title: Platelet-targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow.
abstract: 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.
  
  
  
  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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA.|Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: S
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA.|Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA.|Department of Biochemistry and Molecular Biophysics, University of Pennsylvania, Philadelphia, PA, USA.|Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Welsh
authors.affiliation: Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA.|Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: L F
authors.initials: LF
authors.lastname: Brass
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA.|Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Diamond
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001783:Blood Flow Velocity; D001791:Blood Platelet Disorders / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D005337:Fibrin / Q000737:chemistry; D006439:Hemodynamics; D006801:Humans; D039661:Integrin beta3 / Q000737:chemistry; D000069416:Intravital Microscopy; D051379:Mice; D044085:Microfluidics; D010455:Peptides / Q000737:chemistry; D010973:Platelet Adhesiveness; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013917:Thrombin / Q000737:chemistry; D013927:Thrombosis / Q000378:metabolism
chemical_list: D000906:Antibodies; D039661:Integrin beta3; D010455:Peptides; D013438:Sulfhydryl Compounds; D005337:Fibrin; D013917:Thrombin; D019704:Protein Disulfide-Isomerases
keywords: blood platelets; microfluidics; platelet aggregation; protein disulfide isomerase; thrombosis