - Evaluation of Concizumab in Hemophilia Using Microfluidic Flow Assays

Background: Inhibition of tissue factor pathway inhibitor (TFPI) is an emerging therapeutic strategy for treatment of hemophilia. Concizumab is a monoclonal antibody that binds the Kunitz 2 domain of TFPI and blocks its inhibition of factor Xa thereby extending the initiation phase of coagulation and compensating for lack of factor VIII (FVIII) in hemophilia.

Aims: The objective of this in vitro study was to evaluate how concizumab affects thrombus formation in a microfluidic flow assay in simulated hemophilia A.

Methods: Whole blood was collected into sodium citrate and corn trypsin inhibitor. An anti-FVIII antibody was used to simulate hemophilia A (2% FVIII activity). Recalcification buffer and activated recombinant factor VII (rFVIIa, 25 nM) or concizumab (4000 ng/mL) were continuously mixed into the blood upstream of the flow assay device where the blood was perfused at a wall shear rate of 100 s-1 over collagen peptides and tissue factor (TF) for 20 min. DiOC6 labeled platelets and AlexaFluorTM647-fibrin(ogen) accumulation were measured by spinning disc confocal microscopy. Platelet and fibrin(ogen) kinetics were quantified by (1) lag time (time to fluorescence intensity of 10% of the maximum intensity of the vehicle control), (2) velocity (slope of the linear part of the kinetic curve), and (3) maximum fluorescence.

Results: Induction of haemophilia A resulted in delayed platelet and fibrinogen accumulation. Concizumab and rFVIIa shortened lag time and increased velocity of platelet accumulation during the initial stages ( < 10 min) of thrombus formation (Figure 1A). After platelet aggregation (>10 min), fibrin(ogen) accumulation was increased by concizumab and rFVIIa.

Conclusion(s): These results suggest enhancing thrombin generation during the early stages of thrombus formation via TFPI inhibition rescues platelet accumulation. These larger platelet aggregates provide the biochemical and biophysical conditions to support a thrombin burst and subsequent fibrin accumulation at the later stages of thrombus formation.