Thiol isomerases constitute a family of enzymes that are commonly found in the endoplasmic reticulum (ER). These enzymes mediate disulfide bond formation, reduction, and rearrangements that are essential for protein folding. Platelets do not have an ER, but store and secrete thiol isomerases upon activation. Furthermore, thiol isomerases have been implicated in thrombus formation by facilitating redox remodeling of platelet β3 integrins and tissue factor decryption. Inhibition of thiol isomerase activity has thus been suggested as a potential antithrombotic strategy. However, assays that are suitable for measuring thiol isomerase activity in whole blood under flow condition are scarce. 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 PDI sensor 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 PDI 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 mg/mL corn trypsin inhibitor-treated human blood perfused over collagen (wall shear rate = 100 s-1), PDI-sAb signal increased mostly over the first 200 sec, while platelets continually accumulated for over 500 sec, indicating 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 localized in the core of growing thrombi where platelets displayed P-selectin and were in close proximity to disrupted endothelium. PDI-sAb is a sensitive and real time reporter of extracellular disulfide reduction that targets clots as they form under flow to reveal spatial gradients.

Platelet-targeting thiol reduction sensor detects protein disulfide isomerase activity on activated platelets in mouse and human blood under flow

251st National Meeting (2016)

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