Microfluidic hemophilia models using blood from healthy donors

Abstract

Background: Microfluidic clotting assays permit drug action studies for hemophilia therapeutics under flow. However, limited availability of patient samples and Inter-donor variability limit the application of such assays, especially with many patients on prophylaxis.

Objective: To develop approaches to phenocopy hemophilia using modified healthy blood in microfluidic assays.

Methods: Corn trypsin inhibitor (4 µg/mL)-treated healthy blood was dosed with either anti-factor VIII (FVIII; hemophilia A model) or a recombinant factor IX (FIX) missense variant (FIX-V181T; hemophilia B model). Treated blood was perfused at 100 s^-1 wall shear rate over collagen/tissue factor (TF) or collagen/factor XIa (FXIa).

Results: Anti-FVIII partially blocked fibrin production on collagen/TF, but completely blocked fibrin production on collagen/FXIa, a phenotype reversed with 1 µmol/L bispecific antibody (emicizumab), which binds FIXa and factor X. As expected, emicizumab had no significant effect on healthy blood (no anti-FVIII present) perfused over collagen/FXIa. The efficacy of emicizumab in anti-FVIII-treated healthy blood phenocopied the action of emicizumab in the blood of a patient with hemophilia A perfused over collagen/FXIa. Interestingly, a patient-derived FVIII-neutralizing antibody reduced fibrin production when added to healthy blood perfused over collagen/FXIa. For low TF surfaces, reFIX-V181T (50 µg/mL) fully blocked platelet and fibrin deposition, a phenotype fully reversed with anti-TFPI.

Conclusion: Two new microfluidic hemophilia A and B models demonstrate the potency of anti-TF pathway inhibitor, emicizumab, and a patient-derived inhibitory antibody. Using collagen/FXIa-coated surfaces resulted in reliable and highly sensitive hemophilia models.

Keywords: drug evaluation; fibrin; hemophilia; hemostasis; microfluidics.

Figure 1

Hemophilia A assay. In a microfluidic device (A), whole blood was perfused over collagen/TF surfaces (B) and collagen/FXIa surfaces (C) to form thrombi. Murine anti‐FVIII (4A4) caused no change in platelet deposition (D) and a reduction in fibrin generation (E) on collagen/TF. The same concentrations of the FVIII inhibitory antibody slightly reduced platelet deposition (F) and abolished fibrin accumulation (G) on collagen/FXIa, similar to results obtained with a patient‐derived anti‐FVIII (H, I). Differences between control and treated groups were analyzed with 2‐way analysis of variance with Bonferroni posttest. *P < 0.05. Shaded areas represent standard deviation. CTI, corn trypsin inhibitor; FI, fluorescence intensity; FVIII, factor VIII; FXIa, factor XIa; TF, tissue factor; WB, whole blood

Figure 2

Testing emicizumab in hemophilia A assay. The effect of the bispecific antibody on platelet and fibrin deposition were investigated in healthy blood (A, B) and hemophilia A mimetic blood (C, D). Emicizumab dose‐dependently restored coagulation in the hemophilia model (D), but it only slightly increased fibrin generation in blood from healthy donor (B). Differences between control and treated groups were analyzed with 2‐way analysis of variance with Bonferroni post‐test. *P < 0.05. Shaded areas represent standard deviation. Ab, antibody; FI, fluorescence intensity; FVIII, factor VIII; FXIa, factor XIa; WB, whole blood

Figure 3

Emicizumab restored fibrin generation in hemophilia A assay. At the occlusion time (11 min), the bispecific antibody had no effect on platelet deposition (A), but it increased fibrin polymerization (B) in both healthy and hemophilia A blood. Differences between control and treated groups were analyzed with Student t‐test. *P < 0.05. Shaded areas represent standard deviation. Ab, antibody; FVIII, factor VIII; n.s., nonsignificant

Figure 4

Emicizumab restored fibrin generation in hemophilia A blood. The donor has mild hemophilia A. The bispecific antibody had no effect on platelet deposition (A), but it dose‐dependently rescued fibrin polymerization (B). *P < 0.05. n = 2‐4 clots. Shaded areas represent standard deviation. Ab, antibody; CTI, corn trypsin inhibitor; FI, fluorescence intensity; FXIa, factor XIa; n.s., nonsignificant; WB, whole blood

Figure 5

Hemophilia B assay. Whole blood was perfused over collagen/TF surfaces to form clots. Recombinant FIX variant inhibited platelet deposition (A) and fibrin generation (B). The same concentration of reFIX‐V181T abolished platelet (C) and fibrin accumulation (D) when TF was diluted by 20‐fold. Differences between control and treated groups were analyzed with 2‐way analysis of variance with Bonferroni posttest. Shaded areas represent standard deviation. CTI, corn trypsin inhibitor; DF, dilution factor; FI, fluorescence intensity; TF, tissue factor; WB, whole blood

Figure 6

Testing anti‐TFPI in hemophilia B assay. The effect of anti‐TFPI on platelet and fibrin deposition were investigated in healthy blood (A, B) and hemophilia B mimetic blood (C, D). Anti‐TFPI not only restored coagulation in hemophilia A model (D), but it also has a procoagulant effect in blood with normal FIX activity (B). This is in agreement with results of another anti‐TFPI (concizumab) from a previous clinical trial.34 Differences between control and treated groups were analyzed with 2‐way analysis of variance with Bonferroni posttest. *P < 0.05. Shaded areas represent standard deviation. CTI, corn trypsin inhibitor; FI, fluorescence intensity; n.s., nonsignificant; TF, tissue factor; TFPI, tissue factor pathway inhibitor; WB, whole blood