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J Thromb Haemost. 2012 Nov;10(11):2344-53. doi: 10.1111/j.1538-7836.2012.04928.x.

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

Author information

1
Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering Department of Biochemistry and Molecular Biophysics Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Abstract

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.

KEYWORDS:

fibrin; permeation; platelet; shear rate; thrombin; tissue factor

PMID:
22978514
PMCID:
PMC4082909
DOI:
10.1111/j.1538-7836.2012.04928.x
[Indexed for MEDLINE]
Free PMC Article
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