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 microg/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 micromol/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 microg/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.