Microfluidic hemophilia models using blood from healthy donors.

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 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.