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 (PDIs) may regulate
thrombus formation in vivo, although the sources and targets of PDIs are not fully understood. Methods and results Using click chemistry to link anti-CD61 and a C-terminal azido
disulfide-linked
peptide construct with a quenched reporter, we developed a fluorogenic platelet-targeting antibody (PDI-sAb) for
thiol reductase activity detection in whole blood under flow conditions. PDI-sAb 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/PAR1 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-sAb 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-sAb signal.
Rutin and the PDI blocking antibody RL90 reduced platelet adhesion and the PDI-sAb 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 CD41 PDI-sAb 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-sAb 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.