Microfluidic assay of hemophilic blood clotting: distinct deficits in platelet and fibrin deposition at low factor levels.

BACKGROUND: Coagulation factor deficiencies create a range of bleeding phenotypes. Microfluidic devices offer controlled hemodynamics and defined procoagulant triggers for measurement of clotting under flow. OBJECTIVES: We tested a flow assay of contact pathway-triggered clotting to quantify platelet and fibrin deposition distal of dysfunctional thrombin production. Microfluidic metrics were then compared with PTT or % factor activity assays. METHODS: Whole blood (WB) treated with low level corn trypsin inhibitor (4 µg mL⁻¹) from nine healthy donors and 27 patients (deficient in factor [F] VIII, 19 patients; FIX, one patient; FXI, one patient; VWF, six patients) was perfused over fibrillar collagen at wall shear rate = 100 s⁻¹. RESULTS: Using healthy WB, platelets deposited within 30 s, while fibrin appeared within 6 min. Compared with healthy controls, WB from patients displayed a 50% reduction in platelet deposition only at < 1% factor activity. In contrast, striking defects in fibrin deposition occurred for patients with < 13% factor activity (or PTT > 40 s). Full occlusion of the 60-µm high channel was completely absent over the 15-min test in patients with < 1% factor activity, while an intermediate defect was present in patients with > 1% factor. CONCLUSION: Spontaneous bleeding in patients with < 1% factor activity may be linked to deficits in both platelet and fibrin deposition, a risk known to be mitigated when factor levels are raised to > 1% activity (PTT of ~40-60 s), a level that does not necessarily rescue fibrin formation under flow.

Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse.

BACKGROUND: Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics. METHODS AND RESULTS: An N-terminal-azido thrombin-sensitive fluorescent peptide (ThS-P) with a thrombin-releasable quencher was linked to anti-CD41 using click chemistry to generate a thrombin-sensitive platelet binding sensor (ThS-Ab). Rapid thrombin cleavage of ThS-P (K(m) = 40.3 µm, k(cat) = 1.5 s(-1) ) allowed thrombin monitoring by ThS-P or ThS-Ab in blood treated with 2-25 pm tissue factor (TF). Individual platelets had > 20-fold more ThS-Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s(-1) ), ThS-Ab fluorescence increased between 90 and 450 s for 0.1-1 molecule-TF µm(-2) and co-localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s(-1) ), thrombin and fibrin were detected at the thrombus-collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti-mouse CD41 ThS-Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co-localized and where the thrombus was most mechanically stable. CONCLUSION: ThS-Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients.