In prior microfluidic studies with haemophilic blood perfused over collagen, we found that a severe deficiency (<1% factor level) reduced platelet and fibrin deposition, while a moderate deficiency (1-5%) only reduced fibrin deposition. We investigated: (i) the differential effect of rFVIIa (0.04-20 nm) on platelet and fibrin deposition, and (ii) the contribution of the contact pathway to rFVIIa-induced haemophilic blood clotting. Haemophilic or healthy blood with low and high corn trypsin inhibitor (CTI, 4 or 40 μg mL(-1) ) was perfused over collagen at an initial venous wall shear rate of 100 s(-1) . At 100 s(-1) wall shear rate, where FXIIa leads to thrombin production without added tissue factor, FXI-deficient blood (3%) or severely FVIII-deficient blood (<1%) produced no fibrin at either CTI level. Whereas rFVIIa potently enhanced platelet deposition, fibrin generation was not rescued. Distinct from the high CTI condition, engagement of the contact pathway (low CTI) in moderately FVIII-deficient (3%) or moderately FIX-deficient blood (5%) resulted in enhanced platelet and fibrin deposition following 4 nm rFVIIa supplementation. In mildly FVIII-deficient blood (15%) at <24 h since haemostatic therapy, rFVIIa enhanced both platelet and fibrin generation in either CTI condition although fibrin was produced more quickly and abundantly in low CTI. For tissue factor-free conditions of severe haemophilic blood clotting, we conclude that rFVIIa reliably generates low levels of 'signaling' thrombin sufficient to enhance platelet deposition on collagen, but is insufficient to drive fibrin polymerization unless potentiated by the contact pathway.

Introduction Factor VIII or FIX-deficient hemophilic patients display deficits in platelet and fibrin deposition under flow detectable in microfluidics. Compared to fibrin generation, decreased platelet deposition in hemophilic blood flow is more easily rescued with recombinant factor VIIa (rFVIIa), whereas rFVIIa requires FXIIa participation to generate fibrin when tissue factor (TF) is absent. Aims Perfusion of hemophilic whole blood over collagen/TF surfaces was used to determine if rFVIIa/TF was sufficient to bypass poor FIXa/FVIIIa function in blood from hemophilia A and B patients. Methods Whole blood (WB) treated with high dose corn trypsin inhibitor (40 μg/ml) from 7 healthy donors and 10 patients was perfused over fibrillar collagen presenting low or high TF (TFlow or TFhigh) at wall shear rate of 100 s−1. Results With WB from healthy controls, platelet deposition and fibrin accumulation increased as TF was increased. Factor-deficient WB (1–3% of normal) displayed striking deficits in platelet deposition and fibrin formation at either TFlow or TFhigh. In contrast, mildly factor-deficient WB (14–32%) supported fibrin formation under flow on TFhigh/collagen. With either TFlow or TFhigh, exogenously added rFVIIa (20 nM) increased platelet deposition and fibrin accumulation in WB from factor-deficient patients (1–3% of normal) to levels commensurate with untreated healthy WB. Conclusion The absence of FIXa/FVIIIa in severe hemophilic patients results in deficits in fibrin formation that cannot be rescued by wall-derived TF ex vivo. The effects of rFVIIa on platelet adhesion and rFVIIa/TF can act together to reinforce thrombin generation, platelet deposition, and fibrin formation under flow.