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Platelet‐targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow
Author(s) -
Zhu S.,
Welsh J. D.,
Brass L. F.,
Diamond S. L.
Publication year - 2016
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/jth.13245
Subject(s) - protein disulfide isomerase , chemistry , platelet , dithiothreitol , thiol , biochemistry , platelet activation , thrombin , microbiology and biotechnology , enzyme , biology , immunology
Essentials Protein disulfide isomerases may have an essential role in thrombus formation. A platelet‐binding sensor (PDI‐sAb) was developed to detect thiol reductase activity under flow. Primary human platelet adhesion to collagen at 200 s −1 was correlated with the PDI‐sAb signal. Detected thiol reductase activity was localized in the core of growing thrombi at the site of injury in mice.Summary Background Protein disulfide isomerases ( PDI s) may regulate thrombus formation in vivo , although the sources and targets of PDI s are not fully understood. Methods and results Using click chemistry to link anti‐ CD 61 and a C‐terminal azido disulfide‐linked peptide construct with a quenched reporter, we developed a fluorogenic platelet‐targeting antibody ( PDI ‐ sA b) for thiol reductase activity detection in whole blood under flow conditions. PDI ‐ sA b was highly responsive to various exogenous reducing agents (dithiothreitol, glutathione and recombinant PDI ) and detected thiol reductase activity on P‐selectin/phosphatidylserine‐positive platelets activated with convulxin/ PAR 1 agonist peptide, a signal partially blocked by PDI inhibitors and antibody. In a microfluidic thrombosis model using 4 μg mL −1 corn trypsin inhibitor‐treated human blood perfused over collagen (wall shear rate = 100 s −1 ), the PDI ‐ sA b signal increased mostly over the first 200 s, whereas platelets continually accumulated for over 500 s, indicating that primary adhesion to collagen, but not secondary aggregation, was correlated with the PDI ‐ sA b signal. Rutin and the PDI blocking antibody RL 90 reduced platelet adhesion and the PDI ‐ sA b signal only when thrombin production was inhibited with PPACK , suggesting limited effects of platelet thiol isomerase activity on platelet aggregation on collagen in the presence of thrombin. With anti‐mouse CD 41 PDI ‐ sA b used in an arteriolar laser injury model, thiol reductase activity was localized in the core of growing thrombi where platelets displayed P‐selectin and were in close proximity to disrupted endothelium. Conclusion PDI ‐ sA b is a sensitive and real‐time reporter of platelet‐ and vascular‐derived disulfide reduction that targets clots as they form under flow to reveal spatial gradients.