Concizumab improves clot formation in hemophilia A under flow

Abstract

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

Objectives: The objective of this in vitro study was to evaluate how concizumab affects clot formation in hemophilia A under flow.

Methods: Blood was collected from normal controls or people with hemophilia A. An anti-FVIII antibody was added to normal controls to simulate hemophilia A with inhibitory antibodies to FVIII. Whole blood and recombinant activated FVII (rFVIIa, 25 nM) or concizumab (200, 1000, and 4000 ng/mL) were perfused at 100 s^-1 over a surface micropatterned with tissue factor (TF) and collagen-related peptide. Platelet and fibrin(ogen) accumulation were measured by confocal microscopy. Static thrombin generation in plasma was measured in response to rFVIIa and concizumab.

Results: Concizumab (1000 and 4000 ng/mL) and rFVIIa both rescued (93%-101%) total platelet accumulation, but only partially rescued (53%-63%) fibrin(ogen) incorporation to normal control levels in simulated hemophilia A. Results using congenital hemophilia A blood confirmed effects of rFVIIa and concizumab. While these 2 agents had similar effect on clot formation under flow, concizumab enhanced thrombin generation in plasma under static conditions to a greater extent than rFVIIa.

Conclusion: TFPI inhibition by concizumab enhanced activation and aggregation of platelets and fibrin clot formation in hemophilia A to levels comparable with that of rFVIIa.

Keywords: blood coagulation; hemophilia A; hemorheology; lipoprotein-associated coagulation inhibitor; microfluidics.

Figure 1.

Schematic for microfluidic flow assay. Whole blood (red) and recalcification buffer (cyan) are injected separately via a syringe pump at volumetric flow rate ratio of 9:1 into microfluidic mixing chip that enhances mixing by reducing length scale for diffusion (~20 μm) of divalent cations into the blood. Recalcified whole blood is then perfused over the procoagulant substrate (yellow) in the microfluidic flow assay device forming a clot (green) that is captured by optical microscopy.

Figure 2.

Micropatterning tissue factor and collagen peptides sequentially. (A) Step 1 - Lipidated tissue factor (TF) was first perfused through the patterning device and allowed to bind to the glutaraldehyde- activated surface. This step was repeated four times. Step 2 - A mixture of collagen-related peptides (CRP) was then perfused through the same patterning device and incubated overnight. Step 3 - An assay device was then placed with channels perpendicular to the patterned area to create a precise procoagulant area within the channel of 250 μm x 500 μm. Patterning the TF liposomes first, followed by the collagen related peptides yielded a substrate with both components: (B) Number of TF liposomes in the procoagulant strip as detected by PE-labeled Annexin V staining and (C) collagen peptide surface coverage based on FAM-labeled CRP-XL fluorescence. (D) % surface area coverage of TF liposomes detected.

Figure 3.

(A) Representative images of DIOC6 labeled platelets and AlexaFluor 647 labeled fibrin(ogen) accumulation after 20 min perfusion over collagen peptide-TF surface at 100 s⁻¹. Scale bar = 50 μm. Kinetics of platelet (B) and fibrin(ogen) (C) accumulation. Fluorescence intensity is normalized by the maximum fluorescence intensity of the vehicle control. SHA = simulated hemophilia A (anti-FVIII antibody); czm = concizumab. Data points represent averages and shaded regions represent standard error of the mean (SEM) of n = 9 donors.

Figure 4.

Metrics of platelet (A-C) and fibrin(ogen) (D-F) kinetics on collagen peptide-TF surfaces at a wall shear rate of 100 s⁻¹. Each data point is an individual donor with mean and standard deviation of n=6–9 donors. P-values denoted as * p<0.05; ** p<0.01; *** p<0.001.

Figure 5.

Static thrombin generation (1 pM TF). (A-C) Kinetics and (D-F) metrics of static thrombin generation in plasma for simulated hemophilia A (SimA, anti-FVIII), mild hemophilia A (FVIII=6%), and FVIII deficient (FVIII<1%) in response to 25 nM rFVIIa and 4000 ng/mL concizumab.