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Platelet‐targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse
Author(s) -
WELSH J. D.,
COLACE T. V.,
MUTHARD R. W.,
STALKER T. J.,
BRASS L. F.,
DIAMOND S. L.
Publication year - 2012
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2012.04928.x
Subject(s) - thrombin , fibrin , chemistry , platelet , biophysics , thrombus , tissue factor , platelet activation , microbiology and biotechnology , coagulation , immunology , biology , medicine
Summary. 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 μ m , k cat = 1.5 s −1 ) allowed thrombin monitoring by ThS‐P or ThS‐Ab in blood treated with 2–25 p m 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.