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 mu M, 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 perpendicular to linked TF (wall shear rate = 100 s(-1)), ThS-Ab fluorescence increased between 90 and 450 s for 0.1-1 molecule-TF mu m(-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.