Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse
Article first published online: 30 OCT 2012
© 2012 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 10, Issue 11, pages 2344–2353, November 2012
How to Cite
WELSH, J. D., COLACE, T. V., MUTHARD, R. W., STALKER, T. J., BRASS, L. F. and DIAMOND, S. L. (2012), Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse. Journal of Thrombosis and Haemostasis, 10: 2344–2353. doi: 10.1111/j.1538-7836.2012.04928.x
- Issue published online: 30 OCT 2012
- Article first published online: 30 OCT 2012
- Accepted manuscript online: 15 SEP 2012 03:43AM EST
- Received 31 May 2012, accepted 31 August 2012
- shear rate;
- tissue factor
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 (Km = 40.3 μm, kcat = 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.